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- EMDB-7321: Integrative Structure and Functional Anatomy of a Nuclear Pore Complex -

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Entry
Database: EMDB / ID: EMD-7321
TitleIntegrative Structure and Functional Anatomy of a Nuclear Pore Complex
Map dataIntegrative Structure and Functional Anatomy of a Nuclear Pore Complex
Sample
  • Complex: Saccharomyces cerevisiae NPC (nuclear pore complex)
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsubtomogram averaging / cryo EM / Resolution: 28.0 Å
AuthorsKim SJ / Fernandez-Martinez J / Nudelman I / Shi Y / Zhang W / Ludtke SJ / Akey CW / Chait BT / Sali A / Rout MP
Funding support United States, 12 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM083960 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM103314 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)U54 DK107981 United States
National Science Foundation (NSF, United States)GRF 1650113 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM080477 United States
National Science Foundation (NSF, United States)CHE-1531823 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)U54 GM103511 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)P50 GM076547 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM080139 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM063834 United States
CitationJournal: Nature / Year: 2018
Title: Integrative structure and functional anatomy of a nuclear pore complex.
Authors: Seung Joong Kim / Javier Fernandez-Martinez / Ilona Nudelman / Yi Shi / Wenzhu Zhang / Barak Raveh / Thurston Herricks / Brian D Slaughter / Joanna A Hogan / Paula Upla / Ilan E Chemmama / ...Authors: Seung Joong Kim / Javier Fernandez-Martinez / Ilona Nudelman / Yi Shi / Wenzhu Zhang / Barak Raveh / Thurston Herricks / Brian D Slaughter / Joanna A Hogan / Paula Upla / Ilan E Chemmama / Riccardo Pellarin / Ignacia Echeverria / Manjunatha Shivaraju / Azraa S Chaudhury / Junjie Wang / Rosemary Williams / Jay R Unruh / Charles H Greenberg / Erica Y Jacobs / Zhiheng Yu / M Jason de la Cruz / Roxana Mironska / David L Stokes / John D Aitchison / Martin F Jarrold / Jennifer L Gerton / Steven J Ludtke / Christopher W Akey / Brian T Chait / Andrej Sali / Michael P Rout /
Abstract: Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full ...Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.
History
DepositionDec 27, 2017-
Header (metadata) releaseFeb 14, 2018-
Map releaseMar 28, 2018-
UpdateAug 12, 2020-
Current statusAug 12, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7321.png

Downloads & links

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Map

FileDownload / File: emd_7321.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIntegrative Structure and Functional Anatomy of a Nuclear Pore Complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.3 Å/pix.
x 300 pix.
= 1590. Å		5.3 Å/pix.
x 300 pix.
= 1590. Å		5.3 Å/pix.
x 300 pix.
= 1590. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.059866104 - 0.1641967
Average (Standard dev.)0.002676172 (±0.011975752)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 1590.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.35.35.3
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z1590.0001590.0001590.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-163-114-126
NX/NY/NZ210124170
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0600.1640.003

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Supplemental data

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Sample components

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Entire : Saccharomyces cerevisiae NPC (nuclear pore complex)

EntireName: Saccharomyces cerevisiae NPC (nuclear pore complex)
Components
  • Complex: Saccharomyces cerevisiae NPC (nuclear pore complex)

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Supramolecule #1: Saccharomyces cerevisiae NPC (nuclear pore complex)

SupramoleculeName: Saccharomyces cerevisiae NPC (nuclear pore complex) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Affinity-purified isolated whole NPCs
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightExperimental: 87 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMHEPES-KOH
50.0 mMpotassium acetateKC2H3O2
2.0 mMmagnesium chlorideMgCl2
20.0 mMsodium chlorideNaCl
0.1 w/vTween-20
10.0 w/vglycerol
1.0 mMdithiothreitolDTT
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300
Details: A medium thick carbon film was used to support the NPCs over the holes. Before use, the grids were glow discharged in air, floated on 5 uL sample drops for 45 minutes and then washed by ...Details: A medium thick carbon film was used to support the NPCs over the holes. Before use, the grids were glow discharged in air, floated on 5 uL sample drops for 45 minutes and then washed by serial transfer on 4 x 20 micro-litre drops of sample buffer without glycerol.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK III
Details: Buffer on the grid was removed by blotting from the bottom with a tool that held a filter paper wedge, using access through the left-hand port. Then 2 micro-litre of freezing buffer was ...Details: Buffer on the grid was removed by blotting from the bottom with a tool that held a filter paper wedge, using access through the left-hand port. Then 2 micro-litre of freezing buffer was added to the grid from the right-hand port and the grid was plunge frozen in liquid ethane after blotting..
DetailsSample isolated in one affinity step - pullout from frozen yeast cell grindate.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: Titan-Krios equipped with a spherical aberration (Cs) corrector
Chromatic aberration corrector: none / Energy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 1 eV / Energy filter - Upper energy threshold: 20 eV
DetailsThe electron gun was an XFEG. A total of 253 tilt series were collected in steps between -60, 0 and 60 degrees in increments of 2.5 - 4 degrees for different tilt series. While the full tilt range was used for tomogram reconstruction, in the final subtomogram averaging step only data up to +/-45 degrees tilt from each sub-volume were included in the final average. A defocus range of -4.6 to -7.5 microns was used.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 1 / Average electron dose: 3.0 e/Å2
Details: The dose target for each tilt series was 90-100 electrons/angstrom squared and followed a cosine alpha dose curve with a flux of 20 electrons/pixel/second, and a dose of 3.5 ...Details: The dose target for each tilt series was 90-100 electrons/angstrom squared and followed a cosine alpha dose curve with a flux of 20 electrons/pixel/second, and a dose of 3.5 electrons/angstrom squared for the zero tilt image.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 9434 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.001 mm / Nominal defocus max: 7.5 µm / Nominal defocus min: 4.6 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C8 (8 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 28.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 (ver. 2.1)
Details: The final map has been fully CTF corrected and filtered based on the estimated local resolution.
Number subtomograms used: 1864
ExtractionNumber tomograms: 121 / Number images used: 6416 / Method: manual selection / Software - Name: EMAN2 (ver. 2.12) / Software - details: e2spt_boxer.py
Details: A low pass filter of 100 Angstroms was applied to binned 3X SIRT tomograms prior to particle picking. Final unbinned sub-volumes of 300 x 300 x 300 were extracted from the original tomograms.
CTF correctionSoftware - Name: EMAN2 (ver. 2.1)
Details: Phase flipping of tilted images with etomo in IMOD running in batchtomo mode. The final reconstruction used 1,864 (of the 6,416 initial) particles. Theoretical CTF curves for the mean ...Details: Phase flipping of tilted images with etomo in IMOD running in batchtomo mode. The final reconstruction used 1,864 (of the 6,416 initial) particles. Theoretical CTF curves for the mean defocus values present in the tomograms were averaged assuming 10% amplitude contrast. The reciprocal of this curve was then applied as a filter to the final uncorrected map.
Final angle assignmentType: OTHER / Software - Name: EMAN2
Software - details: e2spt_classaverage.py in theEMAN2 single particle tomography package
Details: Iterative 3D alignment of sub-volumes to a reference volume, using the C8 symmetry of the NPC.

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Atomic model buiding 1

DetailsThe integrative structure modeling protocol was scripted using the Python Modeling Interface (PMI) package, version 4d97507, a library for modeling macromolecular complexes based on our open-source Integrative Modeling Platform (IMP) package, version 2.6 (https://integrativemodeling.org)
RefinementProtocol: OTHER

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