These scripts demonstrate the use of IMP, MODELLER, FoXS, and AllosMod, and Minimal Ensemble Search in the modeling of the Nup133 protein in the S. cerevisiae Nuclear Pore Complex (NPC). First, MODELLER is used to generate an initial comparative model of Nup133 guided by FoXS fits to SAXS data. Then, the model was subjected to conformational sampling with AllosMod, and finally Minimal Ensemble Search was used to identify four models that together reproduced both the SAXS data and a set of electron microscopy class averages. The final model was also validated against a set of chemical cross-links, that were not used in the modeling.

A full description of the scripts can be found in Integrative structure-function mapping of the nucleoporin Nup133 suggests a conserved mechanism for membrane anchoring of the nuclear pore complex.

Steps

Comparative modeling

Python scripts to generate comparative models of parts of the Nup133 can be found in the MODELLER subdirectory. Each subdirectory corresponds to modeling consistent with a single construct (directory names match the protein IDs in Supplementary Table S1 in the publication, also available as SAXS/Nup133_tableS1.xls). A number of candidate models were generated for each construct, ranked by FoXS score, and the single best model selected for subsequent steps.

• all_sjkim_final.py in MODELLER/23917 generates models consistent with construct 490-1157
• all_sjkim_final_23902a.py in MODELLER/23902 generates models consistent with construct 2-515
• all_sjkim_final_23904.py in MODELLER/23904C takes the best scoring model 23917 and generates models consistent with construct 490-1157
• all_sjkim_final_23904.py in MODELLER/23904 takes the best scoring model 23904C and 23902a and generates models consistent with construct 2-1157 (the complete protein)

AllosMod

The complete ensemble was generated by submission to the AllosMod web server.

Minimal Ensemble Search

Results from running Minimal Ensemble Search can be found in the outputs_foxs_ensemble_new/MES_results directory. new_mes4.log contains the final selected ensemble of four models, also present in this directory. The electron microscopy class averages used are also present in the outputs_foxs_ensemble_new/ISAC_p150_t346_m30 directory.

Validation against cross-links

Intramolecular cross-link data is available in Crosslinks/DSS_EDC_crosslinks.txt. Simple Python scripts to calculate residue-residue distances for the final ensemble can be found in the same directory.

Information

Author(s): Seung Joong Kim

Date: September 2nd, 2014

License: CC BY-SA 4.0 This work is licensed under the Creative Commons Attribution-ShareAlike 4.0 International License.

Last known good IMP version:

Testable: Yes.

Parallelizeable: No

Publications:

• Seung Joong Kim*, Javier Fernandez-Martinez*, Parthasarathy Sampathkumar*, Anne Martel, Tsutomu Matsui, Hiro Tsuruta, Thomas Weiss, Yi Shi, Ane Markina-Inarrairaegui, Jeffery B. Bonanno, J. Michael Sauder, Stephen K. Burley, Brian T. Chait, Steven C. Almo, Michael P. Rout, and Andrej Sali, Integrative structure-function mapping of the nucleoporin Nup133 suggests a conserved mechanism for membrane anchoring of the nuclear pore complex, Molecular & Cellular Proteomics, 2014, mcp.M114.040915.

*These authors contributed equally to this work as co-first authors.