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- PDB-7n9f: Structure of the in situ yeast NPC -

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Entry
Database: PDB / ID: 7n9f
TitleStructure of the in situ yeast NPC
Components
  • Dynein light chain 1, cytoplasmic
  • Nucleoporin 145c
  • Nucleoporin ASM4
  • Nucleoporin NIC96
  • Nucleoporin NSP1
  • Nucleoporin NUP120
  • Nucleoporin NUP133
  • Nucleoporin NUP157
  • Nucleoporin NUP159
  • Nucleoporin NUP170
  • Nucleoporin NUP188
  • Nucleoporin NUP192
  • Nucleoporin NUP49/NSP49
  • Nucleoporin NUP53
  • Nucleoporin NUP57
  • Nucleoporin NUP82
  • Nucleoporin NUP84
  • Nucleoporin NUP85
  • Nucleoporin SEH1
  • Protein transport protein SEC13
  • orphans bound to Nup192 NTD
KeywordsTRANSLOCASE / NPC / nucleocytoplasmic transport
Function / homology
Function and homology information


response to spindle checkpoint signaling / nuclear pore linkers / peroxisomal importomer complex / regulation of protein desumoylation / mRNA export from nucleus in response to heat stress / nuclear pore inner ring / Seh1-associated complex / positive regulation of ER to Golgi vesicle-mediated transport / protein exit from endoplasmic reticulum / COPII-coated vesicle budding ...response to spindle checkpoint signaling / nuclear pore linkers / peroxisomal importomer complex / regulation of protein desumoylation / mRNA export from nucleus in response to heat stress / nuclear pore inner ring / Seh1-associated complex / positive regulation of ER to Golgi vesicle-mediated transport / protein exit from endoplasmic reticulum / COPII-coated vesicle budding / protein localization to nuclear inner membrane / chromosome, subtelomeric region / COPII-mediated vesicle transport / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore localization / adenyl-nucleotide exchange factor activity / nuclear pore central transport channel / regulation of nucleocytoplasmic transport / establishment of mitotic spindle localization / nuclear migration along microtubule / regulation of TORC1 signaling / nuclear pore organization / nuclear pore outer ring / nuclear pore complex assembly / telomere tethering at nuclear periphery / COPII vesicle coat / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery / positive regulation of protein exit from endoplasmic reticulum / nuclear pore cytoplasmic filaments / Transport of Mature mRNA derived from an Intron-Containing Transcript / Regulation of HSF1-mediated heat shock response / nuclear pore nuclear basket / tRNA export from nucleus / SUMOylation of SUMOylation proteins / cytoplasmic dynein complex / protein localization to kinetochore / SUMOylation of RNA binding proteins / structural constituent of nuclear pore / RNA export from nucleus / SUMOylation of chromatin organization proteins / silent mating-type cassette heterochromatin formation / nucleocytoplasmic transport / vacuolar membrane / poly(A)+ mRNA export from nucleus / nuclear localization sequence binding / regulation of mitotic nuclear division / dynein intermediate chain binding / NLS-bearing protein import into nucleus / cytoplasmic microtubule / establishment of mitotic spindle orientation / ribosomal large subunit export from nucleus / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / positive regulation of TOR signaling / subtelomeric heterochromatin formation / mRNA transport / mRNA export from nucleus / nuclear pore / ribosomal small subunit export from nucleus / ERAD pathway / Neutrophil degranulation / protein export from nucleus / positive regulation of TORC1 signaling / cellular response to amino acid starvation / nuclear periphery / cell periphery / chromosome segregation / promoter-specific chromatin binding / molecular condensate scaffold activity / heterochromatin formation / phospholipid binding / protein import into nucleus / transcription corepressor activity / protein transport / double-strand break repair / nuclear envelope / single-stranded DNA binding / cellular response to heat / nuclear membrane / amyloid fibril formation / chromosome, telomeric region / hydrolase activity / cell division / chromatin binding / endoplasmic reticulum membrane / protein-containing complex binding / positive regulation of DNA-templated transcription / structural molecule activity / negative regulation of transcription by RNA polymerase II / endoplasmic reticulum / positive regulation of transcription by RNA polymerase II / DNA binding / RNA binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Nucleoporin Nup159/Nup146, N-terminal / Nucleoporin or Nuclear pore complex subunit NUP214=Nup159 / Nucleoporin NUP120, helical domain / RNA-recognition motif (RRM) Nup35-type domain / Nucleoporin, NUP53 / Nucleoporin NUP88/NUP82 / Nup53/35/40-type RNA recognition motif / RNA-recognition motif (RRM) Nup35-type domain profile. / Nuclear pore protein Nup188, C-terminal / Nuclear pore protein NUP188 C-terminal domain ...Nucleoporin Nup159/Nup146, N-terminal / Nucleoporin or Nuclear pore complex subunit NUP214=Nup159 / Nucleoporin NUP120, helical domain / RNA-recognition motif (RRM) Nup35-type domain / Nucleoporin, NUP53 / Nucleoporin NUP88/NUP82 / Nup53/35/40-type RNA recognition motif / RNA-recognition motif (RRM) Nup35-type domain profile. / Nuclear pore protein Nup188, C-terminal / Nuclear pore protein NUP188 C-terminal domain / Nucleoporin Nup188, N-terminal / Nucleoporin Nup188, N-terminal / Nucleoporin Nup54/Nup57/Nup44 / Nucleoporin p58/p45 / Nucleoporin Nup54, alpha-helical domain / Nucleoporin Nup188 / : / Nucleoporin complex subunit 54 / Nucleoporin Nup188, N-terminal subdomain III / Nucleoporin, NSP1-like, C-terminal / Nucleoporin NSP1/NUP62 / Nsp1-like C-terminal region / Nucleoporin Nup85-like / Nucleoporin Nup120/160 / Nup85 Nucleoporin / Nuclear pore protein 84/107 / Nuclear pore protein 84 / 107 / Nuclear pore complex protein Nup133-like / 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, Nup133/Nup155-like, C-terminal / Non-repetitive/WGA-negative nucleoporin C-terminal / Nucleoporin interacting component Nup93/Nic96 / Nup93/Nic96 / Nucleoporin FG repeat / Nucleoporin FG repeat region / Dynein light chain, type 1/2, conserved site / Dynein light chain type 1 signature. / Nucleoporin, Nup133/Nup155-like, N-terminal / Nup133 N terminal like / Sec13/Seh1 family / Nuclear pore complex protein NUP96, C-terminal domain / Nuclear protein 96 / Dynein light chain, type 1/2 / Nuclear pore complex protein Nup98-Nup96-like, autopeptidase S59 domain / Nuclear pore complex protein Nup98-Nup96-like, autopeptidase S59 domain superfamily / Nucleoporin peptidase S59-like / Nucleoporin autopeptidase / NUP C-terminal domain profile. / Dynein light chain type 1 / Dynein light chain type 1 / Dynein light chain superfamily / RNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily / 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
Nucleoporin NSP1 / Nucleoporin NIC96 / Nucleoporin NUP120 / Nucleoporin NUP133 / Nucleoporin NUP170 / Nucleoporin NUP157 / Nucleoporin NUP82 / Nucleoporin NUP159 / Nucleoporin NUP85 / Nucleoporin NUP192 ...Nucleoporin NSP1 / Nucleoporin NIC96 / Nucleoporin NUP120 / Nucleoporin NUP133 / Nucleoporin NUP170 / Nucleoporin NUP157 / Nucleoporin NUP82 / Nucleoporin NUP159 / Nucleoporin NUP85 / Nucleoporin NUP192 / Nucleoporin NUP57 / Nucleoporin NUP145 / Nucleoporin NUP188 / Nucleoporin NUP84 / Nucleoporin SEH1 / Nucleoporin NUP49/NSP49 / Dynein light chain 1, cytoplasmic / Nucleoporin NUP53 / Protein transport protein SEC13 / Nucleoporin ASM4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 37 Å
AuthorsVilla, E. / Singh, D. / Ludtke, S.J. / Akey, C.W. / Rout, M.P. / Echeverria, I. / Suslov, S.
Funding support United States, 7items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2 GM123494 United States
National Science Foundation (NSF, United States)MRI DBI 1920374 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01 GM121203 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM121443 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM109824 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM121443 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM45377 United States
CitationJournal: Cell / Year: 2022
Title: Comprehensive structure and functional adaptations of the yeast nuclear pore complex.
Authors: Christopher W Akey / Digvijay Singh / Christna Ouch / Ignacia Echeverria / Ilona Nudelman / Joseph M Varberg / Zulin Yu / Fei Fang / Yi Shi / Junjie Wang / Daniel Salzberg / Kangkang Song / ...Authors: Christopher W Akey / Digvijay Singh / Christna Ouch / Ignacia Echeverria / Ilona Nudelman / Joseph M Varberg / Zulin Yu / Fei Fang / Yi Shi / Junjie Wang / Daniel Salzberg / Kangkang Song / Chen Xu / James C Gumbart / Sergey Suslov / Jay Unruh / Sue L Jaspersen / Brian T Chait / Andrej Sali / Javier Fernandez-Martinez / Steven J Ludtke / Elizabeth Villa / Michael P Rout /
Abstract: Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub- ...Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.
History
DepositionJun 17, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 26, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 2, 2022Group: Database references / Derived calculations / Category: citation / pdbx_struct_oper_list
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type
Revision 1.2Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Assembly

Deposited unit
0: Nucleoporin NUP170
1: Nucleoporin NUP157
5: orphans bound to Nup192 NTD
6: orphans bound to Nup192 NTD
A: Nucleoporin NSP1
B: Nucleoporin NUP57
C: Nucleoporin NUP49/NSP49
D: Nucleoporin NSP1
E: Nucleoporin NUP57
F: Nucleoporin NUP49/NSP49
G: Nucleoporin NSP1
H: Nucleoporin NUP57
I: Nucleoporin NUP49/NSP49
J: Nucleoporin NSP1
K: Nucleoporin NUP57
L: Nucleoporin NUP49/NSP49
M: Nucleoporin NUP192
N: Nucleoporin NUP188
O: Nucleoporin NUP192
P: Nucleoporin NUP188
Q: Nucleoporin NIC96
R: Nucleoporin NIC96
S: Nucleoporin NIC96
T: Nucleoporin NIC96
U: Nucleoporin NUP53
V: Nucleoporin ASM4
W: Nucleoporin NUP53
X: Nucleoporin ASM4
Y: Nucleoporin NUP170
Z: Nucleoporin NUP157
u: Nucleoporin NUP82
v: Nucleoporin NUP82
w: Nucleoporin NUP159
x: Nucleoporin NUP159
y: Nucleoporin NSP1
z: Nucleoporin NSP1
h: Nucleoporin NUP120
i: Nucleoporin NUP85
j: Nucleoporin 145c
k: Protein transport protein SEC13
l: Nucleoporin SEH1
m: Nucleoporin NUP84
n: Nucleoporin NUP133
o: Dynein light chain 1, cytoplasmic
p: Dynein light chain 1, cytoplasmic
q: Dynein light chain 1, cytoplasmic
r: Dynein light chain 1, cytoplasmic
s: Dynein light chain 1, cytoplasmic
t: Dynein light chain 1, cytoplasmic
b: Nucleoporin NUP85
c: Nucleoporin 145c
d: Protein transport protein SEC13
e: Nucleoporin SEH1
f: Nucleoporin NUP84
g: Nucleoporin NUP133
a: Nucleoporin NUP120


Theoretical massNumber of molelcules
Total (without water)4,672,33156
Polymers4,672,33156
Non-polymers00
Water00
1
0: Nucleoporin NUP170
1: Nucleoporin NUP157
5: orphans bound to Nup192 NTD
6: orphans bound to Nup192 NTD
A: Nucleoporin NSP1
B: Nucleoporin NUP57
C: Nucleoporin NUP49/NSP49
D: Nucleoporin NSP1
E: Nucleoporin NUP57
F: Nucleoporin NUP49/NSP49
G: Nucleoporin NSP1
H: Nucleoporin NUP57
I: Nucleoporin NUP49/NSP49
J: Nucleoporin NSP1
K: Nucleoporin NUP57
L: Nucleoporin NUP49/NSP49
M: Nucleoporin NUP192
N: Nucleoporin NUP188
O: Nucleoporin NUP192
P: Nucleoporin NUP188
Q: Nucleoporin NIC96
R: Nucleoporin NIC96
S: Nucleoporin NIC96
T: Nucleoporin NIC96
U: Nucleoporin NUP53
V: Nucleoporin ASM4
W: Nucleoporin NUP53
X: Nucleoporin ASM4
Y: Nucleoporin NUP170
Z: Nucleoporin NUP157
u: Nucleoporin NUP82
v: Nucleoporin NUP82
w: Nucleoporin NUP159
x: Nucleoporin NUP159
y: Nucleoporin NSP1
z: Nucleoporin NSP1
h: Nucleoporin NUP120
i: Nucleoporin NUP85
j: Nucleoporin 145c
k: Protein transport protein SEC13
l: Nucleoporin SEH1
m: Nucleoporin NUP84
n: Nucleoporin NUP133
o: Dynein light chain 1, cytoplasmic
p: Dynein light chain 1, cytoplasmic
q: Dynein light chain 1, cytoplasmic
r: Dynein light chain 1, cytoplasmic
s: Dynein light chain 1, cytoplasmic
t: Dynein light chain 1, cytoplasmic
b: Nucleoporin NUP85
c: Nucleoporin 145c
d: Protein transport protein SEC13
e: Nucleoporin SEH1
f: Nucleoporin NUP84
g: Nucleoporin NUP133
a: Nucleoporin NUP120
x 8


Theoretical massNumber of molelcules
Total (without water)37,378,644448
Polymers37,378,644448
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation7
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C8 (8 fold cyclic))

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Components

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Protein , 20 types, 54 molecules 0Y1ZADGJyzBEHKCFILMONPQRSTUWVX...

#1: Protein Nucleoporin NUP170 / Nuclear pore protein NUP170


Mass: 169651.969 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38181
#2: Protein Nucleoporin NUP157 / Nuclear pore protein NUP157


Mass: 156827.484 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40064
#4: Protein
Nucleoporin NSP1 / Nuclear pore protein NSP1 / Nucleoskeletal-like protein / p110


Mass: 86611.672 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P14907
#5: Protein
Nucleoporin NUP57 / Nuclear pore protein NUP57


Mass: 57547.145 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P48837
#6: Protein
Nucleoporin NUP49/NSP49 / Nuclear pore protein NUP49/NSP49


Mass: 49174.762 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q02199
#7: Protein Nucleoporin NUP192 / Nuclear pore protein NUP192


Mass: 191718.125 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P47054
#8: Protein Nucleoporin NUP188 / Nuclear pore protein NUP188


Mass: 188753.281 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P52593
#9: Protein
Nucleoporin NIC96 / 96 kDa nucleoporin-interacting component / Nuclear pore protein NIC96


Mass: 96291.586 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P34077
#10: Protein Nucleoporin NUP53 / Nuclear pore protein NUP53


Mass: 52688.668 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03790
#11: Protein Nucleoporin ASM4 / Nuclear pore protein NUP59


Mass: 58853.902 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q05166
#12: Protein Nucleoporin NUP82 / Nuclear pore protein NUP82


Mass: 82174.047 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40368
#13: Protein Nucleoporin NUP159 / Nuclear pore protein NUP159


Mass: 159067.188 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40477
#14: Protein Nucleoporin NUP120 / Nuclear pore protein NUP120


Mass: 120560.328 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P35729
#15: Protein Nucleoporin NUP85 / Nuclear pore protein NUP85


Mass: 84972.438 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P46673
#16: Protein Nucleoporin 145c


Mass: 81157.852 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P49687
#17: Protein Protein transport protein SEC13


Mass: 33082.965 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04491
#18: Protein Nucleoporin SEH1 / Nuclear pore protein SEH1 / SEC13 homolog 1


Mass: 39170.758 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53011
#19: Protein Nucleoporin NUP84 / Nuclear pore protein NUP84


Mass: 83718.867 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P52891
#20: Protein Nucleoporin NUP133 / Nuclear pore protein NUP133


Mass: 133452.672 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P36161
#21: Protein
Dynein light chain 1, cytoplasmic


Mass: 10456.980 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q02647

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Protein/peptide , 1 types, 2 molecules 56

#3: Protein/peptide orphans bound to Nup192 NTD


Mass: 3081.790 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast)

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Details

Compound detailsfull C8 protomer

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: yeast NPC / Type: ORGANELLE OR CELLULAR COMPONENT / Details: Complete yeast NPC from vitrified cells / Entity ID: all / Source: NATURAL
Molecular weightValue: 52.0 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: W303 yeast cells were harvested during log-phase growth and diluted to an 0.8 x 107 cells/mL in YPD media. Five uL of this diluted sample was applied to glow-discharged 200-mesh, Quantafoil ...Details: W303 yeast cells were harvested during log-phase growth and diluted to an 0.8 x 107 cells/mL in YPD media. Five uL of this diluted sample was applied to glow-discharged 200-mesh, Quantafoil R2/1 grids (Electron Microscopy Sciences), excess media was manually blotted from the grid back side (opposite to the carbon substrate where the cells were deposited) and the grid was plunge frozen in a liquid ethane-propane mixture (50/50 volume, Airgas) then cryogenic, FIB milling was performed in an Aquilos DualBeam (Thermo Fisher Scientific)
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE-PROPANE
Details: A custom-built vitrification device (Max Planck Institute for Biochemistry, Munich)

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Tilt series collection: dose-symmetric & bi-directional
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 42000 X / Nominal defocus max: 6000 nm / Nominal defocus min: 2500 nm / Calibrated defocus min: 2500 nm / Calibrated defocus max: 6000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2 sec. / Electron dose: 4.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Details: Images were collected in movie-mode at ~8 frames per second
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3708

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Processing

EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4EMAN22.9CTF correctione2spt_ctf.py
7UCSF Chimera1.14model fitting
9VMD1.9.3model refinement
13EMAN22.93D reconstruction
Image processingDetails: The tilt images were motion-corrected but not dose-weighted.
CTF correctionDetails: e2spt_ctf.py was used to identify the precise z height of the particle in the tomogram. This precise height was then used for computing the CTF and performing the CTF correction.
Type: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C8 (8 fold cyclic)
3D reconstructionResolution: 37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 518 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
EM volume selectionMethod: Manual boxing
Details: The full nuclear pores were manually identified in the tomograms for the manual boxing.
Num. of tomograms: 293 / Num. of volumes extracted: 577
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation
Details: Manual docking and limited molecular dynamics flexible fitting

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