elife03080.xml

<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN"  "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">03080</article-id><article-id pub-id-type="doi">10.7554/eLife.03080</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Biophysics and structural biology</subject></subj-group></article-categories><title-group><article-title>Cryo-EM structure of the <italic>Plasmodium falciparum</italic> 80S ribosome bound to the anti-protozoan drug emetine</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-13189"><name><surname>Wong</surname><given-names>Wilson</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-4"/><xref ref-type="other" rid="par-6"/><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/><xref ref-type="other" rid="dataro4"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-3345"><name><surname>Bai</surname><given-names>Xiao-chen</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-9"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/><xref ref-type="other" rid="dataro4"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-13190"><name><surname>Brown</surname><given-names>Alan</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-5"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/><xref ref-type="other" rid="dataro4"/></contrib><contrib contrib-type="author" id="author-3438"><name><surname>Fernandez</surname><given-names>Israel S</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="other" rid="par-5"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/><xref ref-type="other" rid="dataro4"/></contrib><contrib contrib-type="author" id="author-13191"><name><surname>Hanssen</surname><given-names>Eric</given-names></name><xref ref-type="aff" rid="aff4"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/><xref ref-type="other" rid="dataro4"/></contrib><contrib contrib-type="author" id="author-13192"><name><surname>Condron</surname><given-names>Melanie</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/><xref ref-type="other" rid="dataro4"/></contrib><contrib contrib-type="author" id="author-13193"><name><surname>Tan</surname><given-names>Yan Hong</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/><xref ref-type="other" rid="dataro4"/></contrib><contrib contrib-type="author" corresp="yes" id="author-13194"><name><surname>Baum</surname><given-names>Jake</given-names></name><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0002-0275-352X</contrib-id><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="fn" rid="pa1">‡</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-6"/><xref ref-type="other" rid="par-7"/><xref ref-type="other" rid="par-8"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/><xref ref-type="other" rid="dataro4"/></contrib><contrib contrib-type="author" corresp="yes" id="author-3361"><name><surname>Scheres</surname><given-names>Sjors HW</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/><xref ref-type="other" rid="dataro4"/></contrib><aff id="aff1"><institution content-type="dept">Division of Infection and Immunity</institution>, <institution>Walter and Eliza Hall Institute of Medical Research</institution>, <addr-line><named-content content-type="city">Melbourne</named-content></addr-line>, <country>Australia</country></aff><aff id="aff2"><institution content-type="dept">Department of Medical Biology</institution>, <institution>University of Melbourne</institution>, <addr-line><named-content content-type="city">Melbourne</named-content></addr-line>, <country>Australia</country></aff><aff id="aff3"><institution content-type="dept">Structural Studies</institution>, <institution>Medical Research Council Laboratory of Molecular Biology</institution>, <addr-line><named-content content-type="city">Cambridge</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff4"><institution content-type="dept">Electron Microscopy Unit</institution>, <institution>Bio21 Molecular Science and Biotechnology Institute, University of Melbourne</institution>, <addr-line><named-content content-type="city">Melbourne</named-content></addr-line>, <country>Australia</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Kühlbrandt</surname><given-names>Werner</given-names></name><role>Reviewing editor</role><aff><institution>Max Planck Institute of Biophysics</institution>, <country>Germany</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>jake.baum@imperial.ac.uk</email> (JB); </corresp><corresp id="cor2"><label>*</label>For correspondence: <email>scheres@mrc-lmb.cam.ac.uk</email> (SHWS)</corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn><fn fn-type="present-address" id="pa1"><label>‡</label><p>Department of Life Sciences, Imperial College London, London, United Kingdom</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>09</day><month>06</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03080</elocation-id><history><date date-type="received"><day>14</day><month>04</month><year>2014</year></date><date date-type="accepted"><day>06</day><month>06</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Wong et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Wong et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife03080.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03080.001</object-id><p>Malaria inflicts an enormous burden on global human health. The emergence of parasite resistance to front-line drugs has prompted a renewed focus on the repositioning of clinically approved drugs as potential anti-malarial therapies. Antibiotics that inhibit protein translation are promising candidates for repositioning. We have solved the cryo-EM structure of the cytoplasmic ribosome from the human malaria parasite, <italic>Plasmodium falciparum</italic>, in complex with emetine at 3.2 Å resolution. Emetine is an anti-protozoan drug used in the treatment of ameobiasis that also displays potent anti-malarial activity. Emetine interacts with the E-site of the ribosomal small subunit and shares a similar binding site with the antibiotic pactamycin, thereby delivering its therapeutic effect by blocking mRNA/tRNA translocation. As the first cryo-EM structure that visualizes an antibiotic bound to any ribosome at atomic resolution, this establishes cryo-EM as a powerful tool for screening and guiding the design of drugs that target parasite translation machinery.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.001">http://dx.doi.org/10.7554/eLife.03080.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03080.002</object-id><title>eLife digest</title><p>Each year, malaria kills more than 600,000 people, mostly children younger than 5 years old. Humans who have been bitten by mosquitoes infected with malaria-causing parasites become ill as the parasites rapidly multiply in blood cells. Although there are several drugs that are currently used to treat malaria, the parasites are rapidly developing resistance to them, setting off an urgent hunt for new malaria drugs.</p><p>Developing new antimalarial medications from scratch is likely to take decades—too long to combat the current public health threat posed by emerging strains of drug-resistant parasites. To speed up the process, scientists are investigating whether drugs developed for other illnesses may also act as therapies for malaria, either when used alone or in combination with existing malaria drugs.</p><p>Certain antibiotics—including one called emetine—have already shown promise as antimalarial drugs. These antibiotics prevent the parasites from multiplying by interfering with the ribosome—the part of a cell that builds new proteins. However, humans become ill after taking emetine for long periods because it also blocks the production of human proteins.</p><p>Tweaking emetine so that it acts only against the production of parasite proteins would make it a safer malaria treatment. To do this, scientists must first map the precise interactions between the drug and the ribosomes in parasites. Wong et al. have now used a technique called cryo-electron microscopy to examine the ribosome of the most virulent form of malaria parasite. This technique uses very cold temperatures to rapidly freeze molecules, allowing scientists to look at molecular-level details without distorting the structure of the molecule—a problem sometimes encountered in other techniques.</p><p>The images of the parasitic ribosome taken by Wong, Bai, Brown et al. show that emetine binds to the end of the ribosome where the instructions for how to assemble amino acids into a protein are copied from strands of RNA. In addition, the images revealed features of the parasitic ribosome that are not found in the human form. Drug makers could exploit these features to improve emetine so that it more specifically targets the production of proteins by the parasite and is less toxic to humans.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.002">http://dx.doi.org/10.7554/eLife.03080.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>malaria</kwd><kwd><italic>Plasmodium falciparum</italic></kwd><kwd>ribosome</kwd><kwd>drug development</kwd><kwd>cryo-EM</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>other</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100000925</institution-id><institution>National Health and Medical Research Council</institution></institution-wrap></funding-source><award-id>APP1024678, APP1053801</award-id><principal-award-recipient><name><surname>Wong</surname><given-names>Wilson</given-names></name><name><surname>Condron</surname><given-names>Melanie</given-names></name><name><surname>Tan</surname><given-names>Yan Hong</given-names></name><name><surname>Baum</surname><given-names>Jake</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100000854</institution-id><institution>Human Frontier Science Program</institution></institution-wrap></funding-source><award-id>RGY007½011</award-id><principal-award-recipient><name><surname>Tan</surname><given-names>Yan Hong</given-names></name><name><surname>Baum</surname><given-names>Jake</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100000265</institution-id><institution>Medical Research Council</institution></institution-wrap></funding-source><award-id>MC_UP_A025_1013, MC_U105184332</award-id><principal-award-recipient><name><surname>Bai</surname><given-names>Xiao-chen</given-names></name><name><surname>Brown</surname><given-names>Alan</given-names></name><name><surname>Fernandez</surname><given-names>Israel S</given-names></name><name><surname>Scheres</surname><given-names>Sjors HW</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>OzeMalar</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Wong</surname><given-names>Wilson</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100004440</institution-id><institution>Wellcome Trust</institution></institution-wrap></funding-source><award-id>WT096570, 100993/Z/13/Z</award-id><principal-award-recipient><name><surname>Brown</surname><given-names>Alan</given-names></name><name><surname>Fernandez</surname><given-names>Israel S</given-names></name><name><surname>Baum</surname><given-names>Jake</given-names></name></principal-award-recipient></award-group><award-group id="par-6"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100000934</institution-id><institution>Department of Industry, Innovation, Science, Research and Tertiary Education, Australian Government</institution></institution-wrap></funding-source><award-id>FT100100112</award-id><principal-award-recipient><name><surname>Wong</surname><given-names>Wilson</given-names></name><name><surname>Baum</surname><given-names>Jake</given-names></name></principal-award-recipient></award-group><award-group id="par-7"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100000923</institution-id><institution>Australian Research Council</institution></institution-wrap></funding-source><award-id>FT100100112</award-id><principal-award-recipient><name><surname>Wong</surname><given-names>Wilson</given-names></name><name><surname>Baum</surname><given-names>Jake</given-names></name></principal-award-recipient></award-group><award-group id="par-8"><funding-source><institution-wrap><institution>Victorian State Government Operational Infrastructure Support</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Baum</surname><given-names>Jake</given-names></name></principal-award-recipient></award-group><award-group id="par-9"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100000780</institution-id><institution>European Commission</institution></institution-wrap></funding-source><award-id>EU FP7 Marie Curie</award-id><principal-award-recipient><name><surname>Bai</surname><given-names>Xiao-chen</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>The molecular mechanism behind how emetine inhibits the ribosome of the human malaria parasite, along with structural details of the complex formed, is revealed at high resolution using cryo-electron microscopy.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Malaria is responsible for an estimated 627,000 annual deaths worldwide, with the majority of victims being children under 5 years of age (<xref ref-type="bibr" rid="bib56">WHO, 2012</xref>). At present there is no licensed malaria vaccine and parasites have developed resistance to all front-line anti-malarial drugs. As such, there is an urgent need for novel therapeutics that can be used as monotherapies or as partner drugs for combinatorial regimes (<xref ref-type="bibr" rid="bib33">Kremsner and Krishna, 2004</xref>). An alternative to novel candidates is the repurposing or repositioning of clinically approved drugs that can be used in combination with known anti-malarials, such as chloroquine, antifolates, and artemisinin, to increase their useable lifespan by reducing resistance (<xref ref-type="bibr" rid="bib23">Grimberg and Mehlotra, 2011</xref>).</p><p>The etiological agents for malaria are a family of unicellular protozoan pathogens of the genus <italic>Plasmodium</italic>. The parasite has a complex two-host lifecycle with a sexual stage occurring in the mosquito vector and an asexual stage in the human host. It is during the asexual blood stage that disease symptoms in humans first appear, including those associated with severe malaria, and it is often at this stage that the need for clinical intervention becomes apparent (<xref ref-type="bibr" rid="bib41">Miller et al., 2002</xref>). Much of malaria pathology is the result of exponential growth of the parasite within erythrocytes, and given the critical role that protein synthesis plays in this, the translational machinery is an attractive drug target.</p><p>Protein translation in the parasite is focused on three centers (<xref ref-type="bibr" rid="bib27">Jackson et al., 2011</xref>): the cytoplasmic ribosome, responsible for the vast majority of protein synthesis, and organellar ribosomes of the mitochondrion and non-photosynthetic plastid, termed the apicoplast (<xref ref-type="bibr" rid="bib40">McFadden et al., 1996</xref>). In addition, and unusually for a eukaryotic cell, <italic>Plasmodium</italic> species have two distinct types of cytoplasmic ribosome that differ in their ribosomal RNA (rRNA) composition. These are expressed at different stages of the lifecycle, one predominantly in the mosquito vector and the other in the mammalian host, with evidence that both can occur simultaneously for limited periods (<xref ref-type="bibr" rid="bib55">Waters et al., 1989</xref>).</p><p>Antibiotics known to target the apicoplast ribosome, such as the macrolide azithromycin, demonstrate a delayed-death effect, whereby treated parasites die in the second generation of drug exposure, and therefore have slow clinical onset (<xref ref-type="bibr" rid="bib15">Dahl and Rosenthal, 2007</xref> ; <xref ref-type="bibr" rid="bib21">Goodman et al., 2007</xref> ). However, because anti-malarial treatment at the blood-stage requires rapid intervention, we focused on the dominant, blood stage-specific cytoplasmic ribosome from the most virulent form of <italic>Plasmodium, P. falciparum</italic> (<italic>Pf</italic>80S) (<xref ref-type="bibr" rid="bib55">Waters et al., 1989</xref>), as inhibition of cytosolic translation would be expected to be direct and fast-acting. <italic>Pf</italic>80S is both a candidate for development of novel therapeutics that target specific differences between itself and its counterpart in the human cytosol, and also for repurposing of anti-protozoan inhibitors, such as emetine (<xref ref-type="bibr" rid="bib39">Matthews et al., 2013</xref>).</p><p>In this present study, we solved the structure of <italic>Pf</italic>80S–emetine complex at 3.2 Å resolution and built a fully-refined all-atom model. This represents, to our knowledge, the first structure of an entire eukaryotic ribosome at atomic resolution solved by electron cryo-microscopy (cryo-EM). <italic>Pf</italic>80S has a broad distribution of <italic>Pf</italic>-specific elements across its surface, with particularly long rRNA expansion segments (ESs) in the small subunit. The atomic structure of <italic>Pf</italic>80S in complex with emetine reveals the molecular basis of this clinically relevant anti-protozoan translation inhibitor. In doing so, we establish cryo-EM as a powerful tool for structure-based drug design.</p></sec><sec id="s2" sec-type="results"><title>Results</title><p>Cytoplasmic ribosomes were isolated from the 3D7 strain of <italic>P. falciparum</italic> parasites maintained in human erythrocytes (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>). Limitations in parasite culture volume, yielding ∼10<sup>10</sup> parasitized red blood cells and low yield of sample material (1 g of parasites yielded 0.35 mg <italic>Pf</italic>80S), precluded an ability to crystallize <italic>Pf</italic>80S to solve the structure by conventional X-ray crystallography. We therefore exploited recent advances in direct electron detection and statistical image processing (<xref ref-type="bibr" rid="bib4">Bai et al., 2013</xref>; <xref ref-type="bibr" rid="bib1">Allegretti et al., 2014</xref>) to determine the structure by cryo-EM at an overall resolution of 3.2 Å (<xref ref-type="fig" rid="fig1">Figure 1C–E</xref>, <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03080.003</object-id><label>Figure 1.</label><caption><title>Cryo-EM data and processing.</title><p>(<bold>A</bold>) Sucrose gradient purification of <italic>Pf</italic>80S ribosomes. (<bold>B</bold>) Representative electron micrograph showing <italic>Pf</italic>80S particles. (<bold>C</bold>) Fourier Shell Correlation (FSC) curves indicating the overall resolutions of unmasked (red), <italic>Pf</italic>40S masked (green) and <italic>Pf</italic>60S masked (blue) reconstructions of the <italic>Pf</italic>80S–emetine complex. (<bold>D</bold>) Representative density with built models of a β-strand with well-resolved side chains (left), an RNA segment with separated bases (middle), and a magnesium ion (green sphere) that is coordinated by RNA backbone phosphates. (<bold>E</bold>) Density maps colored according to local resolution for the unmasked <italic>Pf</italic>80S (left) and masked <italic>Pf</italic>40S and <italic>Pf</italic>60S subunits (right).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.003">http://dx.doi.org/10.7554/eLife.03080.003</ext-link></p></caption><graphic xlink:href="elife03080f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03080.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>FSC curves between the final refined atomic model and the reconstructions from all particles (black); between the model refined in the reconstruction from only half of the particles and the reconstruction from that same half (FSC<sub>work</sub>, red); and between that same model and the reconstruction from the other half of the particles (FSC<sub>test</sub>, green), for <italic>Pf</italic>40S (A) and <italic>Pf</italic>60S (B).</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.004">http://dx.doi.org/10.7554/eLife.03080.004</ext-link></p></caption><graphic xlink:href="elife03080fs001"/></fig></fig-group></p><p>Protein side chains and RNA bases were clearly resolved in our maps (<xref ref-type="fig" rid="fig1">Figure 1D</xref>). The use of model building and refinement tools that were adapted from X-ray crystallography (<xref ref-type="bibr" rid="bib2">Amunts et al., 2014</xref>) led to a near-complete atomic model with excellent geometrical properties (<xref ref-type="fig" rid="fig2">Figure 2A,B</xref>; <xref ref-type="table" rid="tbl1">Table 1</xref>). The ribosome model comprises the large (<italic>Pf</italic>60S) and small subunit (<italic>Pf</italic>40S) with a total of 74 proteins (<xref ref-type="table" rid="tbl2 tbl3">Tables 2 and 3</xref>) as well as the 5S, 5.8S, 18S, and 28S rRNAs and a tRNA bound at the E-site. The head region of <italic>Pf</italic>40S has weaker density than the rest of the ribosome due to the inherent flexibility at the neck (centered around h28). This meant that eS31, located in the beak of the 40S head (<xref ref-type="bibr" rid="bib48">Rabl et al., 2011</xref>), could not be positioned accurately, and has therefore been omitted from the final model. Using base-pair information extracted directly from the atomic model it was possible to revise secondary structure diagrams for <italic>P. falciparum</italic> rRNA (<xref ref-type="fig" rid="fig2s1 fig2s2 fig2s3">Figure 2—figure supplements 1–3</xref>), facilitating comparison with rRNA of other species.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03080.005</object-id><label>Figure 2.</label><caption><title>Structure of the <italic>Pf</italic>80S ribosome.</title><p>Overview of <italic>Pf</italic>80S atomic model showing views facing (<bold>A</bold>) tRNA entry side and (<bold>B</bold>) tRNA exit side. rRNAs are shown in gray, proteins numbered according to <xref ref-type="bibr" rid="bib5">Ban et al. (2014)</xref>. (<bold>C</bold> and <bold>D</bold>) <italic>Pf</italic>40S and <italic>Pf</italic>60S subunits are colored in yellow and blue respectively. Flexible regions are shown in red and at a resolution of 6 Å. <italic>Pf</italic>-specific expansion segments (ESs) relative to human ribosomes are labeled. Their numbering is as described for the human cytoplasmic ribosome (<xref ref-type="bibr" rid="bib3">Anger et al., 2013</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.005">http://dx.doi.org/10.7554/eLife.03080.005</ext-link></p></caption><graphic xlink:href="elife03080f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03080.006</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Secondary structure of <italic>Pf</italic>18S rRNAs.</title><p><italic>Pf</italic>-specific ESs are highlighted in a labeled red box. Regions not built in the atomic model are colored in blue text. The secondary structure was modified from the CRW site (<xref ref-type="bibr" rid="bib10">Cannone et al., 2002</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.006">http://dx.doi.org/10.7554/eLife.03080.006</ext-link></p></caption><graphic xlink:href="elife03080fs002"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03080.007</object-id><label>Figure 2—figure supplement 2.</label><caption><title>Secondary structure of the 5′ half of <italic>Pf</italic> 28S rRNA.</title><p><italic>Pf</italic>-specific ESs are highlighted in a labeled red box. Regions not built in the atomic model are colored in blue text. The secondary structure was modified from the CRW site (<xref ref-type="bibr" rid="bib10">Cannone et al., 2002</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.007">http://dx.doi.org/10.7554/eLife.03080.007</ext-link></p></caption><graphic xlink:href="elife03080fs003"/></fig><fig id="fig2s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03080.008</object-id><label>Figure 2—figure supplement 3.</label><caption><title>Secondary structure of the 3′ half of <italic>Pf</italic>28S rRNA.</title><p><italic>Pf</italic>-specific ESs are highlighted in a labeled red box. Regions not built in the atomic model are colored in blue text. The secondary structure was modified from the CRW site (<xref ref-type="bibr" rid="bib10">Cannone et al., 2002</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.008">http://dx.doi.org/10.7554/eLife.03080.008</ext-link></p></caption><graphic xlink:href="elife03080fs004"/></fig></fig-group><table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03080.009</object-id><label>Table 1.</label><caption><p>Refinement and model statistics</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.009">http://dx.doi.org/10.7554/eLife.03080.009</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th/><th><italic>Pf80S–emetine</italic></th></tr></thead><tbody><tr><td>Data collection</td><td/></tr><tr><td> Particles</td><td>105,247</td></tr><tr><td> Pixel size (Å)</td><td>1.34</td></tr><tr><td> Defocus range (μm)</td><td>0.8–3.8</td></tr><tr><td> Voltage (kV)</td><td>300</td></tr><tr><td> Electron dose (e<sup>−</sup> Å<sup>−2</sup>)</td><td>20</td></tr></tbody></table><table frame="hsides" rules="groups"><thead><tr><th/><th><italic>Pf</italic>60S</th><th><italic>Pf</italic>40S</th></tr></thead><tbody><tr><td>Model composition</td><td/><td/></tr><tr><td> Non-hydrogen atoms</td><td>124,509</td><td>68,858</td></tr><tr><td> Protein residues</td><td>6,244</td><td>4,106</td></tr><tr><td> RNA bases</td><td>3,460</td><td>1,682</td></tr><tr><td> Ligands (Zn<sup>2+</sup>/Mg<sup>2+</sup>/emetine)</td><td>5/163/0</td><td>1/67/1</td></tr><tr><td>Refinement</td><td/><td/></tr><tr><td> Resolution used for refinement (Å)</td><td>3.1</td><td>3.3</td></tr><tr><td> Map sharpening B-factor (Å<sup>2</sup>)</td><td>−60.3</td><td>−79.9</td></tr><tr><td> Average B factor (Å<sup>2</sup>)</td><td>113.1</td><td>143.2</td></tr><tr><td> Rfactor<xref ref-type="table-fn" rid="tblfn1">*</xref></td><td>0.2294</td><td>0.257</td></tr><tr><td> Fourier Shell Correlation<xref ref-type="table-fn" rid="tblfn2">†</xref></td><td>0.86</td><td>0.854</td></tr><tr><td>Rms deviations</td><td/><td/></tr><tr><td> Bonds (Å)</td><td>0.006</td><td>0.007</td></tr><tr><td> Angles (°)</td><td>1.20</td><td>1.29</td></tr><tr><td>Validation (proteins)</td><td/><td/></tr><tr><td> Molprobity score</td><td>2.45 (96<sup>th</sup> percentile)</td><td>2.73 (95<sup>th</sup> percentile)</td></tr><tr><td> Clashscore, all atoms</td><td>3.65 (100<sup>th</sup> percentile)</td><td>4.23 (100<sup>th</sup> percentile)</td></tr><tr><td> Good rotamers (%)</td><td>90.0</td><td>86.0</td></tr><tr><td>Ramachandran plot</td><td/><td/></tr><tr><td> Favored (%)</td><td>90.4</td><td>85.4</td></tr><tr><td> Outliers (%)</td><td>2.4</td><td>4.2</td></tr><tr><td>Validation (RNA)</td><td/><td/></tr><tr><td> Correct sugar puckers (%)</td><td>97.3</td><td>97.5</td></tr><tr><td> Good backbone conformations (%)</td><td>71.1</td><td>70.0</td></tr></tbody></table><table-wrap-foot><fn id="tblfn1"><label>*</label><p>Rfactor = Σ||F<sub>obs</sub>| − ||F<sub>calc</sub>|/Σ|F<sub>obs</sub>|.</p></fn><fn id="tblfn2"><label>†</label><p>FSC<sub>overall</sub> = Σ(N<sub>shell</sub> FSC<sub>shell</sub>)/Σ(N<sub>shell</sub>), where FSC<sub>shell</sub> is the FSC in a given shell, N<sub>shell</sub> is the number of ‘structure factors’ in the shell. FSC<sub>shell</sub> = Σ(F<sub>model</sub> F<sub>EM</sub>)/(√(Σ(|F|<sup>2</sup><sub>model</sub>)) √(Σ(F<sup>2</sup><sub>EM</sub>)).</p></fn></table-wrap-foot></table-wrap><table-wrap id="tbl2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03080.010</object-id><label>Table 2.</label><caption><p>Ribosomal proteins of the <italic>Pf</italic>40S subunit</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.010">http://dx.doi.org/10.7554/eLife.03080.010</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Protein names</th><th>Uniprot ID</th><th>PlasmoDB ID</th><th>Chain ID</th><th>Built residues</th><th>Extensions compared to human</th><th>Total number of residues</th></tr></thead><tbody><tr><td>eS1</td><td>RS3A_PLAF7</td><td>PF3D7_0322900</td><td>B</td><td>24–233</td><td>245–262</td><td>262</td></tr><tr><td>uS2</td><td>RSSA_PLAF7</td><td>PF3D7_1026800</td><td>C</td><td>10–204</td><td>–</td><td>263</td></tr><tr><td>uS3</td><td>Q8IKH8_PLAF7</td><td>PF3D7_1465900</td><td>D</td><td>4–39; 65–78; 97–193; 207–216</td><td>–</td><td>221</td></tr><tr><td>uS4</td><td>Q8I3R0_PLAF7</td><td>PF3D7_0520000</td><td>E</td><td>2–186</td><td>–</td><td>189</td></tr><tr><td>eS4</td><td>Q8IIU8_PLAF7</td><td>PF3D7_1105400</td><td>F</td><td>2–258</td><td>–</td><td>261</td></tr><tr><td>uS5</td><td>Q8IL02_PLAF7</td><td>PF3D7_1447000</td><td>G</td><td>39–262</td><td>–</td><td>272</td></tr><tr><td>eS6</td><td>Q8IDR9_PLAF7</td><td>PF3D7_1342000</td><td>H</td><td>1–160; 170–213</td><td>249–306</td><td>306</td></tr><tr><td>uS7</td><td>Q8IBN5_PLAF7</td><td>PF3D7_0721600</td><td>I</td><td>7–118; 128–195</td><td>–</td><td>195</td></tr><tr><td>eS7</td><td>Q8IET7_PLAF7</td><td>PF3D7_1302800</td><td>J</td><td>3–190</td><td>–</td><td>194</td></tr><tr><td>uS8</td><td>O77395_PLAF7</td><td>PF3D7_0316800</td><td>K</td><td>2–130</td><td>–</td><td>130</td></tr><tr><td>eS8</td><td>Q8IM10_PLAF7</td><td>PF3D7_1408600</td><td>L</td><td>5–120; 161–213; 216–218</td><td>154–163</td><td>218</td></tr><tr><td>uS9</td><td>Q8IAX5_PLAF7</td><td>PF3D7_0813900</td><td>M</td><td>6–143</td><td>–</td><td>144</td></tr><tr><td>uS10</td><td>Q8IK02_PLAF7</td><td>PF3D7_1003500</td><td>N</td><td>21–118</td><td>–</td><td>118</td></tr><tr><td>eS10</td><td>Q8IBQ5_PLAF7</td><td>PF3D7_0719700</td><td>O</td><td>11–89</td><td>–</td><td>137</td></tr><tr><td>uS11</td><td>Q8I3U6_PLAF7</td><td>PF3D7_0516200</td><td>P</td><td>25–151</td><td>–</td><td>151</td></tr><tr><td>uS12</td><td>O97248_PLAF7</td><td>PF3D7_0306900</td><td>Q</td><td>2–145</td><td>–</td><td>145</td></tr><tr><td>eS12</td><td>RS12_PLAF7</td><td>PF3D7_0307100</td><td>R</td><td>22–78; 85–100; 111–135</td><td>10–16</td><td>141</td></tr><tr><td>uS13</td><td>Q8IIA2_PLAF7</td><td>PF3D7_1126200</td><td>S</td><td>12–139</td><td>–</td><td>156</td></tr><tr><td>uS14</td><td>C0H4K8_PLAF7</td><td>PF3D7_0705700</td><td>T</td><td>7–54</td><td>–</td><td>54</td></tr><tr><td>uS15</td><td>Q8IDB0_PLAF7</td><td>PF3D7_1358800</td><td>U</td><td>3–151</td><td>–</td><td>151</td></tr><tr><td>uS17</td><td>O77381_PLAF7</td><td>PF3D7_0317600</td><td>V</td><td>6–25; 36–161</td><td>–</td><td>161</td></tr><tr><td>eS17</td><td>Q8I502_PLAF7</td><td>PF3D7_1242700</td><td>W</td><td>3–83; 97–110</td><td>–</td><td>137</td></tr><tr><td>uS19</td><td>C0H5C2_PLAF7</td><td>PF3D7_1317800</td><td>X</td><td>21–95; 103–123</td><td>–</td><td>145</td></tr><tr><td>eS19</td><td>Q8IFP2_PLAF7</td><td>PF3D7_0422400</td><td>Y</td><td>15–168</td><td>1–19</td><td>170</td></tr><tr><td>eS21</td><td>Q8IHS5_PLAF7</td><td>PF3D7_1144000</td><td>Z</td><td>11–82</td><td>–</td><td>82</td></tr><tr><td>eS24</td><td>Q8I3R6_PLAF7</td><td>PF3D7_0519400</td><td>1</td><td>3–122</td><td>–</td><td>133</td></tr><tr><td>eS25</td><td>Q8ILN8_PLAF7</td><td>PF3D7_1421200</td><td>2</td><td>35–42; 58–84; 97–102</td><td>–</td><td>105</td></tr><tr><td>eS26</td><td>O96258_PLAF7</td><td>PF3D7_0217800</td><td>3</td><td>2–96</td><td>–</td><td>107</td></tr><tr><td>eS27</td><td>Q8IEN2_PLAF7</td><td>PF3D7_1308300</td><td>4</td><td>7–82</td><td>–</td><td>82</td></tr><tr><td>eS28</td><td>Q8IKL9_PLAF7</td><td>PF3D7_1461300</td><td>5</td><td>2–29; 37–66</td><td>–</td><td>67</td></tr><tr><td>eS30</td><td>RS30_PLAF7</td><td>PF3D7_0219200</td><td>6</td><td>6–48</td><td>–</td><td>58</td></tr><tr><td>eS31</td><td>Q8IM64_PLAF7</td><td>PF3D7_1402500</td><td>–</td><td>Not built</td><td>–</td><td>149</td></tr></tbody></table></table-wrap><table-wrap id="tbl3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03080.011</object-id><label>Table 3.</label><caption><p>Ribosomal proteins of the <italic>Pf</italic>60S subunit</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.011">http://dx.doi.org/10.7554/eLife.03080.011</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Protein names</th><th>Uniprot ID</th><th>PlasmoDB ID</th><th>Chain ID</th><th>Built residues</th><th>Extensions compared to human</th><th>Total number of residues</th></tr></thead><tbody><tr><td>uL2</td><td>Q8I3T9_PLAF7</td><td>PF3D7_0516900</td><td>D</td><td>2–248</td><td>–</td><td>260</td></tr><tr><td>uL3</td><td>Q8IJC6_PLAF7</td><td>PF3D7_1027800</td><td>E</td><td>2–381</td><td>–</td><td>386</td></tr><tr><td>uL4</td><td>Q8I431_PLAF7</td><td>PF3D7_0507100</td><td>F</td><td>6–395</td><td>373–411</td><td>411</td></tr><tr><td>uL5</td><td>Q8IBQ6_PLAF7</td><td>PF3D7_0719600</td><td>G</td><td>8–51; 64–85; 92–106; 124–166</td><td>–</td><td>173</td></tr><tr><td>uL6</td><td>Q8IE85_PLAF7</td><td>PF3D7_1323100</td><td>H</td><td>2–186</td><td>–</td><td>190</td></tr><tr><td>eL6</td><td>Q8IDV1_PLAF7</td><td>PF3D7_1338200</td><td>I</td><td>9–151; 158–221</td><td>110–118; 139–143; 174–182</td><td>221</td></tr><tr><td>eL8</td><td>Q8ILL2_PLAF7</td><td>PF3D7_1424400</td><td>J</td><td>40–46; 54–131; 147–283</td><td>11–24;279–283</td><td>283</td></tr><tr><td>uL13</td><td>Q8IJZ7_PLAF7</td><td>PF3D7_1004000</td><td>K</td><td>1–201</td><td>–</td><td>202</td></tr><tr><td>eL13</td><td>Q8IAX6_PLAF7</td><td>PF3D7_0814000</td><td>L</td><td>2–212</td><td>134–141; 168–174</td><td>215</td></tr><tr><td>uL14</td><td>Q8IE09_PLAF7</td><td>PF3D7_1331800</td><td>M</td><td>8–139</td><td>–</td><td>139</td></tr><tr><td>eL14</td><td>Q8ILE8_PLAF7</td><td>PF3D7_1431700</td><td>N</td><td>5–150</td><td>1–18</td><td>165</td></tr><tr><td>uL15</td><td>C6KT23_PLAF7</td><td>PF3D7_0618300</td><td>O</td><td>2–148</td><td>–</td><td>148</td></tr><tr><td>eL15</td><td>C0H4A6_PLAF7</td><td>PF3D7_0415900</td><td>P</td><td>2–205</td><td>–</td><td>205</td></tr><tr><td>uL16</td><td>Q8ILV2_PLAF7</td><td>PF3D7_1414300</td><td>Q</td><td>2–101; 118–206</td><td>–</td><td>219</td></tr><tr><td>uL18</td><td>Q8ILL3_PLAF7</td><td>PF3D7_1424100</td><td>R</td><td>5–126; 141–185; 189–250; 271–293</td><td>–</td><td>294</td></tr><tr><td>eL18</td><td>C0H5G3_PLAF7</td><td>PF3D7_1341200</td><td>U</td><td>5–184</td><td>–</td><td>184</td></tr><tr><td>eL19</td><td>C6KSY6_PLAF7</td><td>PF3D7_0614500</td><td>T</td><td>2–182</td><td>–</td><td>182</td></tr><tr><td>eL20</td><td>Q8IDS6_PLAF7</td><td>PF3D7_1341200</td><td>S</td><td>2–187</td><td>–</td><td>184</td></tr><tr><td>eL21</td><td>Q8ILK3_PLAF7</td><td>PF3D7_1426000</td><td>V</td><td>4–158</td><td>–</td><td>161</td></tr><tr><td>uL22</td><td>Q8IDI5_PLAF7</td><td>PF3D7_1351400</td><td>W</td><td>4–154; 197–215</td><td>–</td><td>203</td></tr><tr><td>eL22</td><td>Q8IB51_PLAF7</td><td>PF3D7_0821700</td><td>X</td><td>40–136</td><td>4–18; 34–38</td><td>139</td></tr><tr><td>uL23</td><td>Q8IE82_PLAF7</td><td>PF3D7_1323400</td><td>Y</td><td>88–188</td><td>13–34; 57–67</td><td>190</td></tr><tr><td>uL24</td><td>O77364_PLAF7</td><td>PF3D7_0312800</td><td>Z</td><td>2–122</td><td>–</td><td>126</td></tr><tr><td>eL24</td><td>Q8IEM3_PLAF7</td><td>PF3D7_1309100</td><td>0</td><td>8–69</td><td>–</td><td>162</td></tr><tr><td>eL27</td><td>Q8IKM5_PLAF7</td><td>PF3D7_1460700</td><td>1</td><td>2–126;132–146</td><td>–</td><td>146</td></tr><tr><td>eL28</td><td>Q8IHU0_PLAF7</td><td>PF3D7_1142500</td><td>2</td><td>2–69; 77–82; 86–98; 103–119</td><td>–</td><td>127</td></tr><tr><td>uL29</td><td>Q8IIB4_PLAF7</td><td>PF3D7_1124900</td><td>3</td><td>3–121</td><td>–</td><td>124</td></tr><tr><td>eL29</td><td>C6S3J6_PLAF7</td><td>PF3D7_1460300</td><td>4</td><td>2–67</td><td>–</td><td>67</td></tr><tr><td>uL30</td><td>O97250_PLAF7</td><td>PF3D7_0307200</td><td>5</td><td>35–257</td><td>–</td><td>257</td></tr><tr><td>eL30</td><td>Q8IJK8_PLAF7</td><td>PF3D7_1019400</td><td>6</td><td>8–105</td><td>–</td><td>108</td></tr><tr><td>eL31</td><td>Q8I463_PLAF7</td><td>PF3D7_0503800</td><td>7</td><td>15–88; 95–116</td><td>–</td><td>120</td></tr><tr><td>eL32</td><td>Q8I3B0_PLAF7</td><td>PF3D7_0903900</td><td>8</td><td>2–126</td><td>–</td><td>131</td></tr><tr><td>eL33</td><td>Q8IHT9_PLAF7</td><td>PF3D7_1142600</td><td>9</td><td>35–137</td><td>1–35</td><td>140</td></tr><tr><td>eL34</td><td>Q8IBY4_PLAF7</td><td>PF3D7_0710600</td><td>a</td><td>2–107</td><td>–</td><td>150</td></tr><tr><td>eL36</td><td>Q8I713_PLAF7</td><td>PF3D7_1109900</td><td>b</td><td>2–27; 38–106</td><td>5–10</td><td>112</td></tr><tr><td>eL37</td><td>C0H4L5_PLAF7</td><td>PF3D7_0706400</td><td>c</td><td>2–90</td><td>–</td><td>92</td></tr><tr><td>eL38</td><td>Q8II62_PLAF7</td><td>PF3D7_1130100</td><td>d</td><td>2–31; 36–77</td><td>–</td><td>87</td></tr><tr><td>eL39</td><td>C0H4H3_PLAF7</td><td>PF3D7_0611700</td><td>e</td><td>2–30; 38–51</td><td>–</td><td>51</td></tr><tr><td>eL40</td><td>Q8ID50_PLAF7</td><td>PF3D7_1365900</td><td>f</td><td>1–51</td><td>–</td><td>52</td></tr><tr><td>eL41</td><td>C6S3G4_PLAF7</td><td>PF3D7_1144300</td><td>g</td><td>3–39</td><td>1–14</td><td>39</td></tr><tr><td>eL43</td><td>RL37A_PLAF7</td><td>PF3D7_0210100.1</td><td>h</td><td>2–86</td><td>–</td><td>96</td></tr><tr><td>eL44</td><td>RL44_PLAF7</td><td>PF3D7_0304400</td><td>i</td><td>2–96</td><td>–</td><td>104</td></tr></tbody></table></table-wrap></p><p>Currently, high resolution structures of eukaryotic ribosomes have been solved using X-ray crystallography and are limited to just three structures; the individual subunits from a ciliated protozoan, <italic>Tetrahymena thermophila</italic> (<xref ref-type="bibr" rid="bib31">Klinge et al., 2011</xref>; <xref ref-type="bibr" rid="bib48">Rabl et al., 2011</xref>), and the complete 80S ribosome from the yeast <italic>Saccharomyces cerevisiae</italic> (<xref ref-type="bibr" rid="bib8">Ben-Shem et al., 2011</xref>). These models have been used to interpret lower resolution structures solved by cryo-EM of other species including the yeast <italic>Kluyveromyces lactis</italic> (<xref ref-type="bibr" rid="bib19">Fernandez et al., 2014</xref>), <italic>Drosophila melanogaster</italic> (<xref ref-type="bibr" rid="bib3">Anger et al., 2013</xref>), <italic>Trypanosoma brucei</italic> (<xref ref-type="bibr" rid="bib25">Hashem et al., 2013</xref>), as well as human ribosomes (<xref ref-type="bibr" rid="bib3">Anger et al., 2013</xref>) and provide the basis of the nomenclature used for describing the structures.</p><p>To examine overall architectural differences, we compared the model of <italic>Pf</italic>80S to yeast 80S (<xref ref-type="bibr" rid="bib8">Ben-Shem et al., 2011</xref>). Perhaps the largest difference is the absence of RACK1 (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>), which associates with the head of the 40S in the vicinity of the mRNA exit channel (<xref ref-type="bibr" rid="bib52">Sengupta et al., 2004</xref>; <xref ref-type="bibr" rid="bib48">Rabl et al., 2011</xref>) and has been identified in all eukaryotic ribosome structures solved to-date. RACK1 serves as a signaling scaffold that can recruit other proteins to the ribosome and may link the ribosome with signal transduction pathways, thus allowing translation regulation in response to stimuli. It has also been proposed that RACK1 promotes the docking of ribosomes at sites where local translation is required (<xref ref-type="bibr" rid="bib45">Nilsson et al., 2004</xref>).</p><p><italic>Pf</italic>RACK1 is conserved with its human homolog with an identity of 60%. The binding site on the ribosome, which comprises eS17, uS3, and 18S rRNA helices h39 and h40 (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>), also appears highly conserved (<xref ref-type="bibr" rid="bib48">Rabl et al., 2011</xref>). However, the C-terminus of uS3 is not resolved in our structure and probably only becomes ordered upon binding RACK1. The absence of <italic>Pf</italic>RACK1 as an integral member of the small subunit indicates either a different mode of interaction between the ribosome and <italic>Pf</italic>RACK1 in <italic>Plasmodium</italic> compared to humans, or that under the culturing conditions used <italic>Pf</italic>RACK1 is not expressed, or expressed in a form that does not interact with the ribosome. In yeast, RACK1 has been shown to be present in both a ribosome- and a non-ribosome-bound form dependent on growth conditions (<xref ref-type="bibr" rid="bib6">Baum et al., 2004</xref>). If the interaction between <italic>Pf</italic>RACK1 and the <italic>Pf</italic>40S is weaker than in other organisms, the possibility that <italic>Pf</italic>RACK1 dissociated during purification and grid preparation cannot be discounted.</p><p>The yeast 80S structure was also solved in the presence of STM1, a translation repressor protein, that binds to the head region of the 40S and blocks mRNA entry and binding of tRNA to the A- and P-sites (<xref ref-type="bibr" rid="bib48">Rabl et al., 2011</xref>). The human and <italic>D. melanogaster</italic> structures also co-purified with an STM1-like protein (SERBP1 and VIG2 respectively) (<xref ref-type="bibr" rid="bib3">Anger et al., 2013</xref>). <italic>Pf</italic>80S is not bound by a suppressor molecule, as also observed for the <italic>T. brucei</italic> structure (<xref ref-type="bibr" rid="bib25">Hashem et al., 2013</xref>), and hence reflects a ribosome capable of active translation.</p><p><italic>Pf</italic>80S co-purifies with a tRNA bound to the E-site. Although the density is not well resolved, presumably as a result of low and mixed occupancy, it could be interpreted by positioning a model of tRNA<sup>Met</sup>. The presence of tRNA helps to partially stabilise the L1 stalk near the elbow of the tRNA, however the stalk remains considerably flexible and is averaged out of the high-resolution reconstruction.</p><p>Perhaps due to the absence of RACK1 and/or STM1 or the presence of an E-site tRNA, the head of <italic>Pf</italic>40S adopts an orientation with respect to the body that is different to the yeast structure, with uS11 at the beak of the small subunit displaced by more than 10 Å. The root mean square deviation (RMSD) of the two small subunits is 2.9 Å<sup>2</sup>, however if the head and body are superimposed independently this improves to 1.0 Å<sup>2</sup> and 1.5 Å<sup>2</sup> respectively. The structure of <italic>Pf</italic>60S superimposes with the yeast 60S with a RMSD of 1.6 Å<sup>2</sup>. The largest morphological differences in this subunit result from a cluster of rRNA helices (ES7AL, ES15L, and ES7CL) protruding at the solvent side.</p><p>Given the potential of <italic>Pf</italic>80S as a drug target, we sought to describe its detailed structure in comparison to its direct counterpart in the human cytoplasm, where a 4.8 Å cryo-EM 80S structure represents the highest resolution solved to-date (<xref ref-type="bibr" rid="bib3">Anger et al., 2013</xref>). Therefore, all protein extensions and rRNA expansion segments (ESs) are annotated on the basis of comparison with human ribosomes. While the core of the <italic>Pf</italic>80S and human ribosome are conserved, the periphery of the ribosomes differs extensively in the nature and length of rRNA ESs and protein extensions. The constraints on rRNA expansion appear to be fewer than on protein extension, as rRNA contributes greater to the mass difference between species.</p><p>Compared to human ribosomes, <italic>P. falciparum</italic> typically has shorter ESs, some of which are entirely absent in the large subunit (ES7D-HL, ES9AL, ES10L, ES20L, ES30L) (<xref ref-type="table" rid="tbl4">Table 4</xref>). The functions, if any, of many of these ESs are not well known. ES7E, which is highly conserved in vertebrates, is implicated in selenoprotein synthesis by binding the SBP2 protein that specifically recruits the selenocysteine-specific tRNA and elongation factor (<xref ref-type="bibr" rid="bib32">Kossinova et al., 2014</xref>). While <italic>P. falciparum</italic> does utilize selenocysteine, it is incorporated into very few proteins (<xref ref-type="bibr" rid="bib36">Lobanov et al., 2006</xref>) and there is no homolog of SBP2, providing a possible explanation for why ES7E is not present in <italic>Plasmodium</italic>.<table-wrap id="tbl4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03080.012</object-id><label>Table 4.</label><caption><p>Comparison of ESs in <italic>Pf</italic>80S and human cytoplasmic ribosomes</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.012">http://dx.doi.org/10.7554/eLife.03080.012</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>rRNA</th><th>ES</th><th>Helix</th><th>Comparison between <italic>Pf</italic>80S and human ribosomes</th></tr></thead><tbody><tr><td rowspan="14">18S</td><td>ES2S</td><td/><td>Shorter loop in <italic>Pf</italic>80S</td></tr><tr><td>ES3S</td><td>A</td><td>Conserved</td></tr><tr><td/><td>B</td><td>Truncated in <italic>Pf</italic>80S</td></tr><tr><td>ES13S</td><td/><td>Conserved</td></tr><tr><td>ES6S</td><td>A</td><td>Expanded in <italic>Pf</italic>80S</td></tr><tr><td/><td>B</td><td>Truncated in <italic>Pf</italic>80S</td></tr><tr><td/><td>C</td><td>Conserved</td></tr><tr><td/><td>D</td><td>Expanded in <italic>Pf</italic>80S</td></tr><tr><td/><td>E</td><td>Conserved</td></tr><tr><td>ES7S</td><td/><td>Expanded in <italic>Pf</italic>80S</td></tr><tr><td>ES14S</td><td/><td>Conserved</td></tr><tr><td>ES9S</td><td/><td>Expanded in <italic>Pf</italic>80S</td></tr><tr><td>ES10S</td><td/><td>Expanded in <italic>Pf</italic>80S</td></tr><tr><td>ES12S</td><td/><td>Helix truncated in <italic>Pf</italic>80S</td></tr><tr><td rowspan="29">28S</td><td>ES3L</td><td/><td>Conserved</td></tr><tr><td>ES4L</td><td/><td>Conserved</td></tr><tr><td>ES5L</td><td/><td>Conserved</td></tr><tr><td>ES7L</td><td>A</td><td>Truncated in <italic>Pf</italic>80S</td></tr><tr><td/><td>B</td><td>Truncated. Loop in <italic>Pf</italic>80S forms a novel interaction with eL14</td></tr><tr><td/><td>B1</td><td><italic>Pf</italic>-specific ES</td></tr><tr><td/><td>C</td><td>Present</td></tr><tr><td/><td>D–H</td><td>Absent from <italic>Pf</italic>80S</td></tr><tr><td>ES8L</td><td>H28</td><td>Expanded in <italic>Pf</italic>80S</td></tr><tr><td>ES9L</td><td>A</td><td>Absent in <italic>Pf</italic>80S</td></tr><tr><td/><td>H30</td><td>Conserved</td></tr><tr><td/><td>H31</td><td>Conserved</td></tr><tr><td>ES10L</td><td/><td>Absent in <italic>Pf</italic>80S</td></tr><tr><td>ES12L</td><td/><td>Expanded in <italic>Pf</italic>80S</td></tr><tr><td>ES15L</td><td>A</td><td>Truncated in <italic>Pf</italic>80S</td></tr><tr><td>ES19L</td><td/><td>Truncated in <italic>Pf</italic>80S</td></tr><tr><td>ES20L</td><td>A</td><td>Absent in <italic>Pf</italic>80S</td></tr><tr><td/><td>B</td><td>Conserved in <italic>Pf</italic>80S</td></tr><tr><td>ES26L</td><td/><td>Expanded in <italic>Pf</italic>80S</td></tr><tr><td>ES27L</td><td>A–C</td><td>Not present in Pf80S model, predicted divergence between <italic>Pf</italic> and human cytoplasmic ribosomes</td></tr><tr><td>ES30L</td><td/><td>Absent in <italic>Pf</italic>80S</td></tr><tr><td>ES31L</td><td>A</td><td>Conserved</td></tr><tr><td/><td>B</td><td>Expanded in <italic>Pf</italic>80S</td></tr><tr><td/><td>C</td><td>Conserved</td></tr><tr><td>ES34L</td><td/><td><italic>Pf</italic>-specific ES</td></tr><tr><td>ES36L</td><td/><td><italic>Pf</italic>-specific ES</td></tr><tr><td>ES39L</td><td>A</td><td>Conserved; preceding loop in <italic>Pf</italic>80S forms a short helix (3 base pairs) with the 5′ end of the 5.8S rRNA</td></tr><tr><td/><td>B</td><td>Conserved</td></tr><tr><td>ES41L</td><td/><td>Conserved</td></tr></tbody></table></table-wrap></p><p>The largest <italic>Pf-</italic>specific ESs are concentrated in the 18S rRNA, with ES6S and ES9S being particularly extended (<xref ref-type="fig" rid="fig2">Figure 2C,D</xref>; <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). These ESs, like those described in both the human (<xref ref-type="bibr" rid="bib3">Anger et al., 2013</xref>) and <italic>Trypanosoma brucei</italic> (<xref ref-type="bibr" rid="bib25">Hashem et al., 2013</xref>) ribosome structures, are highly flexible and, in our structure, are only partly visible using a map filtered at 6 Å (<xref ref-type="fig" rid="fig2">Figure 2C,D</xref>). We have therefore not included these sections in our atomic model. ES10S is located at the top of the 40S head and has been partially built.</p><p><italic>P. falciparum</italic> ribosomes resemble those of <italic>T. brucei</italic> in that both have large ES6S and ES7S, although these are slightly larger in <italic>T. brucei</italic> (<xref ref-type="bibr" rid="bib25">Hashem et al., 2013</xref>). ES6S is in contact with ribosomal components that form part of the mRNA entry and exit sites and was therefore suggested as being involved in translation initiation (<xref ref-type="bibr" rid="bib29">Jenner et al., 2012</xref>). Recently, ES6/7S have been implicated in binding of the conserved translation initiation factor eIF3 based on superposition with a mammalian 43S complex (<xref ref-type="bibr" rid="bib26">Hashem et al., 2013</xref>). Almost 90 nucleotides of ES6AS are averaged out of our high-resolution reconstruction indicating this stalk is highly flexible, perhaps acting in a manner similar to the P stalk (known as the L7/L12 stalk in prokaryotes) by recruiting factors necessary for translation (in this case eIF3). The other large ES of the 18S rRNA, ES9S, is positioned at the head of the 40S. Given both the intrinsic mobility of the head and presumably the ES itself, there is no density for this ∼150 nucleotide <italic>Pf</italic>-specific element and the role it plays remains unclear.</p><p>The sites of <italic>Pf-</italic>specific elements are broadly distributed across the solvent-accessible surface of the ribosome, although the region surrounding the exit tunnel is conserved (<xref ref-type="bibr" rid="bib31">Klinge et al., 2011</xref>) and undecorated with ESs and protein extensions (<xref ref-type="fig" rid="fig2">Figure 2C,D</xref>). The subunit interface and eukaryotic-specific bridges, which in addition to having structural roles help transmit information to coordinate activity during translation (<xref ref-type="bibr" rid="bib8">Ben-Shem et al., 2011</xref>), are generally highly conserved in <italic>Pf</italic>80S. There are a couple of examples of stabilizing interactions that are not observed in human ribosomes. Firstly, eL41, the smallest ribosomal protein, bridges the two subunits (<xref ref-type="bibr" rid="bib8">Ben-Shem et al., 2011</xref>) and has a 14-residue <italic>Pf</italic>-specific N-terminal extension that reaches into a pocket formed by 18S rRNA of the small subunit and tightly anchors the protein (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). Secondly, an additional small bridge (∼200 Å<sup>2</sup>) is formed between the platform of <italic>Pf</italic>40S and the region around the L1 stalk by the C-terminal helix extension of eL8 interacting with the C-terminal helix of eS1 (<xref ref-type="fig" rid="fig3">Figure 3B</xref>).<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03080.013</object-id><label>Figure 3.</label><caption><title>Details of <italic>Pf</italic>-specific protein extensions and rRNA ESs near the (A and B) subunit interface (C) P stalk and (D) the L1 stalk.</title><p><italic>Pf</italic>-specific elements are shown in red.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.013">http://dx.doi.org/10.7554/eLife.03080.013</ext-link></p></caption><graphic xlink:href="elife03080f003"/></fig></p><p>Further ordered <italic>Pf-</italic>specific elements are concentrated near the L1 and P stalks of <italic>Pf</italic>60S. Directly above the P stalk, the <italic>Pf-</italic>specific ES7B1L forms a diverted part of ES7CL that is stabilized by several electrostatic interactions with a C-terminal helix extension of uL4 (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). Towards the back of the P-stalk, the C-terminal helix extension of eL14 caps the stem loop of ES7BL (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). On the opposite side of the ribosome, near the E-site tRNA, the <italic>Pf-</italic>specific stem loop ES34L is positioned directly above the L1 stalk (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). This ES appears to have caused a 60° rotation of the C-terminal helix of eL13 relative to its position in human ribosomes (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). The tip of the helix is displaced by ∼28 Å away from the L1 stalk and now stabilizes the interaction between ES34L and the loop of h22. Since the L1 stalk is required for coordinating the movement of tRNAs and the P stalk is required for coordinating the movement of translation factors during the various steps of protein synthesis (<xref ref-type="bibr" rid="bib20">Gonzalo and Reboud, 2003</xref>), the expanded mass around the stalks of <italic>Pf</italic>80S may have functional implications for translation in <italic>P. falciparum</italic>.</p><p>The ability to determine atomic-resolution structures of <italic>Pf</italic>80S provides a platform for investigating the action of anti-malarial therapeutics that target the ribosome. The clinically used, broad-spectrum eukaryotic translation inhibitor emetine (<xref ref-type="fig" rid="fig4">Figure 4A</xref>) (<xref ref-type="bibr" rid="bib24">Grollman, 1968</xref>), has been reported to act as a translocation inhibitor targeting the ribosome (<xref ref-type="bibr" rid="bib30">Jimenez et al., 1977</xref>; <xref ref-type="bibr" rid="bib17">Dinos et al., 2004</xref>), although its precise mode of action is unknown. Emetine is a natural product alkaloid from the plant <italic>Carapichea ipecacuanha</italic>, and an approved medicine for the treatment of amoebiasis (<xref ref-type="bibr" rid="bib22">Goodwin et al., 1948</xref>). Although its toxicity associated with chronic usage in humans has limited its clinical use against malaria in its current formulation (<xref ref-type="bibr" rid="bib16">Dempsey and Salem, 1966</xref>), emetine does demonstrate potent antimalarial activity with a 50% inhibitory concentration (IC<sub>50</sub>) of 47 nM against the blood stage of multidrug resistant strains of <italic>P. falciparum</italic> (<xref ref-type="bibr" rid="bib39">Matthews et al., 2013</xref>). Moreover, the immediate therapeutic effect it offers by rapid killing of blood stage parasites may warrant re-consideration of the use of emetine or its derivatives for short periods during acute malaria infection (<xref ref-type="bibr" rid="bib28">James, 1985</xref>).<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03080.014</object-id><label>Figure 4.</label><caption><title>Emetine binds to the E-site of the <italic>Pf</italic>40S subunit.</title><p>(<bold>A</bold>) 2D chemical structure of emetine. (<bold>B</bold>) A 4.5 Å filtered difference map (red density) at 5 standard deviation overlaid with the <italic>Pf</italic>80S map filtered at 6 Å (blue and yellow for <italic>Pf</italic>60S and <italic>Pf</italic>40S respectively), showing the emetine density at the E-site of the <italic>Pf</italic>40S. The emetine binding site in (<bold>C</bold>) empty and (<bold>D</bold>) emetine-bound structures, with (<bold>E</bold>) density for emetine alone at 3.2 Å.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.014">http://dx.doi.org/10.7554/eLife.03080.014</ext-link></p></caption><graphic xlink:href="elife03080f004"/></fig></p><p>Incubation of purified <italic>Pf</italic>80S with a 1 mM emetine solution prior to cryo-EM grid preparation, led to a 3.2 Å resolution structure of the complex. Using soft masking, the resolution for the large subunit improved to 3.1 Å, with the small subunit at 3.3 Å (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). A difference map was calculated from the reconstructions with and without emetine and showed a single, continuous feature near the E-site of <italic>Pf</italic>40S with a shape and size congruent with a single emetine molecule when thresholded at 5 standard deviations, and with a maximum value of 11 standard deviations (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). At this position in our map, the density provided sufficient detail to confidently model the emetine molecule (<xref ref-type="fig" rid="fig4">Figure 4C–E</xref>). The emetine binding pocket is formed at the interface between 18S rRNA helices 23, 24, 45, and the C-terminus of uS11 (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). Comparison with the unliganded map showed that binding of emetine does not induce changes to the pocket (<xref ref-type="fig" rid="fig4">Figure 4C,D</xref>). The benzo[a]quinolizine ring of emetine mimics a base-stacking interaction with G973 of h23 and its ethyl group forms a hydrophobic interaction with C1075 and C1076 of h24, whereas the isoquinoline ring is stacked against the C-terminal Leu151 of uS11 (<xref ref-type="fig" rid="fig5">Figure 5B,C</xref>). The interaction is stabilized by a hydrogen bond formed between the NH group of the isoquinoline ring in emetine and an oxygen atom on the backbone of U2061 of h45 (<xref ref-type="fig" rid="fig5">Figure 5B,C</xref>). Although there is no high-resolution structure of the human cytoplasmic ribosome, comparison of the emetine binding site in <italic>Pf</italic>80S with the equivalent region in the 4.8 Å human structure (<xref ref-type="bibr" rid="bib3">Anger et al., 2013</xref>) revealed that each of the core binding elements are conserved (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>) indicating that emetine likely binds to the cytoplasmic host ribosomes in the same way, potentially accounting for the observed cytotoxicity in humans.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.03080.015</object-id><label>Figure 5.</label><caption><title>Molecular details of the emetine–ribosome interaction.</title><p>(<bold>A</bold>) Overview of emetine at the binding interface formed by the three conserved rRNA helices and uS11. h23 is in green, h24 in cyan, h45 in blue, uS11 in pink, and emetine in yellow. (<bold>B</bold>) 2D representation showing the interaction of emetine with binding residues. Substitution contour represents potential space for chemical modification of emetine. (<bold>C</bold>) Residues in physical contact with emetine. Hydrogen bond is indicated as dashes.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.015">http://dx.doi.org/10.7554/eLife.03080.015</ext-link></p></caption><graphic xlink:href="elife03080f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03080.016</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Comparison of the emetine binding residues between <italic>Pf</italic>80S and human ribosomes.</title><p>Human and <italic>Pf</italic>-specific elements are colored in yellow and cyan respectively, with <italic>Pf</italic> numbering. Emetine is in purple.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.016">http://dx.doi.org/10.7554/eLife.03080.016</ext-link></p></caption><graphic xlink:href="elife03080fs005"/></fig></fig-group></p><p>The identified binding site is consistent with mutations of Arg149 and Arg150 of uS11 in Chinese hamster ovary (CHO) cells that have been found to confer resistance to emetine (<xref ref-type="bibr" rid="bib38">Madjar et al., 1982</xref>). At the emetine-binding pocket, h24 is sandwiched between the apexes of h23 and h45. The C-terminus of uS11 adopts a long coil with seven basic residues (residues 141–151; RKKSGRRGRRL), which form electrostatic interactions with the phosphate backbones of h45, h23 and h24, thereby stabilizing the conformation of this coil together with the 18S rRNA (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). This would explain the molecular basis for resistance whereby mutations of the C-terminal arginine residues of uS11 destabilize h23 and h45, disrupting the binding pocket.</p><p>The mode of binding of emetine resembles the way in which pactamycin, previously thought to be a unique class of antibiotic, binds to the bacterial 30S (<xref ref-type="bibr" rid="bib9">Brodersen et al., 2000</xref>). In both structures the guanine base at the tip of h23 (G973 in <italic>Pf</italic>; G693 in bacteria) forms a stacking interaction with the hydrophobic rings of either compound. Moreover, the two cytosine bases of h24 (C1075 and 1076 in <italic>Pf;</italic> C795 and 796 in bacteria) are each involved in drug binding (<xref ref-type="bibr" rid="bib9">Brodersen et al., 2000</xref>; <xref ref-type="fig" rid="fig6">Figure 6</xref>). The hydrogen bond to the backbone of h45 and the hydrophobic interaction with Leu151 of uS11 are specific to the <italic>Pf</italic>80S–emetine interaction. In the 30S-pactamycin complex, the last base of the E-site codon of the mRNA was displaced 12.5 Å compared to the native path of mRNA (<xref ref-type="bibr" rid="bib9">Brodersen et al., 2000</xref>) thereby blocking mRNA/tRNA entry into the E-site during the translocation step of protein synthesis (<xref ref-type="bibr" rid="bib17">Dinos et al., 2004</xref>). Based on these structures, emetine appears to elicit its inhibitory effect by the same mechanism as pactamycin.<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.03080.017</object-id><label>Figure 6.</label><caption><title>Comparison with pactamycin.</title><p>Superposition of emetine and pactamycin at the <italic>Pf</italic>40S emetine binding pocket. Emetine and pactamycin are shown in yellow and red respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03080.017">http://dx.doi.org/10.7554/eLife.03080.017</ext-link></p></caption><graphic xlink:href="elife03080f006"/></fig></p></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>The resolution revolution in cryo-EM (<xref ref-type="bibr" rid="bib35">Kühlbrandt, 2014</xref>) is the product of a new generation of sensors that detect electrons directly (without first converting to light) and have improved quantum efficiencies. These cameras are fast enough to follow beam-induced movement of the particles caused by irradiation with electrons. Statistical movie processing can compensate for this movement allowing for structures to be solved at atomic precision. We have harnessed these technological advances to determine the first structure of a ribosome from a parasite at atomic resolution. Previously, structures of eukaryotic cytosolic 80S ribosomes at a similar resolution had only been possible using X-ray crystallography (<xref ref-type="bibr" rid="bib8">Ben-Shem et al., 2011</xref>). From the reconstruction of <italic>Pf</italic>80S–emetine complex at 3.2 Å, we determined a stereochemically accurate all-atom model using recent developments in model building, refinement, and validation (<xref ref-type="bibr" rid="bib2">Amunts et al., 2014</xref>).</p><p>The structure of <italic>Pf</italic>80S further demonstrates the diversity of ribosome structures among eukaryotes, especially in terms of the location and nature of ESs at the periphery, while maintaining a conserved core. The observation of <italic>Pf</italic>-specific features could serve as the basis for exploring their functional relevance as an essential, first step towards finding efficacious and clinically safe anti-malarial drugs. An alternative to drug development against <italic>Pf-</italic>specific ribosomal elements is the repurposing of existing antibiotics as anti-malarials. By determining the structure of <italic>Pf</italic>80S in both a liganded and unliganded state, we were able to locate the binding site of the anti-protozoan inhibitor, emetine, using an unbiased difference map. That emetine and pactamycin share a binding pocket in eukaryotic ribosomes could not be predicted based on the chemical structures of the drug molecules only. Pactamycin itself has been shown to have potent antiprotozoal activity against both drug-susceptible and drug-resistant strains of <italic>P. falciparum</italic> (<xref ref-type="bibr" rid="bib46">Otoguro et al., 2010</xref>). Chemical modifications to pactamycin have yielded analogs that maintain antimalarial activity but with reduced cytotoxicity against mammalian cells (<xref ref-type="bibr" rid="bib37">Lu et al., 2011</xref>). Similarly, an emetine derivative, dehydroemetine, which differs by the presence of a double bond next to the ethyl group of benzo[a]quinolizine ring, exhibits less toxic effects than the parental compound while maintaining anti-parasitic properties (<xref ref-type="bibr" rid="bib16">Dempsey and Salem, 1966</xref>; <xref ref-type="bibr" rid="bib13">Chintana et al., 1986</xref>). This suggests that compounds targeting the emetine/pactamycin binding site are amenable to optimization, potentially leading to drugs more suited to clinical use. The <italic>Pf</italic>80S–emetine structure reveals an edge centered on the ethyl group of the molecule that could be subjected to modification to increase the affinity of emetine for the binding pocket (<xref ref-type="fig" rid="fig5">Figure 5B</xref>, labelled as the ‘contour edge’). Although based on the similarity with the binding site in humans it is unlikely that emetine can be structurally modified to not bind the mammalian system, as demonstrated in the case of dehydroemetine modifications can reduce its cytotoxicity. Although the mechanism for such reduced cytotoxicity mediated by pactamycin and emetine analogs is not known, it may be possible that these derived compounds selectively target tumor/parasite cells that are rapidly dividing, whereby protein synthesis is more sensitive to drug action in these cells. As in the case of antibiotics repurposed as antitumor agents, there is a clinical role for eukaryotic antibiotics that target systems with differential rates of translation provided usage is carefully directed. In malaria, eukaryotic antibiotics, such as emetine, could be used in combination with the slow-acting, but more specific apicoplast-targeting antibiotics (<xref ref-type="bibr" rid="bib15">Dahl and Rosenthal, 2007</xref>).</p><p>This work demonstrates the power of contemporary cryo-EM for drug discovery. A drug, with a previously unknown binding site, can be visualized inside a macromolecular complex that is almost 10,000 times larger in molecular weight and at a level of detail comparable to that obtained by X-ray crystallography. By avoiding the need for crystallization one of the bottlenecks of solving a structure is bypassed. It allows structures to be solved from very small sample quantities, with sample heterogeneity improved through image processing. As such, cryo-EM is of particular use for solving the structures of macromolecules in their native state, isolated from pathogenic organisms where culturing large quantities is not possible.</p><p>In summary, our cryo-EM analyses reveal the first structure of a ribosome from a parasite at atomic resolution, along with detailed insights into the molecular basis of a known anti-protozoan translation inhibitor. Finally, it demonstrates that cryo-EM offers an attractive route towards the development of new compounds that target macromolecules by facilitating structure–activity relationships in otherwise intractable biological systems.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Parasite culture and ribosome purification</title><p>Wild-type 3D7 strain of <italic>P. falciparum</italic> parasites were maintained in human erythrocytes (blood group O) at a hematocrit of 4% with 10% Albumax. Saponin lysed parasite pellets were incubated with lysis buffer (20 mM Hepes, pH 7.4, 250 mM KCl, 25 mM Mg(CH<sub>3</sub>COO)<sub>2</sub>, 0.15% Triton, 5 mM 2-mecaptoethanol) at 4°C for 1 hr. Ribosomes were purified by ultracentrifugation initially with a sucrose cushion (20 mM Hepes pH 7.4, 1.1 M sucrose, 40 mM KCH<sub>3</sub>COO, 10 mM NH<sub>4</sub>CH<sub>3</sub>COO, 10 mM Mg(CH<sub>3</sub>COO)<sub>2</sub>, and 5 mM 2-mecaptoethanol) followed by a 10–40% sucrose gradient separation step using the same buffer.</p></sec><sec id="s4-2"><title>Electron microscopy</title><p>Aliquots of 3 μl of purified <italic>Pf</italic>80S at a concentration of ∼160 nM (∼0.5 mg/ml) were incubated for 30 s on glow-discharged holey carbon grids (Quantifoil R1.2/1.3), on which a home-made continuous carbon film (estimated to be ∼30 Å thick) had previously been deposited. Grids were blotted for 2.5 s and flash frozen in liquid ethane using an FEI Vitrobot. For the empty <italic>Pf</italic>80S sample, grids were transferred to an FEI Titan Krios electron microscope that was operated at 300 kV. Images were recorded manually during two non-consecutive days on a back-thinned FEI Falcon II detector at a calibrated magnification of 135,922 (yielding a pixel size of 1.03 Å). Defocus values in the final data set ranged from 0.7 to 3.9 µm.</p><p>To prepare the <italic>Pf</italic>80S–emetine sample, purified <italic>Pf</italic>80S at 160 nM was incubated with a 1 mM solution of emetine in 20 mM Hepes pH7.4, 40 mM KCH<sub>3</sub>COO, 10 mM NH<sub>4</sub>CH<sub>3</sub>COO, 10 mM Mg(CH<sub>3</sub>COO)<sub>2</sub>, and 5 mM 2-mecaptoethanol for 15 min at 25°C prior to blotting and freezing as described above. <italic>Pf</italic>80S–emetine grids were transferred to an FEI Tecnai Polara electron microscope that was operated at 300 kV. Images were recorded manually during two non-consecutive days on a back-thinned FEI Falcon II detector at a calibrated magnification of 104,478 (yielding a pixel size of 1.34 Å). Defocus values in the final data set ranged from 0.8 to 3.8 µm.</p><p>During the data collection sessions of both samples, all images that showed signs of significant astigmatism or drift were discarded. An in-house built system was used to intercept the videos frames from the detector at a rate of 17 s<sup>−1</sup> for the Krios and 16 s<sup>−1</sup> for the Polara microscope.</p></sec><sec id="s4-3"><title>Image processing</title><p>We used RELION (version 1.3-beta) for automated selection of 126,727 particles from 1310 micrographs for the empty <italic>Pf</italic>80S sample; and 158,212 particles from 1081 micrographs for the <italic>Pf</italic>80S–emetine sample. Contrast transfer function parameters were estimated using CTFFIND3 (<xref ref-type="bibr" rid="bib42">Mindell and Grigorieff, 2003</xref>). All 2D and 3D classifications and refinements were performed using RELION (<xref ref-type="bibr" rid="bib51">Scheres, 2012</xref>). To discard bad particles, we used a single round of reference-free 2D class averaging with 100 classes for both data sets, and a single round of 3D classification with four classes for the <italic>Pf</italic>80S–emetine data set. The final refinement for the empty <italic>Pf</italic>80S and <italic>Pf</italic>80S–emetine sample contained 72,293 and 105,247 particles, respectively. A 60 Å low-pass filtered cryo-EM reconstruction of the yeast cytoplasmic 80S ribosome (EMDB-2275 [<xref ref-type="bibr" rid="bib7">Ben-Shem et al., 2010</xref>]) was used as an initial model for the 3D refinement.</p><p>For the correction of beam-induced movements, we used statistical movie processing as described previously (<xref ref-type="bibr" rid="bib4">Bai et al., 2013</xref>), with running averages of five movie frames, and a standard deviation of 1 pixel for the translational alignment. To further increase the accuracy of the movement correction, we used the beta version of RELION-1.3 to fit linear tracks through the optimal translations for all running averages, and included neighboring particles on the micrograph in these fits. In addition, we employed a resolution and dose-dependent model for the radiation damage, where each frame is weighted with a different B-factor as was estimated from single-frame reconstructions. These procedures yielded maps with an overall resolution of 3.4 Å for the empty <italic>Pf</italic>80S and 3.2 Å for <italic>Pf</italic>80S–emetine.</p><p>Reported resolutions are based on the gold-standard FSC = 0.143 criterion (<xref ref-type="bibr" rid="bib12">Chen et al., 2013</xref>) and were corrected for the effects of a soft mask on the FSC curve using high-resolution noise substitution (<xref ref-type="bibr" rid="bib12">Chen et al., 2013</xref>). Soft masks were made by converting atomic models into density maps, binarizing those, and adding cosine-shaped edges. Prior to visualization, all density maps were corrected for the modulation transfer function (MTF) of the detector, and then sharpened by applying a negative B-factor (<xref ref-type="table" rid="tbl1">Table 1</xref>) that was estimated using automated procedures (<xref ref-type="bibr" rid="bib49">Rosenthal and Henderson, 2003</xref>).</p><p>In order to locate emetine in the <italic>Pf</italic>80S–emetine reconstruction, we calculated a difference map between the reconstructions of empty <italic>Pf</italic>80S and <italic>Pf</italic>80S–emetine. To this purpose, the two MTF-corrected and B-factor sharpened maps were aligned with respect to each other using the ‘Fit in Map’ functionality in UCSF Chimera (<xref ref-type="bibr" rid="bib47">Pettersen et al., 2004</xref> ), and the empty <italic>Pf</italic>80S map was re-interpolated on the Cartesian grid of the <italic>Pf80S–emetine</italic> map prior to subtraction of the maps in RELION. For visualization purposes, the resulting difference map was low-pass filtered at 4.5 Å and the threshold was set at 5 standard deviations as calculated within the area of the <italic>Pf</italic>80S ribosome (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). At this threshold, only one continuous U-shaped feature was visible. The highest difference density inside this feature extended to 11 standard deviations in the difference map.</p><p>Local resolution variations in all reconstructions were estimated using ResMap (<xref ref-type="bibr" rid="bib34">Kucukelbir et al., 2014</xref>). Presumably due to unresolved structural heterogeneity the local resolution in the small ribosomal subunit was typically worse than in the large ribosomal subunit. Therefore, for the <italic>Pf80S–emetine</italic> structure, we performed two additional ‘focussed’ refinements, where we masked out the large or the small subunit at every iteration. This gave rise to two maps (<xref ref-type="fig" rid="fig1">Figure 1E</xref>) with improved density for either the small subunit (at an overall resolution of 3.3 Å) or the large ribosomal subunit (at an overall resolution of 3.1 Å), and these maps were used for the refinement of the atomic model as described below.</p></sec><sec id="s4-4"><title>Model building and refinement</title><p>Ribosomal protein sequences from the 3D7 strain of <italic>P. falciparum</italic> were taken from <italic>PlasmoDB</italic> (<xref ref-type="bibr" rid="bib54">The Plasmodium Genome Database Collaborative, 2001</xref>) and used as template sequences to obtain homology models generated from I-TASSER (<xref ref-type="bibr" rid="bib50">Roy et al., 2010</xref>). Homology models were fitted into the reconstructed map of <italic>Pf</italic>80S using Chimera (<xref ref-type="bibr" rid="bib47">Pettersen et al., 2004</xref>). Each protein was then subjected to a jiggle-fit and extensively rebuilt with sidechains placed into the map density using Coot v.0.8 (<xref ref-type="bibr" rid="bib18">Emsley et al., 2010</xref>). The sequences of the <italic>Pf</italic>80S rRNAs were obtained from <italic>PlasmoDB</italic> (<xref ref-type="bibr" rid="bib54">The Plasmodium Genome Database Collaborative, 2001</xref>) and aligned using Clustal Omega (<xref ref-type="bibr" rid="bib53">Sievers et al., 2011</xref>) with the rRNA sequences extracted from the <italic>Saccharomyces cerevisae (Sc)</italic> 80S structure (PDB ID: 3U5B and 3U5D) (<xref ref-type="bibr" rid="bib8">Ben-Shem et al., 2011</xref>). Conserved regions without insertions or deletions were extracted from the yeast structure, mutated and renumbered. These conserved sections were then connected by de novo building of RNA. The complete rRNA was then manually rebuilt in Coot to optimize the fit to density. Building was aided by secondary structure predictions downloaded from the Comparative RNA Website (<xref ref-type="bibr" rid="bib10">Cannone et al., 2002</xref>).</p><p>The model was refined using REFMAC v.5.8, which was modified for structures determined by cryo-EM (<xref ref-type="bibr" rid="bib43">Murshudov et al., 2011</xref>; <xref ref-type="bibr" rid="bib2">Amunts et al., 2014</xref>). The <italic>Pf</italic>80S atomic model was refined as separate 60S and 40S subunits in the two maps that were obtained for either subunit in the focused refinements of the cryo-EM reconstructions. Structure factors for the (Fourier-space) refinement in REFMAC were obtained by cutting out sections of the corresponding maps with a 3 Å radius from the center of each atom in the model, and structure factor phases were not altered during refinement.</p><p>Throughout refinement, reference and secondary structure restraints were applied to the ribosomal proteins using the <italic>Sc</italic>80S structure as a reference model (<xref ref-type="bibr" rid="bib44">Nicholls et al., 2012</xref>). Base pair and parallelization restraints obtained using LIBG were also applied throughout refinement (<xref ref-type="bibr" rid="bib2">Amunts et al., 2014</xref>). The stereochemistry of the rRNA model was further improved using the ERRASER-PHENIX pipeline (<xref ref-type="bibr" rid="bib14">Chou et al., 2013</xref>). Ramachandran restraints were not applied during refinement to preserve backbone dihedral angles for validation.</p><p>The R-factor and average overall Fourier shell correlation were monitored during refinement (<xref ref-type="table" rid="tbl1">Table 1</xref>) and the final model was validated using MolProbity (<xref ref-type="bibr" rid="bib11">Chen et al., 2010</xref>). For cross-validation against over-fitting, we randomly displaced the atoms of our final model (with an RMSD of 0.5 Å) and performed a fully restrained refinement against a map that was reconstructed from only one of the two independent halves of the data that were used in our gold-standard FSC procedure. We then calculated FSC curves between the resulting model and the half-map against which it had been refined (FSC<sub>work</sub>), as well as the FSC curve between that model and the other half-map (FSC<sub>test</sub>). The observation that the FSC<sub>work</sub> and FSC<sub>test</sub> curves nearly overlap demonstrates the absence of overfitting of the model (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank S Ralph, B Sleebs, D Wilson, E Zuccala, G McFadden, A Cowman, J Rayner, A Ruecker, M Delves, R Sinden, S Chen, C Savva, J Grimmett, T Darling, G Murshudov, and P Emsley for helpful discussions and experimental assistance; and V Ramakrishnan for comments on the manuscript.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>WW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>X-B, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>AB, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>ISF, Ribosome purification, Approval of final manuscript, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con5"><p>EH, Conducted preliminary negative stain and cryo-EM data collection, Approval of final manuscript, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con6"><p>MC, Prepared parasite lysates for ribosome purification, Approval of final manuscript, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con7"><p>YHT, Prepared parasite lysates for ribosome purification, Approval of final manuscript, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con8"><p>JB, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con9"><p>SHWS, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><sec sec-type="datasets"><title>Major datasets</title><p>The following datasets were generated:</p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro1"><name><surname>Wong</surname><given-names>W</given-names></name>, <name><surname>Bai</surname><given-names>X-C</given-names></name>, <name><surname>Brown</surname><given-names>A</given-names></name>, <name><surname>Fernandez</surname><given-names>IS</given-names></name>, <name><surname>Hanssen</surname><given-names>E</given-names></name>, <name><surname>Condron</surname><given-names>C</given-names></name>, <name><surname>Tan</surname><given-names>YH</given-names></name>, <name><surname>Baum</surname><given-names>J</given-names></name>, <name><surname>Scheres</surname><given-names>SHW</given-names></name>, <year>2014</year><x>, </x><source>Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/pdbe/entry/EMD-2660">http://www.ebi.ac.uk/pdbe/entry/EMD-2660</ext-link><x>, </x><comment>Publicly available at Electron Microscopy Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro2"><name><surname>Wong</surname><given-names>W</given-names></name>, <name><surname>Bai</surname><given-names>X-C</given-names></name>, <name><surname>Brown</surname><given-names>A</given-names></name>, <name><surname>Fernandez</surname><given-names>IS</given-names></name>, <name><surname>Hanssen</surname><given-names>E</given-names></name>, <name><surname>Condron</surname><given-names>C</given-names></name>, <name><surname>Tan</surname><given-names>YH</given-names></name>, <name><surname>Baum</surname><given-names>J</given-names></name>, <name><surname>Scheres</surname><given-names>SHW</given-names></name>, <year>2014</year><x>, </x><source>Plasmodium falciparum 80S ribosome</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/pdbe/entry/EMD-2661">http://www.ebi.ac.uk/pdbe/entry/EMD-2661</ext-link><x>, </x><comment>Publicly available at Electron Microscopy Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro3"><name><surname>Wong</surname><given-names>W</given-names></name>, <name><surname>Bai</surname><given-names>X-C</given-names></name>, <name><surname>Brown</surname><given-names>A</given-names></name>, <name><surname>Fernandez</surname><given-names>IS</given-names></name>, <name><surname>Hanssen</surname><given-names>E</given-names></name>, <name><surname>Condron</surname><given-names>C</given-names></name>, <name><surname>Tan</surname><given-names>YH</given-names></name>, <name><surname>Baum</surname><given-names>J</given-names></name>, <name><surname>Scheres</surname><given-names>SHW</given-names></name>, <year>2014</year><x>, </x><source>Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine; large subunit</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=3J79">http://www.pdb.org/pdb/explore/explore.do?structureId=3J79</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro4"><name><surname>Wong</surname><given-names>W</given-names></name>, <name><surname>Bai</surname><given-names>X-C</given-names></name>, <name><surname>Brown</surname><given-names>A</given-names></name>, <name><surname>Fernandez</surname><given-names>IS</given-names></name>, <name><surname>Hanssen</surname><given-names>E</given-names></name>, <name><surname>Condron</surname><given-names>C</given-names></name>, <name><surname>Tan</surname><given-names>YH</given-names></name>, <name><surname>Baum</surname><given-names>J</given-names></name>, <name><surname>Scheres</surname><given-names>SHW</given-names></name>, <year>2014</year><x>, </x><source>Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine; small subunit</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=3J7A">http://www.pdb.org/pdb/explore/explore.do?structureId=3J7A</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p>The following previously published datasets were used:</p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro5"><name><surname>Bai</surname><given-names>X-C</given-names></name>, <name><surname>Fernadez</surname><given-names>IS</given-names></name>, <name><surname>McMullan</surname><given-names>G</given-names></name>, <name><surname>Scheres</surname><given-names>SHW</given-names></name>, <year>2013</year><x>, </x><source>Ribosome structures at near-atomic resolution from thirty thousand cryo-EM particles</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/pdbe/entry/EMD-2275">http://www.ebi.ac.uk/pdbe/entry/EMD-2275</ext-link><x>, </x><comment>Publicly available at Electron Microscopy Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro6"><name><surname>Ben-Shem</surname><given-names>A</given-names></name>, <name><surname>Garreau de Loubresse</surname><given-names>N</given-names></name>, <name><surname>Meinikov</surname><given-names>S</given-names></name>, <name><surname>Jenner</surname><given-names>L</given-names></name>, <name><surname>Yusupov</surname><given-names>G</given-names></name>, <name><surname>Yusupov</surname><given-names>M</given-names></name>, <year>2011</year><x>, </x><source>The structure of the eukaryotic ribosome at 3.0 Å resolution</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=3U5B">http://www.pdb.org/pdb/explore/explore.do?structureId=3U5B</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro7"><name><surname>Ben-Shem</surname><given-names>A</given-names></name>, <name><surname>Garreau de Loubresse</surname><given-names>N</given-names></name>, <name><surname>Meinikov</surname><given-names>S</given-names></name>, <name><surname>Jenner</surname><given-names>L</given-names></name>, <name><surname>Yusupov</surname><given-names>G</given-names></name>, <name><surname>Yusupov</surname><given-names>M</given-names></name>, <year>2011</year><x>, </x><source>The structure of the eukaryotic ribosome at 3.0 Å resolution</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=3U5C">http://www.pdb.org/pdb/explore/explore.do?structureId=3U5C</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro8"><name><surname>Ben-Shem</surname><given-names>A</given-names></name>, <name><surname>Garreau de Loubresse</surname><given-names>N</given-names></name>, <name><surname>Meinikov</surname><given-names>S</given-names></name>, <name><surname>Jenner</surname><given-names>L</given-names></name>, <name><surname>Yusupov</surname><given-names>G</given-names></name>, <name><surname>Yusupov</surname><given-names>M</given-names></name>, <year>2011</year><x>, </x><source>The structure of the eukaryotic ribosome at 3.0 Å resolution</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=3U5D">http://www.pdb.org/pdb/explore/explore.do?structureId=3U5D</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro9"><name><surname>Ben-Shem</surname><given-names>A</given-names></name>, <name><surname>Garreau de Loubresse</surname><given-names>N</given-names></name>, <name><surname>Meinikov</surname><given-names>S</given-names></name>, <name><surname>Jenner</surname><given-names>L</given-names></name>, <name><surname>Yusupov</surname><given-names>G</given-names></name>, <name><surname>Yusupov</surname><given-names>M</given-names></name>, <year>2011</year><x>, </x><source>The structure of the eukaryotic ribosome at 3.0 Å resolution</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=3U5E">http://www.pdb.org/pdb/explore/explore.do?structureId=3U5E</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro10"><name><surname>Anger</surname><given-names>AM</given-names></name>, <name><surname>Armache</surname><given-names>JP</given-names></name>, <name><surname>Berninghausen</surname><given-names>O</given-names></name>, <name><surname>Habeck</surname><given-names>M</given-names></name>, <name><surname>Subklewe</surname><given-names>M</given-names></name>, <name><surname>Wilson</surname><given-names>DN</given-names></name>, <name><surname>Beckmann</surname><given-names>R</given-names></name>, <year>2013</year><x>, </x><source>Structure of the human 40S ribosomal proteins</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=3J3A">http://www.pdb.org/pdb/explore/explore.do?structureId=3J3A</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro11"><name><surname>Anger</surname><given-names>AM</given-names></name>, <name><surname>Armache</surname><given-names>JP</given-names></name>, <name><surname>Berninghausen</surname><given-names>O</given-names></name>, <name><surname>Habeck</surname><given-names>M</given-names></name>, <name><surname>Subklewe</surname><given-names>M</given-names></name>, <name><surname>Wilson</surname><given-names>DN</given-names></name>, <name><surname>Beckmann</surname><given-names>R</given-names></name>, <year>2013</year><x>, </x><source>Structure of the human 60S ribosomal proteins</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=3J3B">http://www.pdb.org/pdb/explore/explore.do?structureId=3J3B</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro12"><name><surname>Anger</surname><given-names>AM</given-names></name>, <name><surname>Armache</surname><given-names>JP</given-names></name>, <name><surname>Berninghausen</surname><given-names>O</given-names></name>, <name><surname>Habeck</surname><given-names>M</given-names></name>, <name><surname>Subklewe</surname><given-names>M</given-names></name>, <name><surname>Wilson</surname><given-names>DN</given-names></name>, <name><surname>Beckmann</surname><given-names>R</given-names></name>, <year>2013</year><x>, </x><source>Structure of the H. sapiens 40S rRNA and E-tRNA</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=3J3D">http://www.pdb.org/pdb/explore/explore.do?structureId=3J3D</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro13"><name><surname>Anger</surname><given-names>AM</given-names></name>, <name><surname>Armache</surname><given-names>JP</given-names></name>, <name><surname>Berninghausen</surname><given-names>O</given-names></name>, <name><surname>Habeck</surname><given-names>M</given-names></name>, <name><surname>Subklewe</surname><given-names>M</given-names></name>, <name><surname>Wilson</surname><given-names>DN</given-names></name>, <name><surname>Beckmann</surname><given-names>R</given-names></name>, <year>2013</year><x>, </x><source>Structure of the H. sapiens 60S rRNA</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=3J3F">http://www.pdb.org/pdb/explore/explore.do?structureId=3J3F</ext-link><x>, </x><comment>Publicly available at RCSB 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An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Cryo-EM structure of the <italic>Plasmodium falciparum</italic> 80S ribosome bound to the anti-protozoan drug emetine” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by John Kuriyan (Senior editor) and 3 reviewers, one of whom, Werner Kühlbrandt, is a member of our Board of Reviewing Editors.</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>All three reviewers agree that this is exciting, important, and innovative work that should be published in <italic>eLife</italic> as soon as possible. Two of the reviewers ask for a more detailed description and a more comprehensive comparison of this new structure with the few available structures of eukaryotic ribosomes, which is reasonable given the importance of the work. However, none of them have substantive concerns, so no major revisions are required.</p><p>Minor comments:</p><p><italic>Reviewer #1:</italic></p><p>1) For future reference it would be interesting to know how much material (in terms mg of purified 80S ribosomes) was available, and from roughly how much starting material (in terms of wet weight of cells) it was isolated.</p><p>2) For ease of comparison, please provide the concentration of the ribosome solution used for grid preparation in mg/ml, rather than only in terms of molarity.</p><p>3) Why is the resolution of the emetin complex significantly higher than that of the empty ribosome, even though the pixel size was larger? Is this fully explained by the larger number of particles averaged?</p><p>4) The tunnel exit should be labelled in <xref ref-type="fig" rid="fig2">Figure 2</xref>.</p><p><italic>Reviewer #2:</italic></p><p>1) The resolution is presented as 3.2A in the last paragraph of the Introduction and 3.4A in the first paragraph of the Results section. The authors need to correct or clarify these two apparently competing statements.</p><p>2) In the third paragraph of the Results section, a description of ESs refers to <xref ref-type="fig" rid="fig1">Figure 1C-D</xref>. Should this be <xref ref-type="fig" rid="fig2">Figure 2C-D</xref>?</p><p>3) In the fifth paragraph of the Results section: By “unmodified” are the authors meaning to indicate that this region is conserved?</p><p>4) <xref ref-type="fig" rid="fig3">Figure 3A</xref>. Is the protein shown in this panel eL41? If so, a label or mention in the legend should be added. Also, it is not clear which rRNA is being shown – is it 18S?</p><p>5) It would be helpful in <xref ref-type="fig" rid="fig3">Figure 3</xref> and elsewhere to have an overall view of the 40S, 60S or 80S to orient the reader to the location of the detailed structures shown in the figures.</p><p>6) In the fifth paragraph of the Results: Should “<xref ref-type="fig" rid="fig3">Figure 3C</xref>” be <xref ref-type="fig" rid="fig3">Figure 3B</xref>? What is meant by the “P stalk”? Lastly, “Since the P and L1-stalks are required for coordinating movement of translation factors...” I am not aware of such a function for the L1 stalk.</p><p>7) In the Discussion section: What exactly is the “contour along one edge”? It was hard to tell from <xref ref-type="fig" rid="fig5">Figure 5B</xref> what was meant.</p><p>8) In general, it would be interesting to see more details of the comparison between this structure and the yeast 80S structure. For example, are they identical, apart from the phylogenetic variations? Are there any interesting conformational changes? What is the rmsd difference between residues in the conserved core? The paper is a bit disappointing in this regard, considering that this is the second structure for a complete eukaryotic ribosome.</p><p><italic>Reviewer #3</italic>:</p><p>1) The authors should explicitly mention which compounds are currently used as front line drugs and cite a review.</p><p>2) Helices in the rRNA are labeled with abbreviations “H##”, also RNA nucleotides are named according to the convention “N##”, both of these abbreviations can cause confusion with single letter amino acid codes also used throughout - this should be changed to avoid ambiguity.</p><p>3) The structure of the <italic>P. falciparium</italic> ribosome is poorly described; it is critical to comment on how the basic architecture deviates (or not) from the crystal structures of previously solved eukaryotic ribosomes, Rabl et al 2011, Klinge et al 2011 and Ben-Shem et al 2011, based on which coordinates the <italic>T. burucei</italic> and the human ribosome has been modeled: otherwise talking about the core structure remains largely unreferenced (eL41 observed in Ben-Shem et al, polypeptide exit tunnel in Klinge et al, description of the architecture of the 40S subunit Rabl et al, etc...). The subsequent comparison with human ribosomes makes sense, as is.</p><p>4) Modifications to pactamycin led to compounds that maintain their antimalarial activity but with reduced toxicity. Can the authors comment on the possible mechanistic reasons for this observation?</p><p>5) <xref ref-type="fig" rid="fig2">Figure 2</xref> – the orientation of the 80S is unusual, the authors should use an orientation that places the two subunits next to each other with the active site and the decoding center oriented up, nascent polypeptide exit tunnel down. This will facilitate comparison with other ribosome structures. Same applies in <xref ref-type="fig" rid="fig4">Figure 4</xref>.</p><p>6) The authors have used a currently unreleased version of Relion (1.3-beta) that includes some modifications. Typically, if modifications of previously published methods and software are used in a publication, the authors should describe it in detail and the program made available.</p><p>7) The 2D and 3D classification should be described in more detail (number of classes, particles per class).</p><p>8) The generation of masks for the focused refinement should be described in more detail.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03080.019</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>Minor comments:</italic></p><p>Reviewer #1:</p><p><italic>1) For future reference it would be interesting to know how much material (in terms mg of purified 80S ribosomes) was available, and from roughly how much starting material (in terms of wet weight of cells) it was isolated</italic>.</p><p>We have now stated in the main text how many mg of ribosomes were yielded per gram of parasite.</p><p><italic>2) For ease of comparison, please provide the concentration of the ribosome solution used for grid preparation in mg/ml, rather than only in terms of molarity</italic>.</p><p>We have now included the ribosome concentration in mg/ml.</p><p><italic>3) Why is the resolution of the emetin complex significantly higher than that of the empty ribosome, even though the pixel size was larger? Is this fully explained by the larger number of particles averaged?</italic></p><p>Pixel size is not the limiting factor in our reconstructions. The DQE of the Falcon-II camera is (nearly) flat out to ∼75% of Nyquist. Apparently, there is at least one other, as yet unidentified, bottleneck that restricts resolution before using a smaller pixel.</p><p><italic>4) The tunnel exit should be labelled in</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2</italic></xref>.</p><p>We have labelled the tunnel exit in <xref ref-type="fig" rid="fig2">Figure 2</xref>.</p><p>Reviewer #2:</p><p><italic>1) The resolution is presented as 3.2A in the last paragraph of the Introduction and 3.4A in the first paragraph of the Results section. The authors need to correct or clarify these two apparently competing statements</italic>.</p><p>We have altered the text to clarify that the resolution of <italic>Pf</italic>80S in the absence of emetine was 3.4 Å, but reached 3.2 Å when emetine was present.</p><p><italic>2) In the third paragraph of the Results section, a description of ESs refers to</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1C-D</italic></xref><italic>. Should this be</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2C-D</italic></xref><italic>?</italic></p><p>We have altered the figure reference.</p><p><italic>3) In the fifth paragraph of the Results section: By “unmodified” are the authors meaning to indicate that this region is conserved?</italic></p><p>We have changed ‘unmodified’ to ‘conserved’.</p><p><italic>4)</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3A</italic></xref><italic>. Is the protein shown in this panel eL41? If so, a label or mention in the legend should be added. Also, it is not clear which rRNA is being shown – is it 18S?</italic></p><p>The figure has been altered to improve the labelling.</p><p><italic>5) It would be helpful in</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3</italic></xref> <italic>and elsewhere to have an overall view of the 40S, 60S or 80S to orient the reader to the location of the detailed structures shown in the figures</italic>.</p><p>We have added overall views of <italic>Pf</italic>80S to clearly indicate the positions of the detailed features.</p><p><italic>6) In the fifth paragraph of the Results: Should “</italic><xref ref-type="fig" rid="fig3"><italic>Figure 3C</italic></xref><italic>” be</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3B</italic></xref><italic>? What is meant by the “P stalk”? Lastly, “Since the P and L1-stalks are required for coordinating movement of translation factors...” I am not aware of such a function for the L1 stalk</italic>.</p><p>Yes! We have corrected the figure reference.</p><p>The P stalk is equivalent to the L7/L12 stalk in prokaryotes and is a lateral protuberance from the ribosome involved in the recruitment of translation factors. This is now stated in the text.</p><p>The L1 stalk is involved in coordinating movement of tRNAs during translocation (Trabuco, et al. (2010) J. Mol. Biol., 402(4):741-760). However, to avoid confusion with protein translation factors we have revised this sentence.</p><p><italic>7) In the Discussion section: What exactly is the “contour along one edge”? It was hard to tell from</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5B</italic></xref> <italic>what was meant</italic>.</p><p>In the emetine molecule there is an edge centred on the ethyl group and closest to the ribosome that could potentially be modified to improve the contacts with the ribosome. This is referred to as the ‘substitution contour’ in <xref ref-type="fig" rid="fig5">Figure 5B</xref>. We have modified the text to make this clearer.</p><p><italic>8) In general, it would be interesting to see more details of the comparison between this structure and the yeast 80S structure. For example, are they identical, apart from the phylogenetic variations? Are there any interesting conformational changes? What is the rmsd difference between residues in the conserved core? The paper is a bit disappointing in this regard, considering that this is the second structure for a complete eukaryotic ribosome</italic>.</p><p>We have now added a substantial discussion of the comparison between the <italic>Plasmodium</italic> and yeast 80S structures focusing particularly on the composition of the two ribosomes and morphological differences in the quaternary structure. This includes a discussion of the absence of RACK1 and a suppressor protein and the inclusion of a tRNA bound at the E-site. We have calculated the RMSD for the conserved cores of the large and small subunits and for the head and body of the small subunit separately. We have also added additional differences between <italic>Pf</italic>80S and the human ribosome, notably the observation of an additional bridge between the platform of the 40S and eL8 in the large subunit.</p><p>Reviewer #3:</p><p><italic>1) The authors should explicitly mention which compounds are currently used as front line drugs and cite a review</italic>.</p><p>We have added the identities of some of the most commonly used anti-malarials that are described in the reference ‘Grimberg BT, Mehlotra RK. Expanding the Antimalarial Drug Arsenal–Now, But How? Pharmaceuticals (Basel). 2011 May 1;4(5):681-712’.</p><p><italic>2) Helices in the rRNA are labeled with abbreviations “H##”, also RNA nucleotides are named according to the convention “N##”, both of these abbreviations can cause confusion with single letter amino acid codes also used throughout – this should be changed to avoid ambiguity</italic>.</p><p>As recommended, we have altered the nomenclature used for labelling rRNA helices from H## to h##. We have also switched to using three-letter amino acid codes in the text.</p><p><italic>3) The structure of the</italic> P. falciparium <italic>ribosome is poorly described; it is critical to comment on how the basic architecture deviates (or not) from the crystal structures of previously solved eukaryotic ribosomes, Rabl et al 2011, Klinge et al 2011 and Ben-Shem et al 2011, based on which coordinates the</italic> T. burucei <italic>and the human ribosome has been modeled: otherwise talking about the core structure remains largely unreferenced (eL41 observed in Ben-Shem et al, polypeptide exit tunnel in Klinge et al, description of the architecture of the 40S subunit Rabl et al, etc...). The subsequent comparison with human ribosomes makes sense, as is</italic>.</p><p>As described in the response to reviewer 2, we have now added a description of how <italic>Pf</italic>80S differs in architecture to the yeast 80S. The features of the core structure have now been referenced accordingly.</p><p><italic>4) Modifications to pactamycin led to compounds that maintain their antimalarial activity but with reduced toxicity. Can the authors comment on the possible mechanistic reasons for this observation?</italic></p><p>We have now commented possible mechanistic reasons for this observation in the Discussion.</p><p><italic>5)</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2</italic></xref> <italic>– the orientation of the 80S is unusual, the authors should use an orientation that places the two subunits next to each other with the active site and the decoding center oriented up, nascent polypeptide exit tunnel down. This will facilitate comparison with other ribosome structures. Same applies in</italic> <xref ref-type="fig" rid="fig4"><italic>Figure 4</italic></xref>.</p><p>We have re-oriented the ribosome in all the relevant figures.</p><p><italic>6) The authors have used a currently unreleased version of Relion (1.3-beta) that includes some modifications. Typically, if modifications of previously published methods and software are used in a publication, the authors should describe it in detail and the program made available</italic>.</p><p>The main features of the new algorithms are described in the Methods section. A more technical description will be presented in a dedicated paper that is currently in preparation. As we have done with all our software in the past, relion-1.3 will be made available as open-source to the community. The stable 1.3-release is planned in 1-2 months, pending updated documentation and final testing.</p><p><italic>7) The 2D and 3D classification should be described in more detail (number of classes, particles per class)</italic>.</p><p>We have added to the Material and methods section a sentence describing how many rounds of 2D and 3D classification were performed for both data sets, and how many classes were used in each case.</p><p><italic>8) The generation of masks for the focused refinement should be described in more detail</italic>.</p><p>An explanation of how the masks were generated has been added to the Materials and methods section.</p></body></sub-article></article>