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- EMDB-41117: Pom34-Pom152 membrane attachment site yeast NPC -

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Basic information

Entry
Database: EMDB / ID: EMD-41117
TitlePom34-Pom152 membrane attachment site yeast NPC
Map datac8 ring reconstructed from multibody volume
Sample
  • Complex: Nuclear Pore ComplexNuclear pore
    • Complex: Pom34-Pom152 Inner ring anchor complex
    • Protein or peptide: Nucleoporin POM152
    • Protein or peptide: Nucleoporin POM34
Keywordsnuclear pore complex / nucleocytoplasmic transport / nucleoporin / membrane protein / translocase / TRANSPORT PROTEIN
Function / homology
Function and homology information


nuclear pore transmembrane ring / spindle pole body duplication / nuclear pore organization / nuclear outer membrane / structural constituent of nuclear pore / nucleocytoplasmic transport / nuclear envelope lumen / mRNA transport / nuclear pore / protein-membrane adaptor activity ...nuclear pore transmembrane ring / spindle pole body duplication / nuclear pore organization / nuclear outer membrane / structural constituent of nuclear pore / nucleocytoplasmic transport / nuclear envelope lumen / mRNA transport / nuclear pore / protein-membrane adaptor activity / nuclear periphery / cell periphery / protein import into nucleus / nuclear envelope / nuclear membrane / mitochondrion
Similarity search - Function
Nuclear pore complex component / Nuclear pore complex component / Nucleoporin Pom152
Similarity search - Domain/homology
Nucleoporin POM152 / Nucleoporin POM34
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.0 Å
AuthorsAkey CW / Echeverria I / Ouch C / Fernandez-Martinez J / Rout MP
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH R01 GM45377 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH P41 GM109824 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH R01 GM112108 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH GM117212 United States
CitationJournal: Mol Cell / Year: 2023
Title: Implications of a multiscale structure of the yeast nuclear pore complex.
Authors: Christopher W Akey / Ignacia Echeverria / Christna Ouch / Ilona Nudelman / Yi Shi / Junjie Wang / Brian T Chait / Andrej Sali / Javier Fernandez-Martinez / Michael P Rout /
Abstract: Nuclear pore complexes (NPCs) direct the nucleocytoplasmic transport of macromolecules. Here, we provide a composite multiscale structure of the yeast NPC, based on improved 3D density maps from ...Nuclear pore complexes (NPCs) direct the nucleocytoplasmic transport of macromolecules. Here, we provide a composite multiscale structure of the yeast NPC, based on improved 3D density maps from cryogenic electron microscopy and AlphaFold2 models. Key features of the inner and outer rings were integrated into a comprehensive model. We resolved flexible connectors that tie together the core scaffold, along with equatorial transmembrane complexes and a lumenal ring that anchor this channel within the pore membrane. The organization of the nuclear double outer ring reveals an architecture that may be shared with ancestral NPCs. Additional connections between the core scaffold and the central transporter suggest that under certain conditions, a degree of local organization is present at the periphery of the transport machinery. These connectors may couple conformational changes in the scaffold to the central transporter to modulate transport. Collectively, this analysis provides insights into assembly, transport, and NPC evolution.
History
DepositionJun 24, 2023-
Header (metadata) releaseOct 11, 2023-
Map releaseOct 11, 2023-
UpdateOct 11, 2023-
Current statusOct 11, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41117.png

Downloads & links

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Map

FileDownload / File: emd_41117.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationc8 ring reconstructed from multibody volume
Voxel sizeX=Y=Z: 2.66 Å
Density
Contour LevelBy AUTHOR: 0.85
Minimum - Maximum-0.6009241 - 1.6932685
Average (Standard dev.)0.0015935651 (±0.034889795)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Additional map: extracted TM density for Ndc1

Fileemd_41117_additional_1.map
Annotationextracted TM density for Ndc1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: extracted region of micelle ring at lower resolution for the Poms.

Fileemd_41117_additional_2.map
Annotationextracted region of micelle ring at lower resolution for the Poms.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: half map of multibody volume

Fileemd_41117_additional_3.map
Annotationhalf map of multibody volume
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: half map of multibody volume

Fileemd_41117_additional_4.map
Annotationhalf map of multibody volume
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: c2 map of multibody volume

Fileemd_41117_additional_5.map
Annotationc2 map of multibody volume
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map c8 ring from multibody

Fileemd_41117_half_map_1.map
Annotationhalf map c8 ring from multibody
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map c8 ring from multibody

Fileemd_41117_half_map_2.map
Annotationhalf map c8 ring from multibody
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : Nuclear Pore Complex

EntireName: Nuclear Pore ComplexNuclear pore
Components
  • Complex: Nuclear Pore ComplexNuclear pore
    • Complex: Pom34-Pom152 Inner ring anchor complex
    • Protein or peptide: Nucleoporin POM152
    • Protein or peptide: Nucleoporin POM34

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Supramolecule #1: Nuclear Pore Complex

SupramoleculeName: Nuclear Pore Complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Protein A tagged Mlp1 pullout of NPC
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Strain: MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 MLP1-PPX-ProteinA::HIS5
Organelle: nucleus / Location in cell: nuclear envelope

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Supramolecule #2: Pom34-Pom152 Inner ring anchor complex

SupramoleculeName: Pom34-Pom152 Inner ring anchor complex / type: complex / ID: 2 / Parent: 1
Details: native complex resolved by multibody image processing
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Strain: MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 MLP1-PPX-ProteinA::HIS5
Organelle: nucleus / Location in cell: nuclear envelope

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Macromolecule #1: Nucleoporin POM152

MacromoleculeName: Nucleoporin POM152 / type: protein_or_peptide / ID: 1
Details: Pore outer membrane protein and anchor for the lumenal ring
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: MATa ade2-1 ura3-1 his3-11
Molecular weightTheoretical: 151.833891 KDa
SequenceString: MEHRYNVFND TPRGNHWMGS SVSGSPRPSY SSRPNVNTTR RFQYSDDEPA EKIRPLRSRS FKSTESNISD EKSRISERDS KDRYINGDK KVDIYSLPLI STDVLEISKQ RTFAVILFLI IQCYKIYDLV ILKSGLPLSG LLFKNYRFNF ISKYFIIDSF F LYVLPSFN ...String:
MEHRYNVFND TPRGNHWMGS SVSGSPRPSY SSRPNVNTTR RFQYSDDEPA EKIRPLRSRS FKSTESNISD EKSRISERDS KDRYINGDK KVDIYSLPLI STDVLEISKQ RTFAVILFLI IQCYKIYDLV ILKSGLPLSG LLFKNYRFNF ISKYFIIDSF F LYVLPSFN IPRLTFKPWV VYLQILAMLL LNIFISSDHE FVLISLIMTT WRKLYTKELS VTGSAINHHR IFDSSAHFKG AL TIKILPE NTAMFNPLHE SYCLPMDTNL FKINSIDVPI RINSTEEIEY IELEYRDLYT NSVELRSLSK KDFKIIDNPK SFL KKDQSV LKSHSNDFEE GSTIRYLAVT LQDIGFYQIK KIVDSKKLNL KIHQSHLVVP YCPIASITGT GSNDRCIGDS DNVS FEIQG VPPMKLAYSK IVNGQTFSYV DSSLQPEYFE SPLQSSKSKQ SFTQGELNDL KWGRNQPVNI NLDSSITQDG KFAYK IDKI TDGLGNVVDF TSLPEELKKR YDLSYNFNVH EVPRAALEER FDPKSPTKRS IAIVFEEIKN WISDIPYVIS LSYTDA QDK SKKIMNVTTD SLTKVLQADL PGSYNLEYIE SKFCPGEIVG KSNVLVTMPV APTMEVKSFP ILDQCVGQVG LNFELSF TG APPYYYNTKI YKLENGERKL YDAKRYTSEG TRNRFSYSPP KEGNYEIVFD TVSNKLFTEP IKLEPVKEYT FKTSMRVK P SASLKLHHDL KLCLGDHSSV PVALKGQGPF TLTYDIIETF SSKRKTFEIK EIKTNEYVIK TPVFTTGGDY ILSLVSIKD STGCVVGLSQ PDAKIQVRRD IPSAAFNFFE PIKEAKIKHG SVTEIPLKLS GEGPFTVKFK HMDYDGNIVK EFENKFQNSY KPALKVSKE GLYQLVDIRD SSCQGNVIYR NSLYKVSFLE KPKFAIQDNH HITKVTENLF SKEEVCQGME GTVDLALFGS P PFILEYDL MAPNGHISTK KIQVATKYAS LKLPNQIPGE YITTIKAIFD GNYGESDIHF REHQSELIIK QTVHPIPDVA FA DGGKTLR ACAANVDQIS FLEPINLKFL QGESPFSITF SVYHESTSRT DQYTIDNIDS ENFSFEKLYE GMKLGNHAIT IDS VVDANG CVNSLISGPR NQILVSITDA PKIHILDPST EYCVGDYVAY QLNGVAPFMI KYEFNGIPLK SKERSSQFVR LASE PGIIS ITSLQDSSSQ CIVDFTNPKL KSEFDDLSLN IHPIPSVTVS QGNYVTEDIR EGDQAEVIFS FEGTPPFSLT YVRTE ETDG KHGKRRSQVV ETHKVTDIYS HEYKVITSLQ GTYEAIEITD AYCFAKNDLF FNN

UniProtKB: Nucleoporin POM152

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Macromolecule #2: Nucleoporin POM34

MacromoleculeName: Nucleoporin POM34 / type: protein_or_peptide / ID: 2 / Details: Pore membrane protein and inner ring anchor / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: 15 trp1-1 leu2-3
Molecular weightTheoretical: 34.279301 KDa
SequenceString: MKIQAGQLGL DDNDVPGPLP DTDSKPSSQS QNDTPMFKLG NFESPVLKEL SRRTVNKEME TQRIMTNVIA FAFWNLLVKF IKFFWNNTH VGRQFCNRLS RIHLYMLTFH TLKKANIIYH TTFSWLNAEL LDYLFHLLIS LNILFSLWKL LSTVKVSDLN L TDRQKKLL ...String:
MKIQAGQLGL DDNDVPGPLP DTDSKPSSQS QNDTPMFKLG NFESPVLKEL SRRTVNKEME TQRIMTNVIA FAFWNLLVKF IKFFWNNTH VGRQFCNRLS RIHLYMLTFH TLKKANIIYH TTFSWLNAEL LDYLFHLLIS LNILFSLWKL LSTVKVSDLN L TDRQKKLL GVDMQSSVDT GLQPQHPHYV STSKISQMAQ NKTHIPQTNL KNHPAYLFKG LETPLKARQR EMAEEQTKLQ SQ SLHTKNV FGTLQRHSGI SSTLVSANND NNSPHTPVTR KGYIPSSKYA YMMNSQSPRG KI

UniProtKB: Nucleoporin POM34

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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

Concentration0.1 mg/mL
BufferpH: 7.5
Details: 20mM HEPES,50mM Potassium acetate,20mM NaCl,2mM MgCl2,1mM DTT
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 5 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK III
DetailsOne step affinity purified

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 37651 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsPreliminary grid screening done manually with individual images of low magnification montages of candidate meshes.
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 - Frames/image: 2-40 / Number grids imaged: 1 / Number real images: 4015 / Average electron dose: 40.0 e/Å2 / Details: 3218 images retained after triage
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 26049
Startup modelType of model: OTHER
Details: C8 symmetrized NPC map segmented in CHIMERA with Segger to provide initial 3D map of the inner ring, which was low pass filtered for the first reference.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7)
Details: C1 symmetry used during refinements with a C2 enforced 3D reference volume
Final 3D classificationSoftware - Name: RELION (ver. 3.0.7)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7)
Details: C1 symmetry used with a suitable mask to focus on the Pom34-Pom152 membrane anchor complex for the inner ring after isolation of the spoke by multibody processing.
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.7) / Details: local resolution for the TMDs 10.5-12 Angstroms / Number images used: 208392
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: none
DetailsRigid body docking and manual building with Chimera and coot
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation
Output model

PDB-8t9l:
Pom34-Pom152 membrane attachment site yeast NPC

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