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- PDB-7fik: The cryo-EM structure of the CR subunit from X. laevis NPC -

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Entry
Database: PDB / ID: 7fik
TitleThe cryo-EM structure of the CR subunit from X. laevis NPC
Components
  • (Nuclear pore complex protein ...Nuclear pore) x 4
  • MGC154553 protein
  • MGC83295 protein
  • MGC83926 protein
  • Nuclear pore complex proteinNuclear pore
  • Nucleoporin Nup88A
  • Nucleoporin SEH1-B
  • Nup155-prov protein
  • Nup358 complex, clamps
  • Nup98
  • Protein SEC13 homolog
  • outer Nup133
  • outer Nup160
KeywordsSTRUCTURAL PROTEIN / CR / NPC / nucleoporin / Nup358 / Nup93 / Y complex
Function / homology
Function and homology information


GATOR2 complex / nephron development / nuclear pore inner ring / protein localization to nuclear inner membrane / COPII-coated vesicle budding / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore organization / nuclear pore outer ring / nuclear pore complex assembly / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery ...GATOR2 complex / nephron development / nuclear pore inner ring / protein localization to nuclear inner membrane / COPII-coated vesicle budding / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore organization / nuclear pore outer ring / nuclear pore complex assembly / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery / COPII vesicle coat / attachment of mitotic spindle microtubules to kinetochore / structural constituent of nuclear pore / RNA export from nucleus / poly(A)+ mRNA export from nucleus / mitotic metaphase chromosome alignment / NLS-bearing protein import into nucleus / cellular response to nutrient levels / ribosomal large subunit export from nucleus / mRNA transport / ribosomal small subunit export from nucleus / mRNA export from nucleus / nuclear pore / positive regulation of TORC1 signaling / cellular response to amino acid starvation / nuclear periphery / GTPase activator activity / kinetochore / protein import into nucleus / protein transport / nuclear membrane / lysosomal membrane / cell division / structural molecule activity / positive regulation of DNA-templated transcription / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Protein Sec13 / Nucleoporin Nup88 / Nuclear pore component / Nucleoporin NUP88/NUP82 / Nuclear pore complex protein NUP98-NUP96 / Nucleoporin, Nup155-like, C-terminal, subdomain 3 / Nucleoporin Nup37 / Nucleoporin Nup85-like / Nup85 Nucleoporin / Nuclear pore protein 84/107 ...Protein Sec13 / Nucleoporin Nup88 / Nuclear pore component / Nucleoporin NUP88/NUP82 / Nuclear pore complex protein NUP98-NUP96 / Nucleoporin, Nup155-like, C-terminal, subdomain 3 / Nucleoporin Nup37 / Nucleoporin Nup85-like / Nup85 Nucleoporin / Nuclear pore protein 84/107 / Nuclear pore protein 84 / 107 / Nuclear pore complex protein Nup133-like / Nup358/RanBP2 E3 ligase domain / Nup358/RanBP2 E3 ligase domain / Nucleoporin, Nup155-like / Nucleoporin, Nup155-like, C-terminal, subdomain 1 / Nucleoporin, Nup155-like, C-terminal, subdomain 2 / Nucleoporin Nup186/Nup192/Nup205 / Nuclear pore complex scaffold, nucleoporins 186/192/205 / Nucleoporin interacting component Nup93/Nic96 / Nup93/Nic96 / Nucleoporin, Nup133/Nup155-like, C-terminal / Nucleoporin FG repeat / Nucleoporin FG repeat region / Non-repetitive/WGA-negative nucleoporin C-terminal / Nucleoporin, Nup133/Nup155-like, N-terminal / Nup133 N terminal like / Sec13/Seh1 family / Nuclear pore complex protein NUP96, C-terminal domain / Nuclear protein 96 / Nuclear pore complex protein Nup98-Nup96-like, autopeptidase S59 domain / Nuclear pore complex protein Nup98-Nup96-like, autopeptidase S59 domain superfamily / Nucleoporin autopeptidase / NUP C-terminal domain profile. / Nucleoporin peptidase S59-like / Ran binding protein RanBP1-like / Ran binding domain / RanBP1 domain / Ran binding domain type 1 profile. / Ran-binding domain / Zinc finger domain / Zn-finger in Ran binding protein and others / Zinc finger RanBP2 type profile. / Zinc finger RanBP2-type signature. / Zinc finger, RanBP2-type superfamily / Zinc finger, RanBP2-type / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / Tetratricopeptide-like helical domain superfamily / PH-like domain superfamily / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Nuclear pore complex protein Nup133 / Nuclear pore complex protein Nup98-Nup96 / RANBP2-like and GRIP domain-containing protein 3 isoform X2 / Nuclear pore complex protein / MGC154553 protein / MGC83295 protein / MGC83926 protein / Nuclear pore complex protein Nup85 / Nucleoporin SEH1-B / Protein SEC13 homolog ...Nuclear pore complex protein Nup133 / Nuclear pore complex protein Nup98-Nup96 / RANBP2-like and GRIP domain-containing protein 3 isoform X2 / Nuclear pore complex protein / MGC154553 protein / MGC83295 protein / MGC83926 protein / Nuclear pore complex protein Nup85 / Nucleoporin SEH1-B / Protein SEC13 homolog / Nucleoporin Nup88A / Nucleoporin 155kDa L homeolog / Nuclear pore complex protein Nup93
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsShi, Y. / Huang, G. / Zhan, X.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Science / Year: 2022
Title: Structure of the cytoplasmic ring of the nuclear pore complex.
Authors: Xuechen Zhu / Gaoxingyu Huang / Chao Zeng / Xiechao Zhan / Ke Liang / Qikui Xu / Yanyu Zhao / Pan Wang / Qifan Wang / Qiang Zhou / Qinghua Tao / Minhao Liu / Jianlin Lei / Chuangye Yan / Yigong Shi /
Abstract: INTRODUCTION The nuclear pore complex (NPC) resides on the nuclear envelope (NE) and mediates nucleocytoplasmic cargo transport. As one of the largest cellular machineries, a vertebrate NPC consists ...INTRODUCTION The nuclear pore complex (NPC) resides on the nuclear envelope (NE) and mediates nucleocytoplasmic cargo transport. As one of the largest cellular machineries, a vertebrate NPC consists of cytoplasmic filaments, a cytoplasmic ring (CR), an inner ring, a nuclear ring, a nuclear basket, and a luminal ring. Each NPC has eight repeating subunits. Structure determination of NPC is a prerequisite for understanding its functional mechanism. In the past two decades, integrative modeling, which combines x-ray structures of individual nucleoporins and subcomplexes with cryo-electron tomography reconstructions, has played a crucial role in advancing our knowledge about the NPC. The CR has been a major focus of structural investigation. The CR subunit of human NPC was reconstructed by cryo-electron tomography through subtomogram averaging to an overall resolution of ~20 Å, with local resolution up to ~15 Å. Each CR subunit comprises two Y-shaped multicomponent complexes known as the inner and outer Y complexes. Eight inner and eight outer Y complexes assemble in a head-to-tail fashion to form the proximal and distal rings, respectively, constituting the CR scaffold. To achieve higher resolution of the CR, we used single-particle cryo-electron microscopy (cryo-EM) to image the intact NPC from the NE of oocytes. Reconstructions of the core region and the Nup358 region of the CR subunit had been achieved at average resolutions of 5 to 8 Å, allowing identification of secondary structural elements. RATIONALE Packing interactions among the components of the CR subunit were poorly defined by all previous EM maps. Additional components of the CR subunit are strongly suggested by the EM maps of 5- to 8-Å resolution but remain to be identified. Addressing these issues requires improved resolution of the cryo-EM reconstruction. Therefore, we may need to enhance sample preparation, optimize image acquisition, and develop an effective data-processing strategy. RESULTS To reduce conformational heterogeneity of the sample, we spread the opened NE onto the grids with minimal force and used the chemical cross-linker glutaraldehyde to stabilize the NPC. To alleviate orientation bias of the NPC, we tilted sample grids and imaged the sample with higher electron dose at higher angles. We improved the image-processing protocol. With these efforts, the average resolutions for the core and the Nup358 regions have been improved to 3.7 and 4.7 Å, respectively. The highest local resolution of the core region reaches 3.3 Å. In addition, a cryo-EM structure of the N-terminal α-helical domain of Nup358 has been resolved at 3.0-Å resolution. These EM maps allow the identification of five copies of Nup358, two copies of Nup93, two copies of Nup205, and two copies of Y complexes in each CR subunit. Relying on the EM maps and facilitated by AlphaFold prediction, we have generated a final model for the CR of the NPC. Our model of the CR subunit includes 19,037 amino acids in 30 nucleoporins. A previously unknown C-terminal fragment of Nup160 was found to constitute a key part of the vertex, in which the short arm, long arm, and stem of the Y complex meet. The Nup160 C-terminal fragment directly binds the β-propeller proteins Seh1 and Sec13. Two Nup205 molecules, which do not contact each other, bind the inner and outer Y complexes through distinct interfaces. Conformational elasticity of the two Nup205 molecules may underlie their versatility in binding to different nucleoporins in the proximal and distal CR rings. Two Nup93 molecules, each comprising an N-terminal extended helix and an ACE1 domain, bridge the Y complexes and Nup205. Nup93 and Nup205 together play a critical role in mediating the contacts between neighboring CR subunits. Five Nup358 molecules, each in the shape of a shrimp tail and named "the clamp," hold the stems of both Y complexes. The innate conformational elasticity allows each Nup358 clamp to adapt to a distinct local environment for optimal interactions with neighboring nucleoporins. In each CR subunit, the α-helical nucleoporins appear to provide the conformational elasticity; the 12 β-propellers may strengthen the scaffold. CONCLUSION Our EM map-based model of the CR subunit substantially expands the molecular mass over the reported composite models of vertebrate CR subunit. In addition to the Y complexes, five Nup358, two Nup205, and two Nup93 molecules constitute the key components of the CR. The improved EM maps reveal insights into the interfaces among the nucleoporins of the CR. [Figure: see text].
History
DepositionJul 31, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 9, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: MGC83295 protein
B: Nuclear pore complex protein Nup85
C: MGC154553 protein
D: Nucleoporin SEH1-B
E: outer Nup160
F: MGC83926 protein
G: Nuclear pore complex protein Nup98-Nup96
H: Protein SEC13 homolog
I: Nuclear pore complex protein
J: nuclear pore complex protein Nup133
K: Nup358 complex, clamps
L: Nup358 complex, clamps
M: Nup358 complex, clamps
N: Nup358 complex, clamps
O: Nup358 complex, clamps
P: Nuclear pore complex protein Nup93
S: Nuclear pore complex protein Nup93
W: Nup155-prov protein
X: Nup98
Y: Nucleoporin Nup88A
i: Nuclear pore complex protein
s: Nuclear pore complex protein Nup93
a: MGC83295 protein
b: Nuclear pore complex protein Nup85
c: MGC154553 protein
d: Nucleoporin SEH1-B
e: outer Nup160
f: MGC83926 protein
g: Nuclear pore complex protein Nup98-Nup96
h: Protein SEC13 homolog
j: outer Nup133
p: Nuclear pore complex protein Nup93


Theoretical massNumber of molelcules
Total (without water)4,309,50532
Polymers4,309,50532
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 12 types, 23 molecules AaCcDdEeFfHhIiKLMNOWXYj

#1: Protein MGC83295 protein / Nup205


Mass: 227854.141 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q642R6
#3: Protein MGC154553 protein / outer Nup43


Mass: 41744.512 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q05AW3
#4: Protein Nucleoporin SEH1-B / GATOR complex protein SEH1-B / Nup107-160 subcomplex subunit seh1-B


Mass: 39798.602 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q6GNF1
#5: Protein outer Nup160


Mass: 162658.234 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog)
#6: Protein MGC83926 protein


Mass: 36588.625 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q66IZ6
#8: Protein Protein SEC13 homolog / GATOR complex protein SEC13


Mass: 35361.309 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q6GNX0
#9: Protein Nuclear pore complex protein / Nuclear pore


Mass: 105398.547 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A2RV69
#11: Protein
Nup358 complex, clamps


Mass: 322784.344 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A0A1L8HGL2
#13: Protein Nup155-prov protein


Mass: 154922.422 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q7ZWL0
#14: Protein Nup98


Mass: 13719.902 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: The putative protein Nup98 was from the same gene of the chains g/G, whose corresponding peptide is post-translationally cleaved into two separate proteins.
Source: (natural) Xenopus laevis (African clawed frog)
#15: Protein Nucleoporin Nup88A


Mass: 82570.148 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q707N0
#16: Protein outer Nup133


Mass: 127551.250 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A0A1L8H1I9

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Nuclear pore complex protein ... , 4 types, 9 molecules BbGgJPSsp

#2: Protein Nuclear pore complex protein Nup85 / Nuclear pore / 85 kDa nucleoporin / Nucleoporin Nup85


Mass: 75160.047 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q68FJ0
#7: Protein Nuclear pore complex protein Nup98-Nup96 / Nuclear pore


Mass: 191067.188 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A0A1L8HBE3
#10: Protein nuclear pore complex protein Nup133 / / nuclear pore complex protein Nup133-like


Mass: 111296.109 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: This entity was identical with chain j. However, N-terminal region cannot be assigned due to disorder.
Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A0A1L8H1I9
#12: Protein
Nuclear pore complex protein Nup93 / Nuclear pore / 93 kDa nucleoporin / An4a / Nucleoporin Nup93


Mass: 93565.156 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q7ZX96

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Xenopus laevis Nuclear Pore Complex (NPC) / Type: COMPLEX / Entity ID: #1-#13, #16, #15, #14 / Source: NATURAL
Source (natural)Organism: Xenopus laevis (African clawed frog)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 75 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1279270 / Symmetry type: POINT
Atomic model buildingSpace: REAL

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