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| <?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">03035</article-id><article-id pub-id-type="doi">10.7554/eLife.03035</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><subj-group subj-group-type="heading"><subject>Cell biology</subject></subj-group></article-categories><title-group><article-title>A structural model of the active ribosome-bound membrane protein insertase YidC</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-13162"><name><surname>Wickles</surname><given-names>Stephan</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-6"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" id="author-13163"><name><surname>Singharoy</surname><given-names>Abhishek</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="other" rid="par-8"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" id="author-13164"><name><surname>Andreani</surname><given-names>Jessica</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" id="author-13165"><name><surname>Seemayer</surname><given-names>Stefan</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-13"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" id="author-13166"><name><surname>Bischoff</surname><given-names>Lukas</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-6"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" id="author-13167"><name><surname>Berninghausen</surname><given-names>Otto</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" id="author-13168"><name><surname>Soeding</surname><given-names>Johannes</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-7"/><xref ref-type="other" rid="par-10"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" id="author-8940"><name><surname>Schulten</surname><given-names>Klaus</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="other" rid="par-9"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" corresp="yes" id="author-13169"><name><surname>van der Sluis</surname><given-names>Eli O</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-12"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" corresp="yes" id="author-7830"><name><surname>Beckmann</surname><given-names>Roland</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-5"/><xref ref-type="other" rid="par-11"/><xref ref-type="other" rid="par-12"/><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><aff id="aff1"><institution content-type="dept">Gene Center Munich, Department of Biochemistry</institution>, <institution>Ludwig-Maximilians-Universität München</institution>, <addr-line><named-content content-type="city">Munich</named-content></addr-line>, <country>Germany</country></aff><aff id="aff2"><institution content-type="dept">Center for Integrated Protein Science Munich, Department of Biochemistry</institution>, <institution>Ludwig-Maximilians-Universität München</institution>, <addr-line><named-content content-type="city">Munich</named-content></addr-line>, <country>Germany</country></aff><aff id="aff3"><institution content-type="dept">Beckman Institute for Advanced Science and Technology</institution>, <institution>University of Illinois at Urbana-Champaign</institution>, <addr-line><named-content content-type="city">Urbana</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Hegde</surname><given-names>Ramanujan S</given-names></name><role>Reviewing editor</role><aff><institution>MRC Laboratory of Molecular Biology</institution>, <country>United Kingdom</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>vandersluis@lmb.uni-muenchen.de</email> (EOS); </corresp><corresp id="cor2"><label>*</label>For correspondence:<email>beckmann@lmb.uni-muenchen.de</email> (RB)</corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>10</day><month>07</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03035</elocation-id><history><date date-type="received"><day>08</day><month>04</month><year>2014</year></date><date date-type="accepted"><day>08</day><month>07</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Wickles et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Wickles et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">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="elife03035.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03035.001</object-id><p>The integration of most membrane proteins into the cytoplasmic membrane of bacteria occurs co-translationally. The universally conserved YidC protein mediates this process either individually as a membrane protein insertase, or in concert with the SecY complex. Here, we present a structural model of YidC based on evolutionary co-variation analysis, lipid-versus-protein-exposure and molecular dynamics simulations. The model suggests a distinctive arrangement of the conserved five transmembrane domains and a helical hairpin between transmembrane segment 2 (TM2) and TM3 on the cytoplasmic membrane surface. The model was used for docking into a cryo-electron microscopy reconstruction of a translating YidC-ribosome complex carrying the YidC substrate F<sub>O</sub>c. This structure reveals how a single copy of YidC interacts with the ribosome at the ribosomal tunnel exit and identifies a site for membrane protein insertion at the YidC protein-lipid interface. Together, these data suggest a mechanism for the co-translational mode of YidC-mediated membrane protein insertion.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.001">http://dx.doi.org/10.7554/eLife.03035.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03035.002</object-id><title>eLife digest</title><p>Cells are surrounded by a plasma membrane that acts like a barrier to help to keep the cell intact. Proteins are embedded in this plasma membrane; and some of these membrane proteins act as channels that allow molecules to enter and leave the cell, while others allow the cell to communicate with its surroundings.</p><p>Like all proteins, membrane proteins are chains of amino acids that are joined together by a molecular machine called a ribosome. Most membrane proteins are inserted into the membrane as they are being built. All bacteria contain a protein called YidC that inserts proteins into the plasma membrane of bacterial cells. However, the mechanism behind this activity and the parts of the YidC protein that interact with the ribosome and plasma membrane are unknown.</p><p>Wickles et al. have now used data from a range of sources to predict the three-dimensional structure of the YidC protein taken from a bacterium called <italic>E. coli</italic>. The model shows how the YidC protein is threaded back-and-forth through the membrane, a total of five times. Some of the protein also extends into the inside of the bacterial cell. Wickles et al. then used a technique called cyro-electron microscopy to look at the structure of a YidC protein bound to a ribosome that is building a new protein. Fitting the more detailed model of YidC into this overall structure of the whole complex revealed how a single YidC protein might interact with the ribosome to insert a newly built protein into a membrane.</p><p>Wickles et al. then used a combination of theoretical modeling and other experiments to identify the amino acids in the YidC protein that bind to the ribosome: as expected, the binding takes place where the newly formed protein chain exits the ribosome. Further experiments also identified the amino acids in the YidC protein that interact with the newly built membrane protein, thus revealing where it might leave the YidC protein and be inserted into the membrane. The next challenge will be to investigate how the YidC protein assists the folding of new membrane proteins into their own highly specific three-dimensional structure.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.002">http://dx.doi.org/10.7554/eLife.03035.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>ribosome</kwd><kwd>YidC</kwd><kwd>cryo-EM</kwd><kwd>bioinformatics</kwd><kwd>molecular dynamics</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>E. coli</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001659</institution-id><institution>Deutsche Forschungsgemeinschaft</institution></institution-wrap></funding-source><award-id>GRK1721, SFB646, QBM</award-id><principal-award-recipient><name><surname>Soeding</surname><given-names>Johannes</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/10.13039/100005156</institution-id><institution>Alexander von Humboldt-Stiftung</institution></institution-wrap></funding-source><award-id>Research Fellowship</award-id><principal-award-recipient><name><surname>Andreani</surname><given-names>Jessica</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/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>Center for Macromolecular Modeling and Bioinformatics, 9P41GM104601</award-id><principal-award-recipient><name><surname>Singharoy</surname><given-names>Abhishek</given-names></name><name><surname>Schulten</surname><given-names>Klaus</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000001</institution-id><institution>National Science Foundation</institution></institution-wrap></funding-source><award-id>Center for the Physics of Living Cells, PHY-0822613</award-id><principal-award-recipient><name><surname>Singharoy</surname><given-names>Abhishek</given-names></name><name><surname>Schulten</surname><given-names>Klaus</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/10.13039/501100000781</institution-id><institution>European Research Council</institution></institution-wrap></funding-source><award-id>CRYOTRANSLATION</award-id><principal-award-recipient><name><surname>Beckmann</surname><given-names>Roland</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/10.13039/501100004189</institution-id><institution>Max-Planck-Gesellschaft</institution></institution-wrap></funding-source><award-id>International Max Planck Research School</award-id><principal-award-recipient><name><surname>Wickles</surname><given-names>Stephan</given-names></name><name><surname>Bischoff</surname><given-names>Lukas</given-names></name></principal-award-recipient></award-group><award-group id="par-7"><funding-source><institution-wrap><institution>Bavarian Network for Molecular Biosystems (BioSysNet)</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Andreani</surname><given-names>Jessica</given-names></name><name><surname>Soeding</surname><given-names>Johannes</given-names></name></principal-award-recipient></award-group><award-group id="par-8"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100005471</institution-id><institution>Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana–Champaign</institution></institution-wrap></funding-source><award-id>Postdoctoral Fellowship</award-id><principal-award-recipient><name><surname>Singharoy</surname><given-names>Abhishek</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/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>Center for Macromolecular Modeling and Bioinformatics, R01-GM67887</award-id><principal-award-recipient><name><surname>Schulten</surname><given-names>Klaus</given-names></name></principal-award-recipient></award-group><award-group id="par-10"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100002347</institution-id><institution>Bundesministerium für Bildung und Forschung</institution></institution-wrap></funding-source><award-id>CoreSys</award-id><principal-award-recipient><name><surname>Soeding</surname><given-names>Johannes</given-names></name></principal-award-recipient></award-group><award-group id="par-11"><funding-source><institution-wrap><institution>Centre for Integrated Protein Science (CIPSM)</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Beckmann</surname><given-names>Roland</given-names></name></principal-award-recipient></award-group><award-group id="par-12"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001659</institution-id><institution>Deutsche Forschungsgemeinschaft</institution></institution-wrap></funding-source><award-id>FOR967</award-id><principal-award-recipient><name><surname>Beckmann</surname><given-names>Roland</given-names></name><name><surname>van der Sluis</surname><given-names>Eli O</given-names></name></principal-award-recipient></award-group><award-group id="par-13"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001659</institution-id><institution>Deutsche Forschungsgemeinschaft</institution></institution-wrap></funding-source><award-id>GRK1721</award-id><principal-award-recipient><name><surname>Seemayer</surname><given-names>Stefan</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><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>A cryo-EM structure combined with bioinformatics provide a structural view on a conserved co-translational membrane protein biogenesis pathway.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>At present, a mechanistic understanding of the function of YidC, as well as its mitochondrial and chloroplast counterparts Oxa1 and Alb3, respectively, is limited by a lack of structural information (<xref ref-type="bibr" rid="bib22">Kol et al., 2008</xref>; <xref ref-type="bibr" rid="bib5">Dalbey et al., 2011</xref>). High resolution structures are available only for the first periplasmic domain (P1) of <italic>Escherichia coli</italic> YidC (<xref ref-type="fig" rid="fig1">Figure 1A</xref>; <xref ref-type="bibr" rid="bib31">Oliver and Paetzel, 2008</xref>; <xref ref-type="bibr" rid="bib36">Ravaud et al., 2008</xref>), however, this domain is poorly conserved, only present in Gram-negative bacteria and not essential for functionality (<xref ref-type="bibr" rid="bib13">Jiang et al., 2003</xref>). Furthermore, the region(s) of YidC mediating the interaction with the ribosome have not been identified, and the oligomeric state of YidC during co-translational translocation remains controversial (<xref ref-type="bibr" rid="bib21">Kohler et al., 2009</xref>; <xref ref-type="bibr" rid="bib11">Herrmann, 2013</xref>; <xref ref-type="bibr" rid="bib17">Kedrov et al., 2013</xref>). Hence, we set out to determine a molecular model of ribosome-bound YidC during co-translational translocation of the substrate F<sub>O</sub>c (<xref ref-type="bibr" rid="bib44">van der Laan et al., 2004</xref>), an integral membrane subunit of the ATP synthase complex.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03035.003</object-id><label>Figure 1.</label><caption><title>Evolutionary covariation based structural model of <italic>E. coli</italic> YidC.</title><p>(<bold>A</bold>) Membrane topology of YidC, with helix coloring as in all subsequent Figures. (<bold>B</bold>) Matrix of coupling strengths between pairs of YidC residues based on an alignment of 2366 non-redundant sequences. Helix–helix pairs with posterior probabilities higher than 57% are outlined in boxes; the 50 residue–residue pairs with highest coupling coefficients are indicated with red crosses. (<bold>C</bold>) Overall arrangement of TM helices viewed from the cytoplasm based on the prediction of helix–helix pairs (black lines) and exposure to lipid (yellow) or protein (green). The first residue of each helix is indicated with an asterisk. (<bold>D</bold>) Linear representation of YidC with the seven most probable helix–helix pairs indicated by arches, with thicknesses approximating posterior probabilities. (<bold>E</bold> and <bold>F</bold>) Side view and cytoplasmic view, respectively, of the <italic>E. coli</italic> YidC model based on covariation analysis, with predicted residue–residue pairs indicated by yellow pseudobonds.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.003">http://dx.doi.org/10.7554/eLife.03035.003</ext-link></p></caption><graphic xlink:href="elife03035f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03035.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Evaluation of possible helix-helix contacts.</title><p>(<bold>A</bold>) Calibration plots for the prediction of helix–helix interactions. Calibration plots for dataset #2 (left), dataset #3 (middle) and combined datasets #2 and #3 (right). The empirical fraction of true positives is plotted depending on the uncalibrated probability (raw score) obtained from our method. Points correspond to empirical averages over bins of 60 predictions (ordered by increasing uncalibrated probability). Lines correspond to maximum likelihood fits of the calibration plots using a transformed Bernoulli distribution with 4 parameters. (<bold>B</bold>) Histogram of posterior probabilities for helix–helix interactions. Distribution of predicted calibrated posterior probabilities for YidC (TM2–TM6) which contains seven predicted helices, thus 21 possible helix–helix contacts. The histogram of predicted probabilities shows the specificity of the predictions: there is a large gap between 15% and 55% probability and most possible contacts have probability <15%.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.004">http://dx.doi.org/10.7554/eLife.03035.004</ext-link></p></caption><graphic xlink:href="elife03035fs001"/></fig></fig-group></p></sec><sec id="s2" sec-type="results"><title>Results</title><p>In order to build an initial structural model of YidC, we predicted contacts between pairs of residues based on covariation analysis (<xref ref-type="bibr" rid="bib27">Marks et al., 2011</xref>; <xref ref-type="bibr" rid="bib12">Hopf et al., 2012</xref>). For that purpose, we constructed a multiple sequence alignment of <italic>E. coli</italic> YidC excluding the nonconserved first transmembrane helix (TM1) and the P1 domain (<xref ref-type="fig" rid="fig1">Figure 1A</xref>) and computed direct evolutionary couplings between pairs of YidC residues (<xref ref-type="bibr" rid="bib16">Kamisetty et al., 2013</xref>). The resulting matrix of coupling strengths (<xref ref-type="fig" rid="fig1">Figure 1B</xref>) contains several diagonal and anti-diagonal patterns of stronger coupling coefficients, which are indicative of parallel or anti-parallel helix–helix pairs, respectively. We computed probabilities for each possible helix–helix contact by aggregating the evidence of stronger coupling coefficients over the expected interaction patterns and calibrating the resulting raw scores on an independent dataset of helix–helix interactions to obtain accurate interaction probabilities. Seven helix–helix contacts attained probabilities above 57% (<xref ref-type="fig" rid="fig1">Figure 1B–D</xref>) while all other possible contacts scored below 15%, demonstrating the specificity of the method (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>).</p><p>We roughly positioned the five TM helices of <italic>E. coli</italic> YidC relative to each other using the predicted helix–helix contacts as constraints, and rotated them according to their predicted lipid or protein exposure (<xref ref-type="bibr" rid="bib24">Lai et al., 2013</xref>; <xref ref-type="fig" rid="fig1">Figure 1C</xref>). Next, we used MODELLER (<xref ref-type="bibr" rid="bib7">Eswar et al., 2008</xref>) to create full length models based on the TM core, secondary structure prediction and the 50 residue–residue contacts with the highest coupling coefficients (39 excluding intrahelical contacts, indels and topology violations). In the resulting model (<xref ref-type="fig" rid="fig1">Figure 1E,F</xref>), the conserved membrane integrated core of YidC forms a helical bundle arranged like the vertices of a pentagon, in the order 4-5-3-2-6 (clockwise) when viewed from the cytoplasm (<xref ref-type="fig" rid="fig1">Figure 1F</xref>). Notably, all the predicted interactions between TM domains can be explained by monomeric YidC suggesting that dimer or oligomer formation may not be strictly required for YidC activity (see also below).</p><p>Outside the membrane region, strong helix–helix contacts were predicted within the cytoplasmic loop between TM2 and TM3, which can be explained the by formation of a helical hairpin (<xref ref-type="fig" rid="fig1">Figure 1F</xref>). The base of this ‘helical paddle domain’ (HPD) is structurally constrained by predicted contacts with TM3, its tip on the other hand is more mobile and appears to interact with lipid headgroups (see below).</p><p>While this manuscript was under review, two crystal structures were published of <italic>Bacillus halodurans</italic> YidC2 (BhYidC2, 34% sequence identity with <italic>E. coli</italic> YidC) (<xref ref-type="bibr" rid="bib23">Kumazaki et al., 2014</xref>), providing us with a unique opportunity to directly assess the accuracy of our model. Overall, the root mean square deviation (RMSD) between the TM helices of our model and those of BhYidC2 is 7.5 Å (3WO6) and 7.3 Å (3WO7) (<xref ref-type="table" rid="tbl1">Table 1</xref>), which is within the resolution limits of our method. The global arrangement of TM helices is the same as in BhYidC2, yet, their tilt angle relative to the plane of the membrane is slightly different (<xref ref-type="fig" rid="fig2">Figure 2</xref>). The tilt angle of the HPD also differs, as well as its side that faces the membrane (<xref ref-type="other" rid="video1">Video 1</xref>), which may be indicative of a high degree of flexibility of this domain, consistent with its high crystallographic B-factors (<xref ref-type="bibr" rid="bib23">Kumazaki et al., 2014</xref>). Notably, the HPD is not essential for YidC function in <italic>E. coli</italic> since the deletion of the entire domain is possible without compromising cell viability (<xref ref-type="bibr" rid="bib13">Jiang et al., 2003</xref>).<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03035.005</object-id><label>Table 1.</label><caption><p>Deviations among YidC structures</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.005">http://dx.doi.org/10.7554/eLife.03035.005</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th/><th/><th>RMSD (Å)</th><th>RMSD (Å) (TM core)</th></tr></thead><tbody><tr><td>3WO6</td><td>3WO7</td><td align="char" char=".">3.1</td><td align="char" char=".">1.8</td></tr><tr><td>3WO6</td><td>model</td><td align="char" char=".">9.4</td><td align="char" char=".">7.5</td></tr><tr><td>3WO7</td><td>model</td><td align="char" char=".">9.8</td><td align="char" char=".">7.3</td></tr></tbody></table><table-wrap-foot><fn><p>Overall root mean square deviations (RMSD) between (the TM helices of) our model of <italic>E. coli</italic> YidC and the two BhYidC2 crystal forms.</p></fn></table-wrap-foot></table-wrap><fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03035.006</object-id><label>Figure 2.</label><caption><title>Covariation-based model vs homology model.</title><p>Comparison of the <italic>E. coli</italic> YidC covariation-based model (<bold>A</bold> and <bold>B</bold>) to a homology model of <italic>E. coli</italic> YidC based on the crystal structure of BhYidC2 (3WO6) (<bold>C</bold> and <bold>D</bold>). Predicted residue–residue pairs are indicated by yellow pseudobonds. Note that extracellular helix 1 (white) was not present in our multiple sequence alignment and is thus not included in the model.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.006">http://dx.doi.org/10.7554/eLife.03035.006</ext-link></p></caption><graphic xlink:href="elife03035f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03035.007</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Local deviations among YidC structures.</title><p>Smoothed Cα distances between the two BhYidC2 crystal forms (3WO6 vs 3WO7, red), between our model of <italic>E. coli</italic> YidC and 3WO6 (green) and between our model and 3WO7 (blue).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.007">http://dx.doi.org/10.7554/eLife.03035.007</ext-link></p></caption><graphic xlink:href="elife03035fs002"/></fig></fig-group><media content-type="glencoe play-in-place height-250 width-310" id="video1" mime-subtype="mov" mimetype="video" xlink:href="elife03035v001.mov"><object-id pub-id-type="doi">10.7554/eLife.03035.008</object-id><label>Video 1.</label><caption><title>Conformational states of YidC.</title><p>Animation showing conformational differences in YidC starting from BhYidC2 crystal form 1 (3WO6), towards crystal form 2 (3WO7) and ending with our covariation based YidC model. Views are from within the membrane (left) and from the cytoplasm (right). Note the movement of the HPD and the closing of the hydrophilic groove between TM3 (orange) and TM5 (green).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.008">http://dx.doi.org/10.7554/eLife.03035.008</ext-link></p></caption></media></p><p>A qualitative difference between our model and BhYidC2 that may have more mechanistic importance is the relative position of TM3. In the structure of BhYidC2 a hydrophilic groove is formed on the cytoplasmic side of the TM bundle that has been proposed to form a binding site for YidC substrates (<xref ref-type="bibr" rid="bib23">Kumazaki et al., 2014</xref>). Interestingly, the opening state of this groove differs between the two crystal forms, that is it is more open in 3WO6 than in 3WO7 (<xref ref-type="other" rid="video1">Video 1</xref>), largely due to movement of the N-terminal half of TM3 (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). In our model on the other hand, this hydrophilic groove is even more closed than in 3WO7 because we imposed covariation-based constraints between TM3 and TM5 (Pro<sup>425</sup>-Pro<sup>499</sup>) and between TM3 and TM6 (Cys<sup>423</sup>-Gln<sup>528</sup> & Phe<sup>433</sup>-Thr<sup>524</sup>) (<xref ref-type="fig" rid="fig2">Figure 2</xref>; <xref ref-type="other" rid="video1">Video 1</xref>). Strikingly, in BhYidC2 the distances between the Cβ atoms of these three pairs are outliers compared to other residue–residue pairs (20.5 Å/20.9 Å/14.9 Å vs an average of 8.2 Å, <xref ref-type="table" rid="tbl2">Table 2</xref>). Thus, given that (i) the position of TM3 differs in the two crystal forms, and (ii) that covariation analysis predicts with high accuracy a closer interaction of TM3 with TM6 and one contact with TM5, we conclude that movement of TM3 is a genuine feature of YidC. This movement and the accompanying dynamics of the hydrophilic groove may represent a crucial step in the functional cycle of the YidC insertase.<table-wrap id="tbl2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03035.009</object-id><label>Table 2.</label><caption><p>Top 50 scoring residue–residue pairs in covariation analysis</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.009">http://dx.doi.org/10.7554/eLife.03035.009</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Residue 1</th><th># Residue 1</th><th>Region</th><th/><th>Residue 2</th><th># Residue 2</th><th>Region</th><th>dmodel (Å)</th><th>d3WO6 (Å)</th><th>Reason for exclusion</th></tr></thead><tbody><tr><td><italic>TRP</italic></td><td><italic>354</italic></td><td><italic>TM2</italic></td><td/><td><italic>–</italic></td><td><italic>–</italic></td><td><italic>–</italic></td><td/><td/><td><italic>indel</italic></td></tr><tr><td><italic>GLY</italic></td><td><italic>355</italic></td><td><italic>TM2</italic></td><td/><td><italic>–</italic></td><td>–</td><td>–</td><td/><td/><td><italic>indel</italic></td></tr><tr><td><italic>PHE</italic></td><td><italic>356</italic></td><td><italic>TM2</italic></td><td/><td>–</td><td>–</td><td>–</td><td/><td/><td><italic>indel</italic></td></tr><tr><td><italic>PHE</italic></td><td><italic>356</italic></td><td><italic>TM2</italic></td><td/><td><italic>ARG</italic></td><td><italic>533</italic></td><td><italic>c-term</italic></td><td/><td/><td><italic>topology violation</italic></td></tr><tr><td>ILE</td><td>358</td><td>TM2</td><td><–></td><td>GLY</td><td>512</td><td>Loop5-6</td><td>9.1</td><td>6.1</td><td/></tr><tr><td>ILE</td><td>359</td><td>TM2</td><td><–></td><td>VAL</td><td>519</td><td>TM6</td><td>6.5</td><td>5.2</td><td/></tr><tr><td>ILE</td><td>359</td><td>TM2</td><td><–></td><td>LEU</td><td>515</td><td>TM6</td><td>8.5</td><td>7.9</td><td/></tr><tr><td><italic>ILE</italic></td><td><italic>359</italic></td><td><italic>TM2</italic></td><td/><td>–</td><td>–</td><td>–</td><td/><td/><td><italic>indel</italic></td></tr><tr><td>ILE</td><td>361</td><td>TM2</td><td><–></td><td>LEU</td><td>436</td><td>TM3</td><td>7.9</td><td>8.2</td><td/></tr><tr><td><italic>THR</italic></td><td><italic>362</italic></td><td><italic>TM2</italic></td><td/><td><italic>PRO</italic></td><td><italic>371</italic></td><td><italic>TM2</italic></td><td/><td/><td><italic>intrahelical</italic></td></tr><tr><td>PHE</td><td>363</td><td>TM2</td><td><–></td><td>VAL</td><td>523</td><td>TM6</td><td>5.2</td><td>6.1</td><td/></tr><tr><td>GLY</td><td>367</td><td>TM2</td><td><–></td><td>VAL</td><td>523</td><td>TM6</td><td>6.0</td><td>8.2</td><td/></tr><tr><td>MET</td><td>369</td><td>TM2</td><td><–></td><td>ILE</td><td>432</td><td>TM3</td><td>9.9</td><td>8.4</td><td/></tr><tr><td><italic>Leu</italic></td><td><italic>372</italic></td><td><italic>Loop2-3</italic></td><td/><td><italic>PRO</italic></td><td><italic>510</italic></td><td><italic>Loop5-6</italic></td><td/><td/><td><italic>topology violation</italic></td></tr><tr><td>SER</td><td>379</td><td>Loop2-3</td><td><–></td><td>PRO</td><td>425</td><td>TM3</td><td>10.2</td><td>9.9</td><td/></tr><tr><td>LEU</td><td>386</td><td>Loop2-3</td><td><–></td><td>VAL</td><td>417</td><td>Loop2-3</td><td>7.5</td><td>7.1</td><td/></tr><tr><td>LEU</td><td>386</td><td>Loop2-3</td><td><–></td><td>LEU</td><td>411</td><td>Loop2-3</td><td>6.2</td><td>6.1</td><td/></tr><tr><td><italic>PRO</italic></td><td><italic>388</italic></td><td><italic>Loop2-3</italic></td><td/><td><italic>GLN</italic></td><td><italic>429</italic></td><td><italic>TM3</italic></td><td/><td/><td><italic>topology violation</italic></td></tr><tr><td>LYS</td><td>389</td><td>Loop2-3</td><td><–></td><td>ALA</td><td>414</td><td>Loop2-3</td><td>10.5</td><td>9.8</td><td/></tr><tr><td>LYS</td><td>389</td><td>Loop2-3</td><td><–></td><td>GLU</td><td>415</td><td>Loop2-3</td><td>11.2</td><td>10.0</td><td/></tr><tr><td>ILE</td><td>390</td><td>Loop2-3</td><td><–></td><td>MET</td><td>408</td><td>Loop2-3</td><td>6.8</td><td>6.2</td><td/></tr><tr><td>MET</td><td>393</td><td>Loop2-3</td><td><–></td><td>ILE</td><td>404</td><td>Loop2-3</td><td>7.9</td><td>7.4</td><td/></tr><tr><td>MET</td><td>393</td><td>Loop2-3</td><td><–></td><td>LEU</td><td>411</td><td>Loop2-3</td><td>8.2</td><td>7.7</td><td/></tr><tr><td>ARG</td><td>394</td><td>Loop2-3</td><td><–></td><td>ILE</td><td>404</td><td>Loop2-3</td><td>8.5</td><td>8.1</td><td/></tr><tr><td>ARG</td><td>396</td><td>Loop2-3</td><td><–></td><td>GLU</td><td>407</td><td>Loop2-3</td><td>8.9</td><td>8.4</td><td/></tr><tr><td>CYS</td><td>423</td><td>TM3</td><td><–></td><td>GLN</td><td>528</td><td>TM6</td><td>16.2</td><td>20.9</td><td/></tr><tr><td>PRO</td><td>425</td><td>TM3</td><td><–></td><td>PRO</td><td>499</td><td>TM5</td><td>10.2</td><td>20.5</td><td/></tr><tr><td>PHE</td><td>433</td><td>TM3</td><td><–></td><td>THR</td><td>524</td><td>TM6</td><td>11.0</td><td>14.9</td><td/></tr><tr><td>LEU</td><td>436</td><td>TM3</td><td><–></td><td>GLY</td><td>512</td><td>Loop5-6</td><td>7.6</td><td>8.3</td><td/></tr><tr><td>TYR</td><td>437</td><td>TM3</td><td><–></td><td>LEU</td><td>513</td><td>Loop5-6</td><td>9.8</td><td>6.4</td><td/></tr><tr><td>TRP</td><td>454</td><td>Loop3-4</td><td><–></td><td>ASP</td><td>462</td><td>Loop3-4</td><td>6.6</td><td>7.0</td><td/></tr><tr><td>TRP</td><td>454</td><td>Loop3-4</td><td><–></td><td>PRO</td><td>468</td><td>TM4</td><td>16.0</td><td>11.5</td><td/></tr><tr><td>TRP</td><td>454</td><td>Loop3-4</td><td><–></td><td>SER</td><td>511</td><td>Loop5-6</td><td>9.8</td><td>8.3</td><td/></tr><tr><td>ILE</td><td>455</td><td>Loop3-4</td><td><–></td><td>LEU</td><td>467</td><td>TM4</td><td>9.8</td><td>10.1</td><td/></tr><tr><td>ILE</td><td>455</td><td>Loop3-4</td><td><–></td><td>ILE</td><td>466</td><td>TM4</td><td>11.0</td><td>8.0</td><td/></tr><tr><td>ASP</td><td>462</td><td>Loop3-4</td><td><–></td><td>PRO</td><td>468</td><td>TM4</td><td>12.5</td><td>6.8</td><td/></tr><tr><td>ASP</td><td>462</td><td>Loop3-4</td><td><–></td><td>SER</td><td>511</td><td>Loop5-6</td><td>11.1</td><td>4.2</td><td/></tr><tr><td>TYR</td><td>465</td><td>TM4</td><td><–></td><td>LEU</td><td>507</td><td>TM5</td><td>10.4</td><td>8.7</td><td/></tr><tr><td>LEU</td><td>467</td><td>TM4</td><td><–></td><td>LEU</td><td>515</td><td>TM6</td><td>11.6</td><td>6.6</td><td/></tr><tr><td>PRO</td><td>468</td><td>TM4</td><td><–></td><td>LEU</td><td>513</td><td>TM6</td><td>14.5</td><td>8.8</td><td/></tr><tr><td>LEU</td><td>470</td><td>TM4</td><td><–></td><td>ILE</td><td>518</td><td>TM6</td><td>6.3</td><td>5.4</td><td/></tr><tr><td>MET</td><td>471</td><td>TM4</td><td><–></td><td>PHE</td><td>502</td><td>TM5</td><td>8.8</td><td>4.9</td><td/></tr><tr><td>GLY</td><td>472</td><td>TM4</td><td><–></td><td>THR</td><td>503</td><td>TM5</td><td>6.7</td><td>5.3</td><td/></tr><tr><td><italic>GLY</italic></td><td><italic>472</italic></td><td><italic>TM4</italic></td><td/><td><italic>GLN</italic></td><td><italic>479</italic></td><td><italic>TM4</italic></td><td/><td/><td><italic>intrahelical</italic></td></tr><tr><td>THR</td><td>474</td><td>TM4</td><td><–></td><td>ASN</td><td>521</td><td>TM6</td><td>4.7</td><td>3.7</td><td/></tr><tr><td>THR</td><td>474</td><td>TM4</td><td><–></td><td>ILE</td><td>525</td><td>TM6</td><td>6.7</td><td>7.8</td><td/></tr><tr><td>ILE</td><td>478</td><td>TM4</td><td><–></td><td>ILE</td><td>525</td><td>TM6</td><td>9.0</td><td>5.0</td><td/></tr><tr><td><italic>THR</italic></td><td><italic>485</italic></td><td><italic>Loop4-5</italic></td><td/><td>–</td><td>–</td><td>–</td><td/><td/><td><italic>indel</italic></td></tr><tr><td>PHE</td><td>506</td><td>TM5</td><td><–></td><td>VAL</td><td>514</td><td>TM6</td><td>14.4</td><td>4.2</td><td/></tr><tr><td><italic>GLY</italic></td><td><italic>512</italic></td><td><italic>Loop5-6</italic></td><td/><td><italic>GLN</italic></td><td><italic>532</italic></td><td><italic>TM6</italic></td><td/><td/><td><italic>topology violation</italic></td></tr><tr><td/><td/><td/><td/><td/><td/><td><bold>Ø</bold></td><td><bold>9.3</bold></td><td><bold>8.1</bold></td><td/></tr></tbody></table><table-wrap-foot><fn><p>Table showing the 50 residue–residue pairs with the highest covariation scores, and the distances between the Cβ atoms in the final model of the 39 pairs that were used as constraints for model building. For comparison, the corresponding distances in 3WO6 are also given. The 11 residue–residue pairs that were excluded for model building are in italics, with the reason for their exclusion indicated on the right.</p></fn></table-wrap-foot></table-wrap></p><p>In summary, the overall structure of our YidC model agrees well with the BhYidC2 crystal structure, and a comparison of both structures reveals dynamic regions in YidC that may be of mechanistic importance. This further illustrates the power of covariation analysis not merely for structure prediction but also for obtaining dynamic insights (<xref ref-type="bibr" rid="bib12">Hopf et al., 2012</xref>).</p><p>Next, in order to further characterize and validate our obtained YidC model, we assessed its stability and biochemical properties in the bacterial membrane by employing traditional molecular dynamics (MD) simulations. Overall, the model was found to be very stable during the simulation. While the five TM helices enable a rigid protein core, the polar loop regions tend to swim on the membrane surface (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). An analysis of inter-residue interactions within the TM region (<xref ref-type="fig" rid="fig3">Figure 3B</xref>) provides a firm basis to the observed stability of YidC: hydrophobic residues on the exterior of the TM bundle stabilize interactions with the apolar lipid tails. The YidC core, in turn, is stabilized both via short and long-range interactions between the five helices. Residues towards the cytoplasmic side of the core are primarily polar or charged and, therefore, engaged in strong electrostatic or charge–dipole interactions. In contrast, residues on the periplasmic side are primarily aromatic and involved in stacking and other nonpolar dispersion interactions.<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03035.010</object-id><label>Figure 3.</label><caption><title>Molecular dynamics simulation of the YidC model.</title><p>(<bold>A</bold>) Side view (left) and cytoplasmic view (right) of the stable YidC model after a 500 ns MD simulation in a lipid bilayer composed of 3:1 POPE:POPG. (<bold>B</bold>) Ribbon representation of the stable model according to inter-helix energy (in kcal/mol), blue: −7.5 to −1; white: −1 to −0.002; red: ≥ −0.00.2. Residues that inactivate YidC upon mutagenesis are indicated by spheres. (<bold>C</bold>) Ribbon representation of the stable model according to flexibility (in Å<sup>2</sup>), blue: 0.04 to 0.09; white: 0.09–1; red: ≥1.0. (<bold>D</bold>) <italic>In vivo</italic> complementation assay of YidC mutants T362A (TM2) and Y517A (TM6). (<bold>E</bold>) Thickness of the cytoplasmic and periplasmic leaflet of the simulated bilayer after 500 ns, highlighting the membrane thinning effect in the vicinity of YidC. The membrane surface is defined by positions of polar head groups in the lipids, and thickness at a given point on the surface is taken to be the shortest distance between the head groups from opposite leaflets. The thickness values are averaged over the MD trajectory and presented as a contour plot on the membrane surface with a color-scale from red, indicating thicker region representing bulk bilayer lipids, to blue showing thinned regions close to YidC suggesting hydrophobic mismatch. (<bold>F</bold>) Distribution of hydrophobic (red) and hydrophilic residues (blue) in YidC at various heights of the membrane, highlighting the hydrophilic environment in the center of YidC on the cytoplasmic side.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.010">http://dx.doi.org/10.7554/eLife.03035.010</ext-link></p></caption><graphic xlink:href="elife03035f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03035.011</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Complementation of MD-based mutants.</title><p>In vivo complementation assay of YidC mutants identified as structurally important by MD simulations. Positions in YidC that were also identified by covariation analyses are indicated in the right column.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.011">http://dx.doi.org/10.7554/eLife.03035.011</ext-link></p></caption><graphic xlink:href="elife03035fs003"/></fig><fig id="fig3s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03035.012</object-id><label>Figure 3—figure supplement 2.</label><caption><title>Expression of MD-based mutants.</title><p>Western blot of whole FTL10 cells grown on arabinose or glucose, showing the stable expression of inactive YidC mutants that were identifed by MD simulations.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.012">http://dx.doi.org/10.7554/eLife.03035.012</ext-link></p></caption><graphic xlink:href="elife03035fs004"/></fig></fig-group></p><p>In order to verify the functional relevance of residues suggested by the MD simulations, we created alanine mutants and subjected them to an <italic>in vivo</italic> complementation assay. Some of the most stabilizing residues, T362 in TM2 and Y517 in TM6, both of which are located at the same height in the membrane, completely inactivated YidC when mutated to alanine (<xref ref-type="fig" rid="fig3">Figure 3D</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). Both mutants were stably expressed, indicating that the lack of complementation was not caused by instability of YidC (<xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2</xref>). Several residues close to this pair show intermediate activity levels (F433, M471 and F505), whereas residues further away do not show an effect (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). Taken together, we provide a model for the overall arrangement of the conserved domains of YidC that is in good agreement with our covariation analysis, lipid exposure prediction, MD simulation, <italic>in vivo</italic> complementation analysis as well as the recent crystal structures.</p><p>Interestingly, we observed that YidC induces thinning of the lipid bilayer during the MD simulation. A significant thinning of 7–10 Å results from the hydrophobic mismatch between the TM helices and the membrane (<xref ref-type="fig" rid="fig3">Figure 3E</xref>). The thinning is similar in the upper and lower leaflet, and the thinnest region is in proximity of TM3 and TM5. Since membrane inserting YidC substrates have been chemically cross-linked to both these helices (<xref ref-type="bibr" rid="bib20">Klenner et al., 2008</xref>; <xref ref-type="bibr" rid="bib46">Yu et al., 2008</xref>; <xref ref-type="bibr" rid="bib19">Klenner and Kuhn, 2012</xref>), we argue that thinning of this region in particular may be relevant for the molecular mechanism of YidC-dependent membrane insertion. In addition, the distribution of hydrophilic and hydrophobic residues within YidC revealed the presence of a hydrophilic environment on the cytoplasmic side of the YidC TM bundle (<xref ref-type="fig" rid="fig3">Figure 3F</xref>), which continues into the mentioned hydrophobic cluster of aromatic residues towards the periplasmic side. It is tempting to speculate that this hydrophilic environment may receive the polar termini and loops of YidC substrates during the initiation of translocation, thus facilitating their transfer across the hydrophobic core of the (thinned) lipid bilayer (see below). Notably, essentially the same conclusions have been drawn on the basis of the BhYidC2 crystal structures and accompanying cross-linking studies (<xref ref-type="bibr" rid="bib23">Kumazaki et al., 2014</xref>).</p><p>In order to provide a molecular model of YidC in its active state, we reconstituted purified full length YidC (extended with the C-terminus of <italic>R. baltica</italic> YidC [<xref ref-type="bibr" rid="bib39">Seitl et al., 2014</xref>]) with ribosome nascent chains (RNCs) exposing the first TM helix of F<sub>O</sub>c, and subjected the complex to cryo-EM and single particle analysis to a resolution of ∼8 Å (<xref ref-type="fig" rid="fig4">Figure 4A,B</xref>). In agreement with previous structural studies (<xref ref-type="bibr" rid="bib21">Kohler et al., 2009</xref>; <xref ref-type="bibr" rid="bib39">Seitl et al., 2014</xref>), YidC binds to the ribosomal exit site, however, the improved resolution now allows for a more detailed interpretation. Firstly, we were able to separate the weaker electron density of the detergent micelle from that of YidC (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). Secondly, the presence of elongated structural features (<xref ref-type="fig" rid="fig4">Figure 4D–F</xref>) allowed us to dock our molecular model in a distinct orientation (cross correlation coefficient 0.865). Following placement of the YidC-core model, two prominent densities in the membrane region, one next to TM3 and one next to TM5, remained unaccounted for. These could be attributed to either TM1 of YidC or to the TM helix of the nascent chain (NC) F<sub>O</sub>c. Given that (i) YidC substrates are known to crosslink to TM3 (<xref ref-type="bibr" rid="bib20">Klenner et al., 2008</xref>; <xref ref-type="bibr" rid="bib46">Yu et al., 2008</xref>; <xref ref-type="bibr" rid="bib19">Klenner and Kuhn, 2012</xref>), and (ii) that the density neighboring TM3 is aligned with the ribosomal exit tunnel and (iii) that at the same relative position nascent chains have been observed inside the SecY channel (<xref ref-type="bibr" rid="bib8">Frauenfeld et al., 2011</xref>) (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>), the most plausible assignment to the density near TM3 appeared to be the TM helix of F<sub>O</sub>c. To verify this, and to exclude that the density neighboring TM5 corresponds to the nascent chain, we reconstituted single cysteine mutants of YidC either in TM3 (M430C and P431C) or in TM5 (V500C and T503C) with RNCs of a single cysteine mutant of F<sub>O</sub>c<sup>(G23C)</sup>, and exposed them to disulphide crosslinking. Upon exposure to the oxidator 5,5′-dithiobis-(2-nitrobenzoicacid) (DTNB), only in the TM3 mutants a DTT-sensitive ∼90 kDa product appeared that reacted with antibodies against the nascent chain (NC-tRNA∼30 kDa, <xref ref-type="fig" rid="fig4">Figure 4C</xref>) as well as YidC (∼60 kDa, <xref ref-type="fig" rid="fig4">Figure 4C</xref>). Thus, the adduct represented indeed the inserting F<sub>O</sub>c TM domain crosslinked to TM3 of YidC. RNCs lacking a cysteine in the nascent chain (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>) or YidC mutants with cysteines in TM5 did not yield any crosslinks (<xref ref-type="fig" rid="fig4">Figure 4C</xref>). Hence, we conclude that the unaccounted electron density next to TM3 represents the TM of the nascent chain, and that the density neighboring TM5 represents TM1 of YidC (<xref ref-type="fig" rid="fig4">Figure 4D–F</xref>).<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03035.013</object-id><label>Figure 4.</label><caption><title>Cryo-EM structure of RNC bound YidC and structural model of the active state.</title><p>(<bold>A</bold>) Side view of the ∼8 Å resolution cryo-EM based electron density of the RNC:YidC complex, with the small subunit depicted in yellow, the large subunit in gray, P-site tRNA and nascent chain in green, YidC in red and the detergent micelle in blue. (<bold>B</bold>) As in <bold>A</bold>, but sliced through the ribosomal exit tunnel. (<bold>C</bold>) Validation of the active state model by disulphide crosslinking. RNCs carrying the mutant F<sub>O</sub>c<sup>(G23C)</sup> were reconstituted with the indicated single cysteine YidC mutants, oxidized, applied to a linear sucrose gradient and harvested from the 70S peak before SDS-PAGE and western blotting. Immunodetection was performed with antibodies raised against the HA-tag (located in the nascent chain inside the ribosomal exit tunnel) and anti-YidC antibodies. YidC, nascent chain-tRNA (NC-tRNA) and the expected crosslink product (NC-tRNA x YidC) are indicated. (<bold>D</bold>–<bold>F</bold>) Structural model of YidC during membrane protein insertion, viewed from two sides within the membrane (<bold>D</bold> and <bold>E</bold>) and from the cytoplasm (<bold>F</bold>). The detergent micelle was removed for clarity, the TM helix of F<sub>O</sub>c is depicted in magenta, and the disulphide crosslink between YidC and F<sub>O</sub>c with -SS-.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.013">http://dx.doi.org/10.7554/eLife.03035.013</ext-link></p></caption><graphic xlink:href="elife03035f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03035.014</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Comparison of the active states of YidC and SecY.</title><p>Left: molecular model of YidC during co-translational translocation of F<sub>O</sub>c, and the contour of active SecY. Middle: composite model of active YidC with F<sub>O</sub>c replaced by the hydrophilic part of nascent FtsQ as found in active SecY. Right: molecular model of SecY during co-translational translocation of FtsQ. For clarity, the N-terminal signal anchor of FtsQ was omitted.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.014">http://dx.doi.org/10.7554/eLife.03035.014</ext-link></p></caption><graphic xlink:href="elife03035fs005"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03035.015</object-id><label>Figure 4—figure supplement 2.</label><caption><title>Negative control for RNC-YidC crosslinking.</title><p>Crosslinking was performed with a cysteine-less F<sub>O</sub>c RNC as described in the legend to <xref ref-type="fig" rid="fig3">Figure 3C</xref>. A poorly reproducible unknown product is indicated with an asterisk.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.015">http://dx.doi.org/10.7554/eLife.03035.015</ext-link></p></caption><graphic xlink:href="elife03035fs006"/></fig></fig-group></p><p>We attribute the remaining unaccounted electron density in the periplasmic region to the P1 domain; however, because it is substantially smaller than the crystal structure of P1, we did not include it in our molecular model. Flexibility relative to the conserved membrane region of YidC is the most likely explanation for this finding. We did not observe density for the HPD, in agreement with its flexibility observed in both, the crystal structures of BhYidC2 and the MD simulations (<xref ref-type="fig" rid="fig3">Figure 3C</xref>).</p><p>In order to validate our molecular model of co-translationally active YidC, we mutated residues that would be in direct contact with the ribosome (<xref ref-type="fig" rid="fig5">Figure 5A,B</xref>) and analyzed their effect on functionality in the <italic>in vivo</italic> complementation test. Indeed, mutation of residues Y370A and Y377A (contacting ribosomal RNA helix 59) and D488K (contacting ribosomal protein uL23) severely interfere with YidC activity (<xref ref-type="fig" rid="fig5">Figure 5C</xref>, <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>) thereby emphasizing their functional significance. All these mutants were stably expressed, indicating that the lack of complementation was not caused by instability of YidC (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2</xref>). Given that YidC in general is known to be very tolerant to point mutations (<xref ref-type="bibr" rid="bib13">Jiang et al., 2003</xref>), this provides further support for the overall correctness of our model of ribosome-bound YidC during membrane protein insertion.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.03035.016</object-id><label>Figure 5.</label><caption><title>Contacts between active YidC and the ribosome.</title><p>(<bold>A</bold> and <bold>B</bold>) Close-up views from within the membrane region highlighting the contact between H59 of the ribosome and the 2/3 loop of YidC (<bold>A</bold>) and ribosomal protein uL23 and the 4/5 loop of YidC (<bold>B</bold>). Residues that inactivate YidC upon mutagenesis or deletion are indicated by magenta spheres. (<bold>C</bold>) <italic>In vivo</italic> complementation assay of YidC point mutants D488A, D488K, deletion mutant Δ487-489 and the double mutants Y370A/Y377A and Y370F/Y377F. (<bold>D</bold>) Periplasmic view of the active ribosome-bound YidC model, with the YidC contour outlined in red. The polypeptide exit tunnel is indicated with an asterisk. (<bold>E</bold>) Cartoon based comparison of active SecY (left) and active YidC (right) during membrane insertion of FtsQ and F<sub>O</sub>c, respectively. The ribosome is depicted in gray, the aqueous channel in SecY as well as the hydrophilic environment within YidC are shaded blue, hydrophobic TM domains of the substrates are depicted magenta, hydrophilic parts in green and the P1 domain by a dashed oval.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.016">http://dx.doi.org/10.7554/eLife.03035.016</ext-link></p></caption><graphic xlink:href="elife03035f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03035.017</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Complementation of ribosome interaction mutants.</title><p>In vivo complementation assay of YidC mutants involved in ribosome binding.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.017">http://dx.doi.org/10.7554/eLife.03035.017</ext-link></p></caption><graphic xlink:href="elife03035fs007"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03035.018</object-id><label>Figure 5—figure supplement 2.</label><caption><title>Expression of ribosome interaction mutants.</title><p>Western blot of whole FTL10 cells grown on arabinose or glucose, showing the stable expression of inactive YidC mutants that interact with the ribosome.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03035.018">http://dx.doi.org/10.7554/eLife.03035.018</ext-link></p></caption><graphic xlink:href="elife03035fs008"/></fig></fig-group></p></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>Finally, it is notable that we observe only a single monomer of YidC bound to the active ribosome. This is in agreement with recent literature showing clearly that both YidC (<xref ref-type="bibr" rid="bib11">Herrmann, 2013</xref>; <xref ref-type="bibr" rid="bib17">Kedrov et al., 2013</xref>; <xref ref-type="bibr" rid="bib39">Seitl et al., 2014</xref>) and the SecY complex (<xref ref-type="bibr" rid="bib8">Frauenfeld et al., 2011</xref>; <xref ref-type="bibr" rid="bib32">Park and Rapoport, 2012</xref>; <xref ref-type="bibr" rid="bib42">Taufik et al., 2013</xref>; <xref ref-type="bibr" rid="bib33">Park et al., 2014</xref>) can be fully active as monomers. However, the comparison of models for active YidC and active SecY (<xref ref-type="fig" rid="fig5">Figure 5E</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>) reveals an important difference between the two proteins that has mechanistic implications. While SecY is known to translocate hydrophilic nascent chains through its central aqueous channel (<xref ref-type="bibr" rid="bib2">Cannon et al., 2005</xref>; <xref ref-type="bibr" rid="bib35">Rapoport, 2007</xref>; <xref ref-type="bibr" rid="bib6">Driessen and Nouwen, 2008</xref>) and insert TM domains through a lateral gate (<xref ref-type="bibr" rid="bib43">Van den Berg et al., 2004</xref>; <xref ref-type="bibr" rid="bib9">Gogala et al., 2014</xref>), our model suggests that the YidC substrates are inserted at the protein-lipid interface. Two principal findings of our work suggest how YidC may facilitate this process: (i) it provides a hydrophilic environment within the membrane core for receiving the hydrophilic moieties (termini or loops) of a substrate, and (ii) it reduces the thickness of the lipid bilayer: initial interaction of the hydrophilic moieties of YidC substrates with the hydrophilic environment of YidC would allow for a partial insertion into the membrane, while facilitating exposure of the hydrophobic TM domains to the hydrophobic core of the bilayer. The latter in turn may compensate for the energetic penalty of driving the hydrophilic moieties across the (already thinned) bilayer. Further biochemical and structural studies that capture the earlier stages of this translocation process are eagerly awaited to fully elucidate this mechanism.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Covariation analysis</title><p>We constructed a multiple sequence alignment of YidC excluding the unconserved first transmembrane helix (TM1) and the periplasmic P1 domain. We searched for homologous sequences of <italic>E. coli</italic> YidC starting from the PFAM seed alignment of family PF02096 (<xref ref-type="bibr" rid="bib34">Punta et al., 2012</xref>) and using the sensitive homology detection software HHblits (<xref ref-type="bibr" rid="bib37">Remmert et al., 2012</xref>). First, five iterations of HHblits were run against the clustered Uniprot database with no filtering, to retrieve as many homologous sequences as possible. Then, we post-processed the alignment using HHfilter to generate a non-redundant alignment at 90% sequence identity. This resulted in an alignment containing 2366 sequences aligned across YidC helices TM2-TM6. Using this multiple sequence alignment, we computed direct evolutionary couplings between pairs of YidC residues using the method of <xref ref-type="bibr" rid="bib16">Kamisetty et al. (2013)</xref>.</p><p>To compute probabilities for each possible helix–helix contact, we aggregated the evidence of stronger coupling coefficients over the expected interaction patterns for helix–helix contacts, taking into account the expected periodicity of ∼3.5 residues per alpha helix turn. We built three non-redundant datasets of mainly-alpha proteins from the CATH database (<xref ref-type="bibr" rid="bib41">Sillitoe et al., 2013</xref>). For each protein, we slid a square pattern (of size 17 × 17 residues = 289 cells) over the matrix of coupling strengths. For each pattern position, we used Bayes theorem to calculate the raw probability for a helix–helix interaction, given the 289 coupling strengths. The distributions of coupling strengths for interacting and non-interacting helix residues were fitted on dataset #1 (1118 proteins). We assigned different weights to the pattern cells, depending on their position within the pattern and the direction of the helix–helix interaction (parallel or antiparallel); these weights were optimized on dataset #2 (204 proteins). Finally, we calibrated the resulting raw scores on dataset #3 (85 proteins) to obtain accurate interaction probabilities. For cross-validation purposes, we also performed optimization on dataset #3 and calibration on dataset #2. Optimization on either dataset #2 or dataset #3 results in the same choice of weights for the pattern cells. The final posterior probabilities were obtained as the average of the values calibrated on datasets #2 and #3, weighted by dataset size. The calibration plots for datasets #2 and #3 are shown in <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref>. The histogram of final posterior probabilities obtained for YidC is shown in <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>, which illustrates the specificity of the helix–helix predictions.</p></sec><sec id="s4-2"><title>YidC initial model building</title><p>The conserved TM helices of <italic>E.coli</italic> YidC were positioned according to the covariation based helix–helix contact prediction, and rotated based on their predicted lipid or protein exposure (<xref ref-type="bibr" rid="bib24">Lai et al., 2013</xref>), resulting in a starting model of the conserved TM core of YidC. Additional information based on direct residue–residue interactions (covariance analysis) and secondary structure predictions by Jpred 3 (<xref ref-type="bibr" rid="bib4">Cole et al., 2008</xref>) were used as structural restraints in MODELLER (<xref ref-type="bibr" rid="bib7">Eswar et al., 2008</xref>). From a total of 10 output models that differed mainly in the relative orientation of the loop regions, the model that satisfied the imposed constraints best was used for further studies.</p></sec><sec id="s4-3"><title>Molecular dynamics simulation</title><sec id="s4-3-1"><title>System preparation</title><p>All simulations were performed with the MD software NAMD 2.9 using the CHARMM36 force field for the proteins and lipids (<xref ref-type="bibr" rid="bib18">Klauda et al., 2010</xref>). The TIP3P model is used to simulate water (<xref ref-type="bibr" rid="bib15">Jorgensen et al., 1983</xref>). The YidC model was inserted into the membrane, solvated, and ionized using the Membrane Builder tools on CHARMM-GUI (<xref ref-type="bibr" rid="bib14">Jo et al., 2008</xref>). The lipid composition is chosen to be 3 POPE to 1 POPG, as has been successfully used for modeling bacterial membranes in several past MD simulations (<xref ref-type="bibr" rid="bib1">Ash et al., 2004</xref>; <xref ref-type="bibr" rid="bib29">Mondal et al., 2013</xref>). An initial membrane surface of area 110 Å × 110 Å was constructed along the XY plane. The protein lipid-construct was solvated with 25 Å thick layers of water along the Cartesian Z directions, and ionized to charge neutralization using Monte Carlo sampling of Na<sup>+</sup> and Cl<sup>−</sup> ions at 0.15 M concentration. The overall system size is 0.15 M. Prior to simulation the system was subjected to 10,000 steps of conjugate gradient energy minimization, followed by 100 ps of thermalization and 25 ns of equilibration. During the first 10 ns of the equilibration stage, the protein was kept fixed, allowing the lipids, ions and water molecules to equilibrate. Subsequent 15 ns of equilibration included the protein as well. We then performed 500 ns of MD simulation at 300 K. The final 100 ns was repeated thrice to examine the statistical significance of the result.</p></sec><sec id="s4-3-2"><title>Simulation parameters</title><p>The systems were kept at constant temperature using Langevin dynamics for all non-hydrogen atoms with a Langevin damping coefficient of 5 ps<sup>−1</sup>. A constant pressure of 1 atm was maintained using the Nose-Hoover Langevin piston with a period of 100 fs and damping timescale of 50 fs. Simulations were performed with an integration time step of 1 fs where bonded interactions were computed every time step, short-range non-bonded interactions every two time steps, and long range electrostatic interactions every four time steps. A cutoff of 12 Å was used for van der Waals and short-range electrostatic interactions: a switching function was started at 10 Å for van der Waals interactions to ensure a smooth cutoff. The simulations were performed under periodic boundary conditions, with full-system, long-range electrostatics calculated by using the PME method with a grid point density of 1/Å. The unit cell was large enough so that adjacent copies of the system did not interact via short-range interactions.</p></sec><sec id="s4-3-3"><title>Flexibility analysis</title><p>The overall flexibility of the transmembrane helices relative to their average configuration was compared. Positional variance of the helix residues was quantified as a measure of their flexibility. Positional variance was computed by summing the deviation of individual backbone atom position and dividing by the number of backbone atoms in the loop. This measure is slightly different from the usual root mean square fluctuation (RMSF) as contributions from overall displacements of the helices and their motions relative to the rotation/translation and internal motions of the protein are included to probe flexibility.</p></sec><sec id="s4-3-4"><title>Interaction energy, hydrogen bonds, and membrane thickness analysis</title><p>To further understand the details of the structure and dynamics of the YidC model we performed interaction energy, hydrogen bond, and membrane thinning analysis. These analyses were carried out on the MD trajectory using standard tools available on VMD. In particular, interaction energies were computed for each trajectory frame of the final 100 ns simulation using the NAMD Energy plugin on VMD. The numbers were then time averaged over the entire 100 ns, locally averaged for every residue over a cut-off distance of 10 Å, and plotted on the structure in <xref ref-type="fig" rid="fig3">Figure 3B</xref>. Hydrogen bonds are defined solely on the basis of geometric parameters (bond angle: 20°; bond-length: 3.8 Å) between donors and acceptors. Thickness at a given point on the membrane surface was probed by finding the nearest lipid head group and measuring the minimum distance between the phosphate on that lipid head and one on the opposite leaflet.</p></sec></sec><sec id="s4-4"><title>Purification of ribosome nascent chain complexes (RNCs)</title><p>RNC constructs encoding residues 1–46 of F<sub>O</sub>c (preceded by an N-terminal His-tag and 3C rhinoprotease cleavage site, and followed by an HA-tag and TnaC stalling sequence) were cloned into a pBAD vector (Invitrogen, Life Technologies, Karlsruhe, Germany) by standard molecular biology techniques, and expressed and purified as described before (<xref ref-type="bibr" rid="bib1a">Bischoff et al., 2014</xref>). Briefly, <italic>E.coli</italic> KC6 Δ<italic>smpB</italic>Δ<italic>ssrA</italic> (<xref ref-type="bibr" rid="bib38">Seidelt et al., 2009</xref>) carrying the plasmid for F<sub>O</sub>c was grown in LB with 100 µg/ml ampicilin at 37°C to an OD<sub>600</sub> = 0.5 and expression was induced for 1 hr by adding 0.2% arabinose. Cells were lysed and debris was removed by centrifugation for 20 min at 16.000 rpm in a SS34-rotor (Sorvall). The cleared lysate was spun overnight through a sucrose cushion at 45.000 rpm in a Ti45 rotor (Beckmann), the ribosomal pellet was resuspended for 1 hr at 4°C and RNCs were purified in batch by affinity purification using Talon (Clontech). After washing the Talon beads with high salt buffer the RNCs were eluted and loaded onto a linear 10%–40% sucrose gradient. The 70S peak was collected, RNCs were concentrated by pelleting, resuspended in an appropriate volume of RNC Buffer (20 mM HEPES pH 7.2, 100 mM KOAc, 6 mM MgOAc<sub>2</sub>, 0.05% (wt/vol) dodecyl maltoside), flash frozen in liquid N<sub>2</sub> and stored at −80°C. The complete sequence of the nascent chain is:</p><p>MGHHHHHHHHDYDIPTTLEVLFQGPGTMENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIYPYDVPDYAGPNILHISVTSKWFNIDNKIVDHRP.</p></sec><sec id="s4-5"><title>Purification of YidC</title><p>For purification and reconstitution studies, <italic>E.coli</italic> YidC extended with the C-terminus from <italic>R. baltica</italic> (<xref ref-type="bibr" rid="bib39">Seitl et al., 2014</xref>) was re-cloned into pET-16 (Novagen) with an N-terminal His-tag followed by a 3C rhinovirus protease site. Expression and purification was performed essentially as described (<xref ref-type="bibr" rid="bib26">Lotz et al., 2008</xref>). Briefly, <italic>E.coli</italic> C43(DE3) cells (<xref ref-type="bibr" rid="bib28">Miroux and Walker, 1996</xref>) harboring the YidC construct were grown at 37°C to an OD<sub>600</sub> = 0.6 and expression was induced by adding 0.5 mM IPTG. YidC was solubilized with Cymal-6 (Anatrace) and purified by affinity chromatography using TALON (Clontech). The N-terminal His-tag of the eluted protein was cleaved off with 3C protease during overnight dialysis at 4°C, followed by gel filtration chromatography (Superdex 200; GE Healthcare). Fractions of the monodisperse peak were pooled, concentrated to ∼1 mg/ml in YidC Buffer (20 mM NaPO<sub>4</sub> pH 6.8, 100 mM KOAc, 10% glycerol, 0.05% Cymal-6) and directly used for further structural or biochemical assays.</p></sec><sec id="s4-6"><title>Disulphide crosslinking</title><p>For disulphide crosslink analysis, F<sub>O</sub>c<sup>(G23C)</sup>-RNCs and single cysteine mutants of YidC were purified separately and reconstituted by incubating 100 pmol of RNCs with 500 pmol of freshly purified YidC for 30 min at 37°C. The endogenous cysteine in YidC at position 423 was replaced by serine. Disulphide crosslinking was induced by adding 1 mM 5,5′-dithiobis-(2-nitrobenzoicacid) (DTNB) for 10 min at 4°C and quenched by adding 20 mM N-Ethylmaleimide (NEM) for 20 min at 4°C. Crosslinked RNC-YidC complexes were separated from non-crosslinked YidC using a 10%–40% linear sucrose gradient, and the 70S peak was harvested and analyzed by SDS-PAGE followed by western blotting.</p></sec><sec id="s4-7"><title>Complementation assay</title><p>For <italic>in vivo</italic> complementation studies, wildtype <italic>E. coli</italic> YidC was recloned into pTrc99a (Pharmacia), and mutants were created by standard molecular cloning techniques. <italic>E.coli</italic> FTL10 cells (<xref ref-type="bibr" rid="bib10">Hatzixanthis et al., 2003</xref>) harboring pTrc99a plasmids encoding the YidC variants were grown overnight at 37°C in LB medium supplemented with 100 µg/ml ampiciline, 50 µg/ml kanamycin and 0.2% arabinose. YidC depletion was carried out by transferring the cells to LB medium supplemented with 100 µg/ml ampiciline, 50 µg/ml kanamycin and 0.2% glucose, followed by and additional incubation for 3 hr at 37°C. Cell suspensions of all constructs were adjusted to OD<sub>600</sub> = 0.1 and either loaded onto SDS-PAGE gels for subsequent Western blotting, or further diluted to OD<sub>600</sub> = 10<sup>−5</sup>. Each dilution was spotted on LB agar plates supplemented 100 µg/ml ampiciline, 50 µg/ml kanamycin and either 0.2% arabinose or 0.2% glucose, and incubated overnight at 37°C.</p></sec><sec id="s4-8"><title>Electron microscopy and image processing</title><p>For cryo-EM analysis, F<sub>O</sub>c-RNC:YidC complexes were reconstituted by incubating 10 pmol of RNCs with 100 pmol of freshly purified YidC for 30 min at 37°C in a final volume of 50 µl of RNC buffer. Samples were applied to carbon-coated holey grids according to standard methods (<xref ref-type="bibr" rid="bib45">Wagenknecht et al., 1988</xref>). Micrographs were collected under low-dose conditions on a FEI TITAN KRIOS operating at 200 kV using a 4 k × 4 k TemCam-F416 CMOS camera and a final pixel size of 1.035 Å on the object scale.</p><p>Image processing was done using the SPIDER software package (<xref ref-type="bibr" rid="bib40">Shaikh et al., 2008</xref>). The defocus was determined using the TF ED command in SPIDER followed by automated particle picking using Signature (<xref ref-type="bibr" rid="bib3">Chen and Grigorieff, 2007</xref>). The machine-learning algorithm MAPPOS (<xref ref-type="bibr" rid="bib30">Norousi et al., 2013</xref>) was used to subtract ‘false positive’ particles from the data set and initial alignment was performed using an empty 70S ribosome as reference. The complete data set (876376 particles) was sorted using competitive projection matching in SPIDER followed by focused sorting for ligand density (<xref ref-type="bibr" rid="bib25">Leidig et al., 2013</xref>), and refined to a final resolution of ∼8.0 Å (Fourier shell correlation [FSC] cut-off 0.5). The final dataset consisted of 58,960 particles showing electron density for P-site tRNA and ligand density at the tunnel exit. We have deposited our cryo-EM map at the EMDB under accession number 2705, and the model of the transmembrane domains at the PDB under accession number 4utq.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We would like to thank C Ungewickell for assistance with cryo-electron microscopy, Susan Vorberg for assistance with covariation analyses, T Palmer for providing <italic>E. coli</italic> strain FTL10, A Driessen and A Kuhn for providing YidC antibodies, J Philippou-Massier and U Gaul for use of the robotic high-throughput facility, A Heuer for assistance with animations and B Beckert and A Kedrov for discussions.</p><p>SW and LB were supported by the International Max Planck Research School, SS by grant GRK1721 from the DFG, JA by a Humboldt Research Felloship of the Alexander-von-Humboldt Foundation and the Bavarian Network for Molecular Biosystems (BioSysNet), AS by a Beckman Postdoctoral Fellowship, KS by the Center for Macromolecular Modeling and Bioinformatics (NIH 9P41GM104601, NIH R01-GM67887) and the Center for the Physics of Living Cells (NSF PHY-0822613), JS by the Deutsche Forschungsgemeinschaft (DFG) trough grants SFB646, GRK1721, and QBM, by the Bundesministerium für Bildung und Forschung through grant CoreSys and the Bavarian Network for Molecular Biosystems (BioSysNet), and RB by the Center for Integrated Protein Science, the DFG (FOR967) and the European Research Council (Advanced Grant CRYOTRANSLATION).</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>SW, 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>AS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>JA, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>SS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>LB, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con6"><p>OB, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con7"><p>JS, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con8"><p>KS, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con9"><p>EOS, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con10"><p>RB, 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 dataset</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>Wickles</surname><given-names>S</given-names></name>, <name><surname>Singharoy</surname><given-names>A</given-names></name>, <name><surname>Andreani</surname><given-names>J</given-names></name>, <name><surname>Seemayer</surname><given-names>S</given-names></name>, <name><surname>Bischoff</surname><given-names>L</given-names></name>, <name><surname>Berninghausen</surname><given-names>O</given-names></name>, <name><surname>Soeding</surname><given-names>J</given-names></name>, <name><surname>Schulten</surname><given-names>K</given-names></name>, <name><surname>O van der Sluis</surname><given-names>E</given-names></name>, <name><surname>Beckmann</surname><given-names>R</given-names></name>, <year>2014</year><x>, </x><source>A structural model of the active ribosome-bound membrane protein insertase YidC</source><x>, </x><object-id pub-id-type="art-access-id">2705</object-id><x>; </x><ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/pdbe/entry/EMD-2705">http://www.ebi.ac.uk/pdbe/entry/EMD-2705</ext-link><x>, </x><comment>Publicly available at the EMBL-EBI EMDB.</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>Wickles</surname><given-names>S</given-names></name>, <name><surname>Singharoy</surname><given-names>A</given-names></name>, <name><surname>Andreani</surname><given-names>J</given-names></name>, <name><surname>Seemayer</surname><given-names>S</given-names></name>, <name><surname>Bischoff</surname><given-names>L</given-names></name>, <name><surname>Berninghausen</surname><given-names>O</given-names></name>, <name><surname>Soeding</surname><given-names>J</given-names></name>, <name><surname>Schulten</surname><given-names>K</given-names></name>, <name><surname>O van der Sluis</surname><given-names>E</given-names></name>, <name><surname>Beckmann</surname><given-names>R</given-names></name>, <year>2014</year><x>, </x><source>A structural model of the active ribosome-bound membrane protein insertase YidC</source><x>, </x><object-id pub-id-type="art-access-id">4utq</object-id><x>; </x><ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/pdbe-srv/view/entry/4utq/summary.html">http://www.ebi.ac.uk/pdbe-srv/view/entry/4utq/summary.html</ext-link><x>, </x><comment>Publicly available at the EMBL-EBI Protein Data Bank in 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S</given-names></name><role>Reviewing editor</role><aff><institution>MRC Laboratory of Molecular Biology</institution>, <country>United Kingdom</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). 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 “A structural model of the active ribosome-bound membrane protein insertase YidC” for consideration at <italic>eLife.</italic> Your article has been favorably evaluated by Randy Schekman (Senior editor) and 2 reviewers, one of whom 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>This excellent contribution by Beckmann and co-workers develops a structural model of the active ribosome-bound YidC. They derive a structural model of the YidC monomer using co-variation analysis and molecular dynamics. The Beckmann YidC structural model is very good (with a root mean square deviation between the TM helices of their model of 7.5 Angstroms) when compared to the structure of the <italic>Bacillus halodurans</italic> YidC2 recently published in Nature. Both the <italic>B. h</italic> structure and Beckmann model possess a hydrophilic groove within the membrane embedded region that is open to the cytoplasm and lipid bilayer. However, the Beckmann model is less open (which makes sense) because they imposed TM3/TM5 interactions at Pro425-Pro499 and TM3/TM6 interactions at both Cys423-Gln528 and Phe 433-Thr524. It may represent a different conformational state of YidC than seen in the <italic>B. h</italic> structure, and this could reflect some of the differences in the structures.</p><p>The YidC model was then used to reconstruct the structural features of a translating YidC-ribosome complex with a bound subunit c of ATP synthase (F<sub>o</sub>c), determined by cryo-electron microscopy. The reconstruction model is quite good. Of the two unknown membrane densities in their model, one was suggested to be TM1 of the <italic>E. coli</italic> YidC (not present in their model) and the other was the hydrophobic TM segment of F<sub>o</sub>c. Overall, this work presenting a structural model of the active ribosome-bound F<sub>o</sub>C YidC complex will have a significant impact within the YidC field and the membrane field in general.</p><p>The main essential point for improvement agreed by both referees is the rigorous assignment of TM1 and the TM segment of F<sub>o</sub>c in their structure. This is an important part of this paper, and is worth nailing down. The crosslinking experiment as presented is incomplete as there is no suitable negative control (i.e., a cysteine position that does not crosslink to substrate). In short, the authors have two extra unaccounted densities (near helix 3 and near helix 5). At a minimum, the authors should place a cysteine in either helix 3 or helix 5, and directly compare substrate crosslinks. As currently depicted, one cannot evaluate the specificity of the crosslink and therefore the validity of the assignment of the nascent chain helix in their structure.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03035.020</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>[…] The main essential point for improvement agreed by both referees is the rigorous assignment of TM1 and the TM segment of F</italic><sub><italic>o</italic></sub><italic>c in their structure. This is an important part of this paper, and is worth nailing down. The crosslinking experiment as presented is incomplete as there is no suitable negative control (i.e., a cysteine position that does not crosslink to substrate). In short, the authors have two extra unaccounted densities (near helix 3 and near helix 5). At a minimum, the authors should place a cysteine in either helix 3 or helix 5, and directly compare substrate crosslinks. As currently depicted, one cannot evaluate the specificity of the crosslink and therefore the validity of the assignment of the nascent chain helix in their structure</italic>.</p><p>As suggested by the referees we have performed additional crosslinking experiments, the results of which are now shown in <xref ref-type="fig" rid="fig3">Figure 3c</xref>. Specifically, we have attempted to crosslink F<sub>O</sub>c(G23C)-RNCs to single cysteine mutants of YidC at positions P431 (one residue after the previously shown M430 in TM3), and positions V500 and T503 in TM5. In our YidC model, the latter two positions point away from the electron density that we assigned to the nascent chain, and face the electron density that we assigned to YidC-TM1. Hence, crosslinks to these positions would be expected in case our assignment of the two electron densities would be inverted.</p><p>As a result, in full agreement with our interpretation in the initial submission, YidC mutants V500C and T503C in TM5 do not crosslink to the nascent chain. An additional YidC mutant in TM3 (P431C) on the other hand does crosslink to the nascent chain, and as also expected from our model, it does so with lower efficiency than the neighboring M430C. Thus, these additional crosslinking experiments findings provide strong additional support for the correctness of our model, and we have included the results in the main text accordingly. We have moved the previous version of panel 3c, which contains the negative control with a cystein-less RNC, to <xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2</xref>.</p><p>Taken together, these crosslink experiments indeed validate the assignment of the nascent chain helix in our structure, as requested.</p></body></sub-article></article> |