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- EMDB-3297: Cryo-EM structure of human p97 bound to ATPgS (Conformation I) -

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Basic information

Entry
Database: EMDB / ID: EMD-3297
TitleCryo-EM structure of human p97 bound to ATPgS (Conformation I)
Map dataReconstruction of human p97 bound to ATPgS (conformation I).
Sample
  • Sample: Full-length human p97 bound to ATPgS
  • Protein or peptide: p97/VCP Transitional endoplasmic reticulum ATPase
  • Ligand: Adenosine 5 gamma-thio triphosphate
Keywordscryo-electron microscopy / single-particle / p97 / AAA ATPase
Function / homology
Function and homology information


protein binding / : / flavin adenine dinucleotide catabolic process / VCP-NSFL1C complex / endosome to lysosome transport via multivesicular body sorting pathway / endoplasmic reticulum stress-induced pre-emptive quality control / BAT3 complex binding / cellular response to arsenite ion / protein-DNA covalent cross-linking repair / Derlin-1 retrotranslocation complex ...protein binding / : / flavin adenine dinucleotide catabolic process / VCP-NSFL1C complex / endosome to lysosome transport via multivesicular body sorting pathway / endoplasmic reticulum stress-induced pre-emptive quality control / BAT3 complex binding / cellular response to arsenite ion / protein-DNA covalent cross-linking repair / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / cytoplasm protein quality control / positive regulation of oxidative phosphorylation / : / aggresome assembly / deubiquitinase activator activity / mitotic spindle disassembly / ubiquitin-modified protein reader activity / regulation of protein localization to chromatin / VCP-NPL4-UFD1 AAA ATPase complex / cellular response to misfolded protein / negative regulation of protein localization to chromatin / positive regulation of mitochondrial membrane potential / vesicle-fusing ATPase / K48-linked polyubiquitin modification-dependent protein binding / regulation of aerobic respiration / retrograde protein transport, ER to cytosol / stress granule disassembly / ATPase complex / regulation of synapse organization / ubiquitin-specific protease binding / positive regulation of ATP biosynthetic process / MHC class I protein binding / ubiquitin-like protein ligase binding / RHOH GTPase cycle / polyubiquitin modification-dependent protein binding / autophagosome maturation / negative regulation of hippo signaling / endoplasmic reticulum to Golgi vesicle-mediated transport / HSF1 activation / translesion synthesis / interstrand cross-link repair / ATP metabolic process / proteasomal protein catabolic process / endoplasmic reticulum unfolded protein response / Protein methylation / Attachment and Entry / ERAD pathway / lipid droplet / proteasome complex / viral genome replication / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / negative regulation of smoothened signaling pathway / macroautophagy / positive regulation of protein-containing complex assembly / Hh mutants are degraded by ERAD / establishment of protein localization / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / positive regulation of non-canonical NF-kappaB signal transduction / Translesion Synthesis by POLH / ADP binding / ABC-family proteins mediated transport / autophagy / cytoplasmic stress granule / Aggrephagy / positive regulation of protein catabolic process / azurophil granule lumen / KEAP1-NFE2L2 pathway / Ovarian tumor domain proteases / positive regulation of canonical Wnt signaling pathway / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / double-strand break repair / E3 ubiquitin ligases ubiquitinate target proteins / site of double-strand break / cellular response to heat / Neddylation / ubiquitin-dependent protein catabolic process / secretory granule lumen / protein phosphatase binding / regulation of apoptotic process / ficolin-1-rich granule lumen / proteasome-mediated ubiquitin-dependent protein catabolic process / Attachment and Entry / protein ubiquitination / ciliary basal body / signaling receptor binding / protein domain specific binding / DNA repair / intracellular membrane-bounded organelle / DNA damage response / lipid binding / ubiquitin protein ligase binding / Neutrophil degranulation / endoplasmic reticulum membrane / perinuclear region of cytoplasm / glutamatergic synapse / endoplasmic reticulum / protein-containing complex
Similarity search - Function
AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / : / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / Aspartate decarboxylase-like domain superfamily ...AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / : / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / Aspartate decarboxylase-like domain superfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Transitional endoplasmic reticulum ATPase
Similarity search - Component
Biological speciesHomo sapiens (human) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsBanerjee S / Bartesaghi A / Merk A / Rao P / Bulfer SL / Yan Y / Green N / Mroczkowski B / Neitz RJ / Wipf P ...Banerjee S / Bartesaghi A / Merk A / Rao P / Bulfer SL / Yan Y / Green N / Mroczkowski B / Neitz RJ / Wipf P / Falconieri V / Deshaies RJ / Milne JLS / Huryn D / Arkin M / Subramaniam S
CitationJournal: Science / Year: 2016
Title: 2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition.
Authors: Soojay Banerjee / Alberto Bartesaghi / Alan Merk / Prashant Rao / Stacie L Bulfer / Yongzhao Yan / Neal Green / Barbara Mroczkowski / R Jeffrey Neitz / Peter Wipf / Veronica Falconieri / ...Authors: Soojay Banerjee / Alberto Bartesaghi / Alan Merk / Prashant Rao / Stacie L Bulfer / Yongzhao Yan / Neal Green / Barbara Mroczkowski / R Jeffrey Neitz / Peter Wipf / Veronica Falconieri / Raymond J Deshaies / Jacqueline L S Milne / Donna Huryn / Michelle Arkin / Sriram Subramaniam /
Abstract: p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo-electron microscopy (cryo-EM) structures for adenosine diphosphate ...p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo-electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)-bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5'-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.
History
DepositionJan 12, 2016-
Header (metadata) releaseJan 27, 2016-
Map releaseJan 27, 2016-
UpdateMar 9, 2016-
Current statusMar 9, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5ftl
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3297.png

Downloads & links

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Map

FileDownload / File: emd_3297.map.gz / Format: CCP4 / Size: 78.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of human p97 bound to ATPgS (conformation I).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.68 Å/pix.
x 276 pix.
= 186.576 Å		0.68 Å/pix.
x 276 pix.
= 186.576 Å		0.68 Å/pix.
x 276 pix.
= 186.576 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.676 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0179 / Movie #1: 0.016
Minimum - Maximum-0.03242269 - 0.05889366
Average (Standard dev.)0.00027416 (±0.00494921)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions276276276
Spacing276276276
CellA=B=C: 186.576 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6760.6760.676
M x/y/z276276276
origin x/y/z0.0000.0000.000
length x/y/z186.576186.576186.576
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS276276276
D min/max/mean-0.0320.0590.000

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Supplemental data

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Sample components

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Entire : Full-length human p97 bound to ATPgS

EntireName: Full-length human p97 bound to ATPgS
Components
  • Sample: Full-length human p97 bound to ATPgS
  • Protein or peptide: p97/VCP Transitional endoplasmic reticulum ATPase
  • Ligand: Adenosine 5 gamma-thio triphosphate

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Supramolecule #1000: Full-length human p97 bound to ATPgS

SupramoleculeName: Full-length human p97 bound to ATPgS / type: sample / ID: 1000 / Details: The sample was monodisperse. / Oligomeric state: hexamer / Number unique components: 2
Molecular weightTheoretical: 540 KDa / Method: sequence

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Macromolecule #1: p97/VCP Transitional endoplasmic reticulum ATPase

MacromoleculeName: p97/VCP Transitional endoplasmic reticulum ATPase / type: protein_or_peptide / ID: 1 / Name.synonym: p97 / Number of copies: 1 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: cytoplasm
Molecular weightTheoretical: 540 KDa
Recombinant expressionOrganism: Bacteria (eubacteria) / Recombinant cell: E. coli / Recombinant plasmid: Rosetta2 (DE3)
SequenceUniProtKB: Transitional endoplasmic reticulum ATPase
GO: ATP binding, signaling receptor binding, protein binding, lipid binding

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Macromolecule #2: Adenosine 5 gamma-thio triphosphate

MacromoleculeName: Adenosine 5 gamma-thio triphosphate / type: ligand / ID: 2 / Name.synonym: ATPgS / Number of copies: 12 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: unidentified (others)

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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

Concentration0.9 mg/mL
BufferpH: 8 / Details: 25 mM Tris, 150 mM NaCl, 1 mM MgCl2, 1.0 mM TCEP
GridDetails: 200 mesh Quantifoil R1.2/1/3 grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90.15 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 2.5 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 79.6 K / Max: 79.8 K / Average: 79.7 K
Specialist opticsEnergy filter - Name: Gatan, Inc. / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DetailsParallel beam illumination
DateJan 17, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 628 / Average electron dose: 40 e/Å2
Details: Every image is the average of 40 frames recorded by the direct electron detector.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 36980 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.95 µm / Nominal magnification: 105000
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: OTHER / Software - Name: FREALIGN / Details: Map was corrected using a B-factor of -85 A^2. / Number images used: 33882

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