Determination of the Pseudo-Atomic Structure of Nuclear Pore Complex (NPC) Components by Small Angle X-Ray Scattering (SAXS) and Computational Modeling

Abstract

The Nuclear Pore Complex (NPC, ∼50 MDa) is the sole passageway for the transport of macromolecules across the nuclear envelope. The pore plays a key role in numerous critical cellular processes such as transcription, and many of its components are implicated in human diseases such as cancer. Previous works provided the first description of the macromolecular architecture of the yeast NPC. This structure defined the relative positions and proximities of its 456 constituent nucleoporin (nup) proteins, based on spatial restraints derived from experimental data. Further elucidation of the evolutionary origin and transport mechanism of the NPC will require higher resolution information. To help improve upon the resolution and accuracy of the NPC structure, we obtained small angle x-ray scattering (SAXS) data.We prepared sets of single protein, protein domain, and small NPC sub-complex samples for SAXS analysis, because producing crystal structures for many of the proteins has proven difficult. We generated SAXS profiles for individual proteins or sub-complexes in solution, which provide shape information. This shape information generated by SAXS can in turn be used to improve atomic homology models for individual proteins or complexes.We apply our Integrated Modeling Platform (IMP) software to incorporate a diverse set of experimental data, including SAXS spectra, as spatial restraints, to determine the three dimensional structures of these sub-complexes and proteins by simultaneously minimizing violations of all of the restraints.We specifically focus on components of two sub-complexes, the 7-protein Nup84 sub-complex, and the 4-protein Nup170 sub-complex, for which complementary experimental data are available. For each SAXS profile, we utilize a score that evaluates a model structure based on the deviation between the experimental profile and a calculated profile for the model.