EMDB-14328: Inner/spoke ring of the human nuclear pore complex from isolated ...

Basic information

Entry

Database: EMDB / ID: EMD-14328

• Title: Inner/spoke ring of the human nuclear pore complex from isolated HeLa nuclear envelopes.
• Map data: Inner/spoke ring of the human nuclear pore complex from isolated HeLa nuclear envelopes.

Sample

• Organelle or cellular component: Cytoplasmic ring of the human nuclear pore complex

• Biological species: Homo sapiens (human)
• Method: subtomogram averaging / cryo EM / Resolution: 12.6 Å
• Authors: Mosalaganti S / Obarska-Kosinska A / Siggel M / Taniguchi R / Turonova B / Zimmerli CE / Buczak K / Schmidt FH / Margiotta E / Mackmull MT / Hagen WJH / Hummer G / Kosinski J / Beck M

Funding support

European Union, Germany, 2 items

Organization	Grant number	Country
European Research Council (ERC)	724349	European Union
German Federal Ministry for Education and Research	031L0100	Germany

• Citation: Journal: Science / Year: 2022; Title: AI-based structure prediction empowers integrative structural analysis of human nuclear pores.; Authors: Shyamal Mosalaganti / Agnieszka Obarska-Kosinska / Marc Siggel / Reiya Taniguchi / Beata Turoňová / Christian E Zimmerli / Katarzyna Buczak / Florian H Schmidt / Erica Margiotta / Marie-Therese Mackmull / Wim J H Hagen / Gerhard Hummer / Jan Kosinski / Martin Beck / ; Abstract: INTRODUCTION The eukaryotic nucleus pro-tects the genome and is enclosed by the two membranes of the nuclear envelope. Nuclear pore complexes (NPCs) perforate the nuclear envelope to facilitate nucleocytoplasmic transport. With a molecular weight of ∼120 MDa, the human NPC is one of the larg-est protein complexes. Its ~1000 proteins are taken in multiple copies from a set of about 30 distinct nucleoporins (NUPs). They can be roughly categorized into two classes. Scaf-fold NUPs contain folded domains and form a cylindrical scaffold architecture around a central channel. Intrinsically disordered NUPs line the scaffold and extend into the central channel, where they interact with cargo complexes. The NPC architecture is highly dynamic. It responds to changes in nuclear envelope tension with conforma-tional breathing that manifests in dilation and constriction movements. Elucidating the scaffold architecture, ultimately at atomic resolution, will be important for gaining a more precise understanding of NPC function and dynamics but imposes a substantial chal-lenge for structural biologists. RATIONALE Considerable progress has been made toward this goal by a joint effort in the field. A synergistic combination of complementary approaches has turned out to be critical. In situ structural biology techniques were used to reveal the overall layout of the NPC scaffold that defines the spatial reference for molecular modeling. High-resolution structures of many NUPs were determined in vitro. Proteomic analysis and extensive biochemical work unraveled the interaction network of NUPs. Integra-tive modeling has been used to combine the different types of data, resulting in a rough outline of the NPC scaffold. Previous struc-tural models of the human NPC, however, were patchy and limited in accuracy owing to several challenges: (i) Many of the high-resolution structures of individual NUPs have been solved from distantly related species and, consequently, do not comprehensively cover their human counterparts. (ii) The scaf-fold is interconnected by a set of intrinsically disordered linker NUPs that are not straight-forwardly accessible to common structural biology techniques. (iii) The NPC scaffold intimately embraces the fused inner and outer nuclear membranes in a distinctive topol-ogy and cannot be studied in isolation. (iv) The conformational dynamics of scaffold NUPs limits the resolution achievable in structure determination. RESULTS In this study, we used artificial intelligence (AI)-based prediction to generate an exten-sive repertoire of structural models of human NUPs and their subcomplexes. The resulting models cover various domains and interfaces that so far remained structurally uncharac-terized. Benchmarking against previous and unpublished x-ray and cryo-electron micros-copy structures revealed unprecedented accu-racy. We obtained well-resolved cryo-electron tomographic maps of both the constricted and dilated conformational states of the hu-man NPC. Using integrative modeling, we fit-ted the structural models of individual NUPs into the cryo-electron microscopy maps. We explicitly included several linker NUPs and traced their trajectory through the NPC scaf-fold. We elucidated in great detail how mem-brane-associated and transmembrane NUPs are distributed across the fusion topology of both nuclear membranes. The resulting architectural model increases the structural coverage of the human NPC scaffold by about twofold. We extensively validated our model against both earlier and new experimental data. The completeness of our model has enabled microsecond-long coarse-grained molecular dynamics simulations of the NPC scaffold within an explicit membrane en-vironment and solvent. These simulations reveal that the NPC scaffold prevents the constriction of the otherwise stable double-membrane fusion pore to small diameters in the absence of membrane tension. CONCLUSION Our 70-MDa atomically re-solved model covers >90% of the human NPC scaffold. It captures conforma-tional changes that occur during dilation and constriction. It also reveals the precise anchoring sites for intrinsically disordered NUPs, the identification of which is a prerequisite for a complete and dy-namic model of the NPC. Our study exempli-fies how AI-based structure prediction may accelerate the elucidation of subcellular ar-chitecture at atomic resolution. [Figure: see text].

History

Deposition	Feb 10, 2022	-
Header (metadata) release	Oct 12, 2022	-
Map release	Oct 12, 2022	-
Update	Oct 12, 2022	-
Current status	Oct 12, 2022	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_14328.map.gz / Format: CCP4 / Size: 729 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Inner/spoke ring of the human nuclear pore complex from isolated HeLa nuclear envelopes.
• Voxel size: X=Y=Z: 3.37 Å

Density

Contour Level	By AUTHOR: 3.37
Minimum - Maximum	-17.804152 - 18.708817
Average (Standard dev.)	0.025294362 (±0.5640397)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 576 576 576 Spacing 576 576 576
Cell	A=B=C: 1941.1199 Å α=β=γ: 90.0 °

Supplemental data

Sample components

Entire : Cytoplasmic ring of the human nuclear pore complex

• Entire: Name: Cytoplasmic ring of the human nuclear pore complex

Components

• Organelle or cellular component: Cytoplasmic ring of the human nuclear pore complex

Supramolecule #1: Cytoplasmic ring of the human nuclear pore complex

• Supramolecule: Name: Cytoplasmic ring of the human nuclear pore complex / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#25
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 50 MDa

Experimental details

Structure determination

• Method: cryo EM
• Processing: subtomogram averaging
• Aggregation state: cell

Sample preparation

• Buffer: pH: 7.5
• Vitrification: Cryogen name: ETHANE / Instrument: LEICA EM GP

Electron microscopy

• Microscope: TFS KRIOS
• Image recording: Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 2.93 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 70.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 12.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 30000
• Extraction: Number tomograms: 554 / Number images used: 7711 ; Details: Full pores were extracted, which were subsequently split into asymmetric units (61688 subtomograms).
• Final angle assignment: Type: OTHER