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- EMDB-3104: The human Nuclear Pore Complex with nucleoporin 358 knockdown -

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Basic information

Entry
Database: EMDB / ID: EMD-3104
TitleThe human Nuclear Pore Complex with nucleoporin 358 knockdown
Map dataReconstruction of human nuclear pore complex after Nup358 knockdown
Sample
  • Sample: Human nuclear pore complex with nucleoporin 358 knockdown
  • Protein or peptide: nuclear pore complexNuclear pore
Keywordsnuclear pore complex / Nup358 knockdown
Function / homology
Function and homology information


GATOR2 complex / nephron development / nuclear pore inner ring / Seh1-associated complex / protein localization to nuclear inner membrane / protein exit from endoplasmic reticulum / COPII-coated vesicle budding / nuclear envelope organization / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / telomere tethering at nuclear periphery ...GATOR2 complex / nephron development / nuclear pore inner ring / Seh1-associated complex / protein localization to nuclear inner membrane / protein exit from endoplasmic reticulum / COPII-coated vesicle budding / nuclear envelope organization / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / telomere tethering at nuclear periphery / COPII-coated vesicle cargo loading / nuclear pore organization / nuclear pore complex assembly / nuclear pore outer ring / atrial cardiac muscle cell action potential / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore cytoplasmic filaments / COPII vesicle coat / somite development / Nuclear Pore Complex (NPC) Disassembly / nuclear inclusion body / nuclear pore nuclear basket / paraxial mesoderm development / Transport of Ribonucleoproteins into the Host Nucleus / Regulation of Glucokinase by Glucokinase Regulatory Protein / Defective TPR may confer susceptibility towards thyroid papillary carcinoma (TPC) / Amino acids regulate mTORC1 / miRNA processing / attachment of mitotic spindle microtubules to kinetochore / Transport of the SLBP independent Mature mRNA / Transport of the SLBP Dependant Mature mRNA / NS1 Mediated Effects on Host Pathways / SUMOylation of SUMOylation proteins / Transport of Mature mRNA Derived from an Intronless Transcript / protein-containing complex localization / RNA export from nucleus / positive regulation of mRNA splicing, via spliceosome / structural constituent of nuclear pore / Rev-mediated nuclear export of HIV RNA / Nuclear import of Rev protein / SUMOylation of RNA binding proteins / Transport of Mature mRNA derived from an Intron-Containing Transcript / tRNA processing in the nucleus / NEP/NS2 Interacts with the Cellular Export Machinery / COPII-mediated vesicle transport / Postmitotic nuclear pore complex (NPC) reformation / neural tube development / nucleocytoplasmic transport / poly(A)+ mRNA export from nucleus / lamellipodium assembly / Viral Messenger RNA Synthesis / nuclear localization sequence binding / mitotic metaphase chromosome alignment / female gonad development / macrophage chemotaxis / SUMOylation of ubiquitinylation proteins / Vpr-mediated nuclear import of PICs / cellular response to nutrient levels / SUMOylation of DNA replication proteins / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / positive regulation of TOR signaling / Regulation of HSF1-mediated heat shock response / mRNA transport / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / SUMOylation of DNA damage response and repair proteins / mRNA export from nucleus / nuclear pore / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / negative regulation of TORC1 signaling / positive regulation of TORC1 signaling / Resolution of Sister Chromatid Cohesion / cellular response to amino acid starvation / MHC class II antigen presentation / neurogenesis / nuclear periphery / serine-type peptidase activity / SUMOylation of chromatin organization proteins / HCMV Late Events / chromosome segregation / RHO GTPases Activate Formins / promoter-specific chromatin binding / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / Transcriptional regulation by small RNAs / intracellular protein transport / ER to Golgi transport vesicle membrane / spindle / kinetochore / ISG15 antiviral mechanism / HCMV Early Events / protein import into nucleus / Separation of Sister Chromatids / protein transport / nuclear envelope / snRNP Assembly / nuclear membrane / transcription coactivator activity / nuclear body / nuclear speck / defense response to Gram-positive bacterium
Similarity search - Function
Nucleoporin Nup120/160 / Nup98, Gle2-binding sequence / Nucleoporin, Nup155-like, C-terminal, subdomain 3 / Nucleoporin Nup37 / 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 Nup120/160 / Nup98, Gle2-binding sequence / Nucleoporin, Nup155-like, C-terminal, subdomain 3 / Nucleoporin Nup37 / 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, 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 / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / 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
Nuclear pore complex protein Nup155 / Nuclear pore complex protein Nup98-Nup96 / Protein SEC13 homolog / Nuclear pore complex protein Nup107 / Nuclear pore complex protein Nup160 / Nucleoporin Nup43 / Nucleoporin Nup37 / Nuclear pore complex protein Nup133 / Nucleoporin SEH1 / Nuclear pore complex protein Nup85
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 37.5 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: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3104.map.gz / Format: CCP4 / Size: 17.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of human nuclear pore complex after Nup358 knockdown
Voxel sizeX=Y=Z: 8.6 Å
Density
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-11.19243908 - 19.80205917
Average (Standard dev.)-0.00104026 (±1.6228081)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions168168168
Spacing168168168
CellA=B=C: 1444.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z8.68.68.6
M x/y/z168168168
origin x/y/z0.0000.0000.000
length x/y/z1444.8001444.8001444.800
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/NS168168168
D min/max/mean-11.19219.802-0.001

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Supplemental data

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Sample components

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Entire : Human nuclear pore complex with nucleoporin 358 knockdown

EntireName: Human nuclear pore complex with nucleoporin 358 knockdown
Components
  • Sample: Human nuclear pore complex with nucleoporin 358 knockdown
  • Protein or peptide: nuclear pore complexNuclear pore

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Supramolecule #1000: Human nuclear pore complex with nucleoporin 358 knockdown

SupramoleculeName: Human nuclear pore complex with nucleoporin 358 knockdown
type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 120 MDa / Theoretical: 120 MDa

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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: 34883 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 19500
Specialist opticsEnergy filter - Name: GIF2002 / 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: -44 ° / Tilt series - Axis1 - Max angle: 60 °
DateSep 20, 2014
Image recordingCategory: CCD / Film or detector model: GATAN MULTISCAN / Average electron dose: 100 e/Å2 / Bits/pixel: 16
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: OTHER / Resolution.type: BY AUTHOR / Resolution: 37.5 Å / Resolution method: OTHER / Software - Name: IMOD, TOM, AV3 / Number subtomograms used: 7360
DetailsThe subtomograms were selected manually and further processed by iterative, symmetry-independent averaging.

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