Integrative Structure Determination of the Components of the Nuclear Pore Complex by X-Ray Crystallography, Small Angle X-Ray Scattering, Electron Microscopy, NMR, and Comparative Modeling

Abstract

The Nuclear Pore Complex (NPC, ∼50 MDa) is the sole passageway for the transport of macromolecules across the nuclear envelope. The NPC 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 work (ref 1, 2) defined the relative positions of its 456 constituent proteins (nucleoporin or Nups), based on spatial restraints derived from biophysical, electron microscopy, and proteomic data. Further elucidation of the evolutionary origin, transport mechanism, and assembly of the NPC will require higher resolution information. As part of an effort to improve upon the resolution and accuracy of the NPC structure, we set out to determine the atomic structures of the NPC components. Because it proved difficult to determine the atomic structures of whole Nups by X-ray crystallography alone, we are relying on multiple datasets that are combined computationally by our Integrative Modeling Platform (IMP) package (http://salilab.org/imp). In particular, we developed an integrative modeling approach that benefits from crystallographic structures of fragments of the protein or its homologs, Solution Small Angle X-ray Scattering (SAXS) profiles of the protein and its fragments (ref 3), NMR, and negative stain Electron Microscopy (EM) micrographs of the protein. Each dataset is converted into a set of spatial restraints on the protein structure, followed by finding a model that satisfies the restraints as well as possible using a Monte Carlo / molecular dynamics optimization procedure. The approach will be illustrated by its application to yeast Nup133.1. Alber et al., Nature 450, 683-694 (2007).2. Alber et al., Nature 450, 695-701 (2007).3. Forster et al., J Mol Biol 382 (4), 1089-1106 (2008).