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- EMDB-24231: Double nuclear outer ring from the isolated yeast NPC -

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Basic information

Entry
Database: EMDB / ID: EMD-24231
TitleDouble nuclear outer ring from the isolated yeast NPC
Map datarecombined double nuclear outer ring
Sample
  • Complex: yeast double outer ring
    • Protein or peptide: Nucleoporin NUP188
    • Protein or peptide: unknown
    • Protein or peptide: Nucleoporin NUP120
    • Protein or peptide: Nucleoporin NUP85
    • Protein or peptide: Nucleoporin 145c
    • Protein or peptide: Protein transport protein SEC13Protein targeting
    • Protein or peptide: Nucleoporin SEH1
    • Protein or peptide: Nucleoporin NUP84
    • Protein or peptide: Nucleoporin NUP133
Function / homology
Function and homology information


mRNA export from nucleus in response to heat stress / nuclear pore inner ring / Seh1-associated complex / protein localization to nuclear inner membrane / positive regulation of ER to Golgi vesicle-mediated transport / protein exit from endoplasmic reticulum / COPII-coated vesicle budding / COPII-mediated vesicle transport / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore central transport channel ...mRNA export from nucleus in response to heat stress / nuclear pore inner ring / Seh1-associated complex / protein localization to nuclear inner membrane / positive regulation of ER to Golgi vesicle-mediated transport / protein exit from endoplasmic reticulum / COPII-coated vesicle budding / COPII-mediated vesicle transport / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore central transport channel / nuclear pore localization / telomere tethering at nuclear periphery / regulation of nucleocytoplasmic transport / regulation of TORC1 signaling / nuclear pore organization / nuclear pore outer ring / tRNA export from nucleus / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore cytoplasmic filaments / COPII vesicle coat / positive regulation of protein exit from endoplasmic reticulum / RNA export from nucleus / structural constituent of nuclear pore / silent mating-type cassette heterochromatin formation / nucleocytoplasmic transport / vacuolar membrane / poly(A)+ mRNA export from nucleus / nuclear localization sequence binding / NLS-bearing protein import into nucleus / subtelomeric heterochromatin formation / ribosomal large subunit export from nucleus / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / positive regulation of TOR signaling / mRNA transport / mRNA export from nucleus / nuclear pore / : / positive regulation of TORC1 signaling / cellular response to amino acid starvation / protein export from nucleus / cell periphery / protein import into nucleus / double-strand break repair / protein transport / nuclear envelope / nuclear membrane / chromosome, telomeric region / hydrolase activity / endoplasmic reticulum membrane / structural molecule activity / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II / endoplasmic reticulum / positive regulation of transcription by RNA polymerase II / RNA binding / identical protein binding / nucleus / cytosol
Similarity search - Function
Nuclear pore protein Nup188, C-terminal / Nuclear pore protein NUP188 C-terminal domain / Nucleoporin Nup188, N-terminal / Nucleoporin Nup188, N-terminal / : / Nucleoporin Nup188, N-terminal subdomain III / Nucleoporin Nup188 / Nucleoporin Nup85-like / Nucleoporin Nup120/160 / Nup85 Nucleoporin ...Nuclear pore protein Nup188, C-terminal / Nuclear pore protein NUP188 C-terminal domain / Nucleoporin Nup188, N-terminal / Nucleoporin Nup188, N-terminal / : / Nucleoporin Nup188, N-terminal subdomain III / Nucleoporin Nup188 / 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 FG repeat / Nucleoporin FG repeat region / Nucleoporin, Nup133/Nup155-like, C-terminal / 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 / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Nucleoporin NUP120 / Nucleoporin NUP133 / Nucleoporin NUP85 / Nucleoporin NUP145 / Nucleoporin NUP188 / Nucleoporin NUP84 / Nucleoporin SEH1 / Protein transport protein SEC13
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Baker's yeast (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.6 Å
AuthorsAkey CW / Rout MP / Ouch C / Echevarria I / Fernandez-Martinez J / Nudelman I
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM45377 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM112108 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM109824 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 13, 2021-
Header (metadata) releaseJan 26, 2022-
Map releaseJan 26, 2022-
UpdateFeb 2, 2022-
Current statusFeb 2, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7n84
  • Surface level: 0.01
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7n84
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24231.png

Downloads & links

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Map

FileDownload / File: emd_24231.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationrecombined double nuclear outer ring
Voxel sizeX=Y=Z: 2.66 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.01903938 - 0.1297321
Average (Standard dev.)0.0007293956 (±0.0062733702)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 1276.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.662.662.66
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z1276.8001276.8001276.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.0190.1300.001

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Supplemental data

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Mask #1

Fileemd_24231_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: zoned proximal outer nuclear ring

Fileemd_24231_additional_1.map
Annotationzoned proximal outer nuclear ring
Projections & Slices
AxesZYX

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Histogram (log scale)

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Additional map: zoned proximal outer nuclear ring

Fileemd_24231_additional_2.map
Annotationzoned proximal outer nuclear ring
Projections & Slices
AxesZYX

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Additional map: post processed multibody for double nuclear outer ring

Fileemd_24231_additional_3.map
Annotationpost processed multibody for double nuclear outer ring
Projections & Slices
AxesZYX

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Additional map: zoned Nup188-192 density ring

Fileemd_24231_additional_4.map
Annotationzoned Nup188-192 density ring
Projections & Slices
AxesZYX

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Histogram (log scale)

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Additional map: zoned putative basket density ring

Fileemd_24231_additional_5.map
Annotationzoned putative basket density ring
Projections & Slices
AxesZYX

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Histogram (log scale)

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Half map: half map for double Y multibody

Fileemd_24231_half_map_1.map
Annotationhalf map for double Y multibody
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Half map: half map for double Y multibody

Fileemd_24231_half_map_2.map
Annotationhalf map for double Y multibody
Projections & Slices
AxesZYX

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Sample components

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Entire : yeast double outer ring

EntireName: yeast double outer ring
Components
  • Complex: yeast double outer ring
    • Protein or peptide: Nucleoporin NUP188
    • Protein or peptide: unknown
    • Protein or peptide: Nucleoporin NUP120
    • Protein or peptide: Nucleoporin NUP85
    • Protein or peptide: Nucleoporin 145c
    • Protein or peptide: Protein transport protein SEC13Protein targeting
    • Protein or peptide: Nucleoporin SEH1
    • Protein or peptide: Nucleoporin NUP84
    • Protein or peptide: Nucleoporin NUP133

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Supramolecule #1: yeast double outer ring

SupramoleculeName: yeast double outer ring / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Novel outer ring C8 protomer with two copies of Nup84 complex, one copy of Nup188 and an unknown basket anchor model
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Location in cell: nuclear envelope
Molecular weightTheoretical: 10.4 MDa

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Macromolecule #1: Nucleoporin NUP188

MacromoleculeName: Nucleoporin NUP188 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Baker's yeast (brewer's yeast)
Molecular weightTheoretical: 188.753281 KDa
SequenceString: MATPSFGNSS PQLTFTHVAN FMNDAAADVS AVDAKQLAQI RQFLKANKTN LIESLNTIRQ NVTSSGDHNK LRSTIANLLQ INVDNDPFF AQSEDLSHAV EFFMSERSSR LHIVYSLLVN PDIDLETYSF IDNDRFNVVG KLISIISSVI QNYDIITASS L AHDYNNDQ ...String:
MATPSFGNSS PQLTFTHVAN FMNDAAADVS AVDAKQLAQI RQFLKANKTN LIESLNTIRQ NVTSSGDHNK LRSTIANLLQ INVDNDPFF AQSEDLSHAV EFFMSERSSR LHIVYSLLVN PDIDLETYSF IDNDRFNVVG KLISIISSVI QNYDIITASS L AHDYNNDQ DMFTIVSLVQ LKKFSDLKFI LQILQILNLM ILNTKVPVDI VNQWFLQYQN QFVEFCRNIN STDKSIDTSS LQ LYKFQNF QDLSYLSETL ISRISSLFTI TTILILGLNT SIAQFDIQSP LYMDTETFDT VNSALENDVA TNIVNEDPIF HPM IHYSWS FILYYRRALQ SSESFDDSDI TKFALFAESH DVLQKLNTLS EILSFDPVYT TVITVFLEFS LNFIPITAST SRVF AKIIS KAPEQFIENF LTNDTFEKKL SIIKAKLPLL NESLIPLINL ALIDTEFANF ELKDICSFAV TKSSLNDLDY DLIAD TITN SSSSSDIIVP DLIELKSDLL VAPPLENENS NCLLSIPKST KGKILTIKQQ QQQQQQQNGQ QPPTTSNLII FLYKFN GWS LVGRILQNLL HSYMEKGTQL DDLQHELMIS IIKLVTNVVD PKTSIEKSSE ILSYLSNSLD TSASTINGAS IIQVIFE IF EISLQRKDYT SIVQCCEFMT MLTPNYLHLV SSYLNKSDLL DKYGKTGLSN MILGSVELST GDYTFTIQLL KLTKVFIR E SLSLKNIHIS KRSKIDIINK LILHAIHIFE SYYNWKYNNF LQKFEIAFHL TLIFYDVLHD VFTINPHQKD QLIISSSAN KLLQLFLTPM DSIDLAPNTL TNILISPLNT TTKILGDKIL GNLYSKVMNN SFKLCTLLIA IRGSNRDLKP SNLEKLLFIN SSKLVDVYT LPSYVHFKVQ IIELLSYLVE APWNDDYPFL LSFLGEAKSM AFLKEVLSDL SSPVQDWNLL RSLYIFFTTL L ESKQDGLS ILFLTGQFAS NKKINDESSI DKKSSILTVL QKNSLLLDST PEEVSCKLLE TITYVLNTWT NSKIFIKDPK FV NSLLAKL KDSKKLFQKK ENLTRDETVS LIKKYKLISR IVEIFALCIY NSTDSNSEIL NFLNQEDLFE LVHHFFQIDG FNK TFHDEL NLKFKEKWPS LELQSFQKIP LSRINENENF GYDIPLLDIV LKADRSWNEP SKSQTNFKEE ITDASLNLQY VNYE ISTAK AWGALITTFV KRSTVPLNDG FVDLVEHFLK LNIDFGSDKQ MFTQIYLERI ELSFYILYSF KLSGKLLKEE KIIEL MNKI FTIFKSGEID FIKNIGKSLK NNFYRPLLRS VLVLLELVSS GDRFIELISD QLLEFFELVF SKGVYLILSE ILCQIN KCS TRGLSTDHTT QIVNLEDNTQ DLLLLLSLFK KITNVNPSKN FNVILASSLN EVGTLKVILN LYSSAHLIRI NDEPILG QI TLTFISELCS IEPIAAKLIN SGLYSVLLES PLSVAIQQGD IKPEFSPRLH NIWSNGLLSI VLLLLSQFGI KVLPETCL F VSYFGKQIKS TIYNWGDNKL AVSSSLIKET NQLVLLQKML NLLNYQELFI QPKNSDDQQE AVELVIGLDS EHDKKRLSA ALSKFLTHPK YLNSRIIPTT LEEQQQLEDE SSRLEFVKGI SRDIKALQDS LFKDV

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Macromolecule #2: unknown

MacromoleculeName: unknown / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 5.379623 KDa
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #3: Nucleoporin NUP120

MacromoleculeName: Nucleoporin NUP120 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Baker's yeast (brewer's yeast)
Molecular weightTheoretical: 120.560328 KDa
SequenceString: MACLSRIDAN LLQYYEKPEP NNTVDLYVSN NSNNNGLKEG DKSISTPVPQ PYGSEYSNCL LLSNSEYICY HFSSRSTLLT FYPLSDAYH GKTINIHLPN ASMNQRYTLT IQEVEQQLLV NVILKDGSFL TLQLPLSFLF SSANTLNGEW FHLQNPYDFT V RVPHFLFY ...String:
MACLSRIDAN LLQYYEKPEP NNTVDLYVSN NSNNNGLKEG DKSISTPVPQ PYGSEYSNCL LLSNSEYICY HFSSRSTLLT FYPLSDAYH GKTINIHLPN ASMNQRYTLT IQEVEQQLLV NVILKDGSFL TLQLPLSFLF SSANTLNGEW FHLQNPYDFT V RVPHFLFY VSPQFSVVFL EDGGLLGLKK VDGVHYEPLL FNDNSYLKSL TRFFSRSSKS DYDSVISCKL FHERYLIVLT QN CHLKIWD LTSFTLIQDY DMVSQSDSDP SHFRKVEAVG EYLSLYNNTL VTLLPLENGL FQMGTLLVDS SGILTYTFQN NIP TNLSAS AIWSIVDLVL TRPLELNVEA SYLNLIVLWK SGTASKLQIL NVNDESFKNY EWIESVNKSL VDLQSEHDLD IVTK TGDVE RGFCNLKSRY GTQIFERAQQ ILSENKIIMA HNEDEEYLAN LETILRDVKT AFNEASSITL YGDEIILVNC FQPYN HSLY KLNTTVENWF YNMHSETDGS ELFKYLRTLN GFASTLSNDV LRSISKKFLD IITGELPDSM TTVEKFTDIF KNCLEN QFE ITNLKILFDE LNSFDIPVVL NDLINNQMKP GIFWKKDFIS AIKFDGFTSI ISLESLHQLL SIHYRITLQV LLTFVLF DL DTEIFGQHIS TLLDLHYKQF LLLNLYRQDK CLLAEVLLKD SSEFSFGVKF FNYGQLIAYI DSLNSNVYNA SITENSFF M TFFRSYIIEN TSHKNIRFFL ENVECPFYLR HNEVQEFMFA MTLFSCGNFD QSYEIFQLHD YPEAINDKLP TFLEDLKSE NYHGDSIWKD LLCTFTVPYR HSAFYYQLSL LFDRNNSQEF ALKCISKSAE YSLKEIQIEE LQDFKEKQHI HYLNLLIHFR MFEEVLDVL RLGHECLSDT VRTNFLQLLL QEDIYSRDFF STLLRLCNAH SDNGELYLRT VDIKIVDSIL SQNLRSGDWE C FKKLYCFR MLNKSERAAA EVLYQYILMQ ADLDVIRKRK CYLMVINVLS SFDSAYDQWI LNGSKVVTLT DLRDELRGL

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Macromolecule #4: Nucleoporin NUP85

MacromoleculeName: Nucleoporin NUP85 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Baker's yeast (brewer's yeast)
Molecular weightTheoretical: 84.972438 KDa
SequenceString: MTIDDSNRLL MDVDQFDFLD DGTAQLSNNK TDEEEQLYKR DPVSGAILVP MTVNDQPIEK NGDKMPLKFK LGPLSYQNMA FITAKDKYK LYPVRIPRLD TSKEFSAYVS GLFEIYRDLG DDRVFNVPTI GVVNSNFAKE HNATVNLAME AILNELEVFI G RVKDQDGR ...String:
MTIDDSNRLL MDVDQFDFLD DGTAQLSNNK TDEEEQLYKR DPVSGAILVP MTVNDQPIEK NGDKMPLKFK LGPLSYQNMA FITAKDKYK LYPVRIPRLD TSKEFSAYVS GLFEIYRDLG DDRVFNVPTI GVVNSNFAKE HNATVNLAME AILNELEVFI G RVKDQDGR VNRFYELEES LTVLNCLRTM YFILDGQDVE ENRSEFIESL LNWINRSDGE PDEEYIEQVF SVKDSTAGKK VF ETQYFWK LLNQLVLRGL LSQAIGCIER SDLLPYLSDT CAVSFDAVSD SIELLKQYPK DSSSTFREWK NLVLKLSQAF GSS ATDISG ELRDYIEDFL LVIGGNQRKI LQYSRTWYES FCGFLLYYIP SLELSAEYLQ MSLEANVVDI TNDWEQPCVD IISG KIHSI LPVMESLDSC TAAFTAMICE AKGLIENIFE GEKNSDDYSN EDNEMLEDLF SYRNGMASYM LNSFAFELCS LGDKE LWPV AIGLIALSAT GTRSAKKMVI AELLPHYPFV TNDDIEWMLS ICVEWRLPEI AKEIYTTLGN QMLSAHNIIE SIANFS RAG KYELVKSYSW LLFEASCMEG QKLDDPVLNA IVSKNSPAED DVIIPQDILD CVVTNSMRQT LAPYAVLSQF YELRDRE DW GQALRLLLLL IEFPYLPKHY LVLLVAKFLY PIFLLDDKKL MDEDSVATVI EVIETKWDDA DEKSSNLYET IIEADKSL P SSMATLLKNL RKKLNFKLCQ AFM

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Macromolecule #5: Nucleoporin 145c

MacromoleculeName: Nucleoporin 145c / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 81.157852 KDa
SequenceString: SIWGLVNEED AEIDEDDLSK QEDGGEQPLR KVRTLAQSKP SDKEVILKTD GTFGTLSGKD DSIVEEKAYE PDLSDADFEG IEASPKLDV SKDWVEQLIL AGSSLRSVFA TSKEFDGPCQ NEIDLLFSEC NDEIDNAKLI MKERRFTASY TFAKFSTGSM L LTKDIVGK ...String:
SIWGLVNEED AEIDEDDLSK QEDGGEQPLR KVRTLAQSKP SDKEVILKTD GTFGTLSGKD DSIVEEKAYE PDLSDADFEG IEASPKLDV SKDWVEQLIL AGSSLRSVFA TSKEFDGPCQ NEIDLLFSEC NDEIDNAKLI MKERRFTASY TFAKFSTGSM L LTKDIVGK SGVSIKRLPT ELQRKFLFDD VYLDKEIEKV TIEARKSNPY PQISESSLLF KDALDYMEKT SSDYNLWKLS SI LFDPVSY PYKTDNDQVK MALLKKERHC RLTSWIVSQI GPEIEEKIRN SSNEIEQIFL YLLLNDVVRA SKLAIESKNG HLS VLISYL GSNDPRIRDL AELQLQKWST GGCSIDKNIS KIYKLLSGSP FEGLFSLKEL ESEFSWLCLL NLTLCYGQID EYSL ESLVQ SHLDKFSLPY DDPIGVIFQL YAANENTEKL YKEVRQRTNA LDVQFCWYLI QTLRFNGTRV FSKETSDEAT FAFAA QLEF AQLHGHSLFV SCFLNDDKAA EDTIKRLVMR EITLLRASTN DHILNRLKIP SQLIFNAQAL KDRYEGNYLS EVQNLL LGS SYDLAEMAIV TSLGPRLLLS NNPVQNNELK TLREILNEFP DSERDKWSVS INVFEVYLKL VLDNVETQET IDSLISG MK IFYDQYKHCR EVAACCNVMS QEIVSKILEK NNPSIGDSKA KLLELPLGQP EKAYLRGEFA QDLMKCTYKI

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Macromolecule #6: Protein transport protein SEC13

MacromoleculeName: Protein transport protein SEC13 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Baker's yeast (brewer's yeast)
Molecular weightTheoretical: 33.082965 KDa
SequenceString: MVVIANAHNE LIHDAVLDYY GKRLATCSSD KTIKIFEVEG ETHKLIDTLT GHEGPVWRVD WAHPKFGTIL ASCSYDGKVL IWKEENGRW SQIAVHAVHS ASVNSVQWAP HEYGPLLLVA SSDGKVSVVE FKENGTTSPI IIDAHAIGVN SASWAPATIE E DGEHNGTK ...String:
MVVIANAHNE LIHDAVLDYY GKRLATCSSD KTIKIFEVEG ETHKLIDTLT GHEGPVWRVD WAHPKFGTIL ASCSYDGKVL IWKEENGRW SQIAVHAVHS ASVNSVQWAP HEYGPLLLVA SSDGKVSVVE FKENGTTSPI IIDAHAIGVN SASWAPATIE E DGEHNGTK ESRKFVTGGA DNLVKIWKYN SDAQTYVLES TLEGHSDWVR DVAWSPTVLL RSYLASVSQD RTCIIWTQDN EQ GPWKKTL LKEEKFPDVL WRASWSLSGN VLALSGGDNK VTLWKENLEG KWEPAGEVHQ

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Macromolecule #7: Nucleoporin SEH1

MacromoleculeName: Nucleoporin SEH1 / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Baker's yeast (brewer's yeast)
Molecular weightTheoretical: 39.170758 KDa
SequenceString: MQPFDSGHDD LVHDVVYDFY GRHVATCSSD QHIKVFKLDK DTSNWELSDS WRAHDSSIVA IDWASPEYGR IIASASYDKT VKLWEEDPD QEECSGRRWN KLCTLNDSKG SLYSVKFAPA HLGLKLACLG NDGILRLYDA LEPSDLRSWT LTSEMKVLSI P PANHLQSD ...String:
MQPFDSGHDD LVHDVVYDFY GRHVATCSSD QHIKVFKLDK DTSNWELSDS WRAHDSSIVA IDWASPEYGR IIASASYDKT VKLWEEDPD QEECSGRRWN KLCTLNDSKG SLYSVKFAPA HLGLKLACLG NDGILRLYDA LEPSDLRSWT LTSEMKVLSI P PANHLQSD FCLSWCPSRF SPEKLAVSAL EQAIIYQRGK DGKLHVAAKL PGHKSLIRSI SWAPSIGRWY QLIATGCKDG RI RIFKITE KLSPLASEES LTNSNMFDNS ADVDMDAQGR SDSNTEEKAE LQSNLQVELL SEHDDHNGEV WSVSWNLTGT ILS SAGDDG KVRLWKATYS NEFKCMSVIT AQQ

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Macromolecule #8: Nucleoporin NUP84

MacromoleculeName: Nucleoporin NUP84 / type: protein_or_peptide / ID: 8 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Baker's yeast (brewer's yeast)
Molecular weightTheoretical: 83.718867 KDa
SequenceString: MELSPTYQTE RFTKFSDTLK EFKIEQNNEQ NPIDPFNIIR EFRSAAGQLA LDLANSGDES NVISSKDWEL EARFWHLVEL LLVFRNADL DLDEMELHPY NSRGLFEKKL MQDNKQLYQI WIVMVWLKEN TYVMERPKNV PTSKWLNSIT SGGLKSCDLD F PLRENTNV ...String:
MELSPTYQTE RFTKFSDTLK EFKIEQNNEQ NPIDPFNIIR EFRSAAGQLA LDLANSGDES NVISSKDWEL EARFWHLVEL LLVFRNADL DLDEMELHPY NSRGLFEKKL MQDNKQLYQI WIVMVWLKEN TYVMERPKNV PTSKWLNSIT SGGLKSCDLD F PLRENTNV LDVKDKEEDH IFFKYIYELI LAGAIDEALE EAKLSDNISI CMILCGIQEY LNPVIDTQIA NEFNTQQGIK KH SLWRRTV YSLSQQAGLD PYERAIYSYL SGAIPNQEVL QYSDWESDLH IHLNQILQTE IENYLLENNQ VGTDELILPL PSH ALTVQE VLNRVASRHP SESEHPIRVL MASVILDSLP SVIHSSVEML LDVVKGTEAS NDIIDKPYLL RIVTHLAICL DIIN PGSVE EVDKSKLITT YISLLKLQGL YENIPIYATF LNESDCLEAC SFILSSLEDP QVRKKQIETI NFLRLPASNI LRRTT QRVF DETEQEYSPS NEISISFDVN NIDMHLIYGV EWLIEGKLYV DAVHSIIALS RRFLLNGRVK ALEQFMERNN IGEICK NYE LEKIADNISK DENEDQFLEE ITQYEHLIKG IREYEEWQKS VSLLSSESNI PTLIEKLQGF SKDTFELIKT FLVDLTS SN FADSADYEIL YEIRALYTPF LLMELHKKLV EAAKLLKIPK FISEALAFTS LVANENDKIY LLFQSSGKLK EYLDLVAR T ATLSN

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Macromolecule #9: Nucleoporin NUP133

MacromoleculeName: Nucleoporin NUP133 / type: protein_or_peptide / ID: 9 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Baker's yeast (brewer's yeast)
Molecular weightTheoretical: 133.452672 KDa
SequenceString: MSEKKVHLRL RKELSVPIAV VENESLAQLS YEEESQASLM DISMEQQQLR LHSHFDNSKV FTENNRYIVK TLQTDYSSGF SNDDELNGY IDMQIGYGLV NDHKKVYIWN IHSTQKDTPY ITVPFRSDDN DEIAVAPRCI LTFPATMDES PLALNPNDQD E TGGLIIIK ...String:
MSEKKVHLRL RKELSVPIAV VENESLAQLS YEEESQASLM DISMEQQQLR LHSHFDNSKV FTENNRYIVK TLQTDYSSGF SNDDELNGY IDMQIGYGLV NDHKKVYIWN IHSTQKDTPY ITVPFRSDDN DEIAVAPRCI LTFPATMDES PLALNPNDQD E TGGLIIIK GSKAIYYEDI NSINNLNFKL SEKFSHELEL PINSSGGEKC DLMLNCEPAG IVLSTNMGRI FFITIRNSMG KP QLKLGKL LNKPFKLGIW SKIFNTNSSV VSLRNGPILG KGTRLVYITT NKGIFQTWQL SATNSHPTKL IDVNIYEAIL ESL QDLYPF AHGTLKIWDS HPLQDESSQL FLSSIYDSSC NETYYILSTI IFDSSSNSFT IFSTYRLNTF MESITDTKFK PKIF IPQME NANDTNEVTS ILVMFPNAVV ITQVNSKLDS SYSMRRKWED IVSLRNDIDI IGSGYDSKSL YVLTKQMGVL QFFVK ENEE TNSKPEVGFV KSHVDQAVYF SKINANPIDF NLPPEISLDQ ESIEHDLKLT SEEIFHSNGK YIPPMLNTLG QHLSVR KEF FQNFLTFVAK NFNYKISPEL KLDLIEKFEI LNCCIKFNSI IRQSDVLNDI WEKTLSNYNL TQNEHLTTKT VVINSPD VF PVIFKQFLNH VVFVLFPSQN QNFKLNVTNL INLCFYDGIL EEGEKTIRYE LLELDPMEVD TSKLPWFINF DYLNCINQ C FFDFTFACEE EGSLDSYKEG LLKIVKILYY QFNQFKIWIN TQPVKSVNAN DNFININNLY DDNHLDWNHV LCKVNLKEQ CIQIAEFYKD LSGLVQTLQT LDQNDSTTVS LYETFFNEFP KEFSFTLFEY LIKHKKLNDL IFRFPQQHDV LIQFFQESAP KYGHVAWIQ QILDGSYADA MNTLKNITVD DSKKGESLSE CELHLNVAKL SSLLVEKDNL DINTLRKIQY NLDTIDAEKN I SNKLKKGE VQICKRFKNG SIREVFNILV EELKSTTVVN LSDLVELYSM LDDEESLFIP LRLLSVDGNL LNFEVKKFLN AL VWRRIVL LNASNEGDKL LQHIVKRVFD EELPKNNDFP LPSVDLLCDK SLLTPEYISE TYGRFPIDQN AIREEIYEEI SQV ETLNSD NSLEIKLHST IGSVAKEKNY TINYETNTVE Y

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES
50.0 mMpotassium acetate
20.0 mMsodium chlorideNaClSodium chloride
2.0 mMmagnesium chloride
1.0 mMDTT
0.1 percent wt/wtDeoxyBigChaps
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK III
Detailsdouble outer ring imaged from isolated yeast NPC

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.8000000000000003 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 37651 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 50000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 2-40 / Number grids imaged: 1 / Number real images: 4015 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf (ver. 1.18)
Startup modelType of model: EMDB MAP
EMDB ID:

7321

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 11.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Details: gold standard in RELION / Number images used: 45000
FSC plot (resolution estimation)

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Atomic model buiding 1

Detailsmolecular dynamics flexible fitting
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7n84:
Double nuclear outer ring from the isolated yeast NPC

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