Structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex by cryo-electron microscopy single particle analysis

Abstract

The nuclear pore complex (NPC) exhibits structural plasticity and has only been characterized at local resolutions of up to 15 Å for the cytoplasmic ring (CR). Here we present a single-particle cryo-electron microscopy (cryo-EM) structure of the CR from Xenopus laevis NPC at average resolutions of 5.5-7.9 Å, with local resolutions reaching 4.5 Å. Improved resolutions allow identification and placement of secondary structural elements in the majority of the CR components. The two Y complexes in each CR subunit interact with each other and associate with those from flanking subunits, forming a circular scaffold. Within each CR subunit, the Nup358containing region wraps around the stems of both Y complexes, likely stabilizing the scaffold. Nup205 connects the short arms of the two Y complexes and associates with the stem of a neighboring Y complex. The Nup214-containing region uses an extended coiled-coil to link Nup85 of the two Y complexes and protrudes into the axial pore of the NPC. These previously uncharacterized structural features reveal insights into NPC assembly.

Molecular architecture of the luminal ring of the Xenopus laevis nuclear pore complex

“…To address this issue and to validate the cryo-ET STA reconstruction, we determined the cryo-EM structure of the LR using a completely independent data set through the single particle analysis (SPA) approach. During cryo-EM data collection, the sample grids were tilted at fixed angles of 0°, 30°, 45°and 55°, generating 12,399 good micrographs 34 (Supplementary information, Table S2). The SPA approach resulted in the reconstruction of the LR subunit at an average resolution of 10.7 Å (Supplementary information, Figs.…”

“…Cryo-EM data acquisition Details for the acquisition of cryo-EM data are described in the accompanying manuscript. 34 Briefly, micrographs were recorded on a Titan Krios (FEI) electron microscope, operating at 300 kV and equipped with a Gatan Gif Quantum energy filter (slit width 20 eV). A K2 Summit detector (Gatan Company) in super-resolution mode with a nominal magnification of 64,000× was used, resulting in a calibrated pixel size of 1.111 Å.…”

“…Cryo-EM data analysis The single-particle cryo-EM data was mainly used to generate a reconstruction of the CR at an improved resolution. 34 After completing this task, the same data were analyzed to generate a SPA-based reconstruction of the LR. The strategy for processing of the cryo-EM data toward reconstruction of the LR subunit is presented in Supplementary information, Fig.…”

Integrity of the short arm of nuclear pore Y-complex is required for mouse embryonic stem cell growth and differentiation

“…Alternatively, the short arm of the Y-complex may affect NPC assembly in interphase, by facilitating the recruitment of the Y-complex to the nuclear envelope, for instance, via its interaction with Nup153 (Vollmer et al, 2015). Finally, Nup43 and Seh1 may contribute to the stabilization of the NPC scaffold, by virtue of their direct interactions with neighbouring subunits from either Y-complexes or inner ring complexes (Huang et al, 2020;Kosinski et al, 2016;von Appen et al, 2015). NPCs lacking these stabilizing interactions might then be recognized by one of the recently described quality-control mechanisms, which mediate the removal of some misassembled NPCs from the nuclear envelope (reviewed in Webster and Lusk, 2016).…”

“…4A). Alternatively, Seh1 may establish additional contacts with other nucleoporins in the context of the assembled mammalian NPC (Huang et al, 2020;Kosinski et al, 2016;von Appen et al, 2015).…”