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- EMDB-3107: segment of the nuclear ring of the human nuclear pore complex -

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Basic information

Entry
Database: EMDB / ID: EMD-3107
Titlesegment of the nuclear ring of the human nuclear pore complex
Map datasegment of nuclear ring of the human nuclear pore complex
Sample
  • Sample: Human Nuclear Pore ComplexNuclear pore
  • Protein or peptide: Nuclear Pore ComplexNuclear pore
Keywordsnuclear pore complex / nuclear ring
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 24.1 Å
Authorsvon Appen A / Kosinski J / Sparks L / Ori A / DiGuilio A / Vollmer B / Mackmull M / Banterle N / Parca L / Kastritis P ...von Appen A / Kosinski J / Sparks L / Ori A / DiGuilio A / Vollmer B / Mackmull M / Banterle N / Parca L / Kastritis P / Buczak K / Mosalaganti S / Hagen W / Andres-Pons A / Lemke EA / Bork P / Antonin W / Glavy JS / Bui KH / Beck M
CitationJournal: Nature / Year: 2015
Title: In situ structural analysis of the human nuclear pore complex.
Authors: Alexander von Appen / Jan Kosinski / Lenore Sparks / Alessandro Ori / Amanda L DiGuilio / Benjamin Vollmer / Marie-Therese Mackmull / Niccolo Banterle / Luca Parca / Panagiotis Kastritis / ...Authors: Alexander von Appen / Jan Kosinski / Lenore Sparks / Alessandro Ori / Amanda L DiGuilio / Benjamin Vollmer / Marie-Therese Mackmull / Niccolo Banterle / Luca Parca / Panagiotis Kastritis / Katarzyna Buczak / Shyamal Mosalaganti / Wim Hagen / Amparo Andres-Pons / Edward A Lemke / Peer Bork / Wolfram Antonin / Joseph S Glavy / Khanh Huy Bui / Martin Beck /
Abstract: Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and ...Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations.
History
DepositionJul 21, 2015-
Header (metadata) releaseAug 12, 2015-
Map releaseSep 30, 2015-
UpdateOct 7, 2015-
Current statusOct 7, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 4.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3107.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsegment of nuclear ring of the human nuclear pore complex
Voxel sizeX=Y=Z: 6.84 Å
Density
Contour LevelBy AUTHOR: 4.5 / Movie #1: 4.5
Minimum - Maximum-7.90856171 - 21.457025529999999
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions144144144
Spacing144144144
CellA=B=C: 984.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.846.846.84
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z984.960984.960984.960
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-7.90921.457-0.000

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Supplemental data

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Sample components

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Entire : Human Nuclear Pore Complex

EntireName: Human Nuclear Pore ComplexNuclear pore
Components
  • Sample: Human Nuclear Pore ComplexNuclear pore
  • Protein or peptide: Nuclear Pore ComplexNuclear pore

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Supramolecule #1000: Human Nuclear Pore Complex

SupramoleculeName: Human Nuclear Pore Complex / type: sample / ID: 1000
Details: The sample is purified human nuclear envelope containing nuclear pore complex.
Number unique components: 1
Molecular weightMethod: Absolute Quantitative Mass Spectrometry

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Macromolecule #1: Nuclear Pore Complex

MacromoleculeName: Nuclear Pore Complex / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: Nucleus / Location in cell: Nuclear envelope

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: 20mM Tris, 0.2-0.4% Trehalose
GridDetails: 200 mesh Quantifoil Copper Holey Carbon Grid R2/1
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 42000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 42000
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -45 ° / Tilt series - Axis1 - Max angle: 60 °
DateOct 17, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 110 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flipping of tilt series
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.1 Å / Resolution method: OTHER / Software - Name: IMOD, TOM, AV3 / Number subtomograms used: 17368
DetailsThe subtomograms were picked manually and further processed iterative symmetry independent averaging.
FSC plot (resolution estimation)

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