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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 data
SampleFull-length human p97 bound to ATPgS
  • p97/VCP Transitional endoplasmic reticulum ATPase
  • ligand
Keywordscryo-electron microscopy / single-particle / p97 / AAA ATPase
Function / homology
Function and homology information


protein binding / flavin adenine dinucleotide catabolic process / positive regulation of Lys63-specific deubiquitinase activity / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / ATPase complex / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / deubiquitinase activator activity / endosome to lysosome transport via multivesicular body sorting pathway ...protein binding / flavin adenine dinucleotide catabolic process / positive regulation of Lys63-specific deubiquitinase activity / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / ATPase complex / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / deubiquitinase activator activity / endosome to lysosome transport via multivesicular body sorting pathway / cellular response to arsenite ion / endoplasmic reticulum stress-induced pre-emptive quality control / BAT3 complex binding / Derlin-1 retrotranslocation complex / ERAD pathway / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / NADH metabolic process / aggresome assembly / regulation of protein localization to chromatin / vesicle-fusing ATPase / ER-associated misfolded protein catabolic process / K48-linked polyubiquitin modification-dependent protein binding / stress granule disassembly / positive regulation of mitochondrial membrane potential / retrograde protein transport, ER to cytosol / autophagosome maturation / regulation of aerobic respiration / MHC class I protein binding / regulation of synapse organization / positive regulation of ATP biosynthetic process / negative regulation of smoothened signaling pathway / ubiquitin-specific protease binding / ubiquitin-like protein ligase binding / ATP metabolic process / RHOH GTPase cycle / Attachment and Entry / polyubiquitin modification-dependent protein binding / proteasomal protein catabolic process / endoplasmic reticulum to Golgi vesicle-mediated transport / Protein methylation / translesion synthesis / HSF1 activation / proteasome complex / interstrand cross-link repair / endoplasmic reticulum unfolded protein response / lipid droplet / ubiquitin-dependent ERAD pathway / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / macroautophagy / ADP binding / Hh mutants are degraded by ERAD / Defective CFTR causes cystic fibrosis / Hedgehog ligand biogenesis / Translesion Synthesis by POLH / ABC-family proteins mediated transport / positive regulation of protein catabolic process / positive regulation of protein-containing complex assembly / double-strand break repair / establishment of protein localization / Aggrephagy / cytoplasmic stress granule / autophagy / positive regulation of canonical Wnt signaling pathway / azurophil granule lumen / activation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / site of double-strand break / viral genome replication / Ovarian tumor domain proteases / Attachment and Entry / E3 ubiquitin ligases ubiquitinate target proteins / proteasome-mediated ubiquitin-dependent protein catabolic process / cellular response to heat / secretory granule lumen / protein phosphatase binding / regulation of apoptotic process / protein ubiquitination / ficolin-1-rich granule lumen / lipid binding / : / glutamatergic synapse / DNA repair / signaling receptor binding / protein domain specific binding / intracellular membrane-bounded organelle / cellular response to DNA damage stimulus / ubiquitin protein ligase binding / Neutrophil degranulation / endoplasmic reticulum membrane / perinuclear region of cytoplasm / endoplasmic reticulum / protein-containing complex / RNA binding / extracellular exosome / extracellular region / nucleoplasm / ATP binding / identical protein binding
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 / Vps4 C terminal oligomerisation domain / Vps4 oligomerisation, C-terminal ...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 / Vps4 C terminal oligomerisation domain / Vps4 oligomerisation, C-terminal / Aspartate decarboxylase-like domain superfamily / AAA+ lid domain / AAA ATPase, AAA+ lid domain / AAA-protein family signature. / ATPase, AAA-type, conserved site / 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

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)
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
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 / Details: The sample was monodisperse. / Number of Components: 2 / Oligomeric State: hexamer
MassTheoretical: 540 kDa / Measured by: sequence

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Component #1: protein, p97/VCP Transitional endoplasmic reticulum ATPase

ProteinName: p97/VCP Transitional endoplasmic reticulum ATPase / a.k.a: p97 / Oligomeric Details: hexamer / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 540 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Bacteria (eubacteria) / Vector: Rosetta2 (DE3) / Cell of expression system: E. coli
Source (natural)Location in cell: cytoplasm
External referencesGene Ontology: ATP binding, signaling receptor binding, protein binding, lipid binding
UniProt: Transitional endoplasmic reticulum ATPase

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Component #2: ligand, Adenosine 5 gamma-thio triphosphate

LigandName: Adenosine 5 gamma-thio triphosphate / a.k.a: ATPgS / Oligomeric Details: monomer / Recombinant expression: No / Number of Copies: 12
SourceSpecies: unidentified (others)

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.9 mg/mL
Buffer solution: 25 mM Tris, 150 mM NaCl, 1 mM MgCl2, 1.0 mM TCEP
pH: 8
Support film200 mesh Quantifoil R1.2/1/3 grids
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen Name: ETHANE / Temperature: 90.15 K / Humidity: 100 % / Method: Blot for 2.5 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Jan 17, 2015 / Details: Parallel beam illumination
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 40 e/Å2 / Illumination Mode: FLOOD BEAM
LensMagnification: 105000 X (nominal), 36980 X (calibrated) / Cs: 2.7 mm / Imaging Mode: BRIGHT FIELD / Defocus: 950 - 2700 nm / Energy Filter: Gatan, Inc. / Energy Window: 0-20 eV
Specimen HolderHolder: Liquid nitrogen cooled / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 79.7 (79.6 - 79.8 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of Digital Images: 628
Details: Every image is the average of 40 frames recorded by the direct electron detector.

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Image processing

ProcessingMethod: single particle reconstruction / Applied Symmetry: C6 (6 fold cyclic) / Number of Projections: 33882
3D reconstructionAlgorithm: score minimization / Software: FREALIGN / CTF correction: each particle / Details: Map was corrected using a B-factor of -85 A^2. / Resolution: 3.3 Å / Resolution Method: FSC 0.143, semi-independent

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Atomic model buiding

Output model

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