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- EMDB-3298: Cryo-EM structure of human p97 bound to ATPgS (Conformation II) -

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Basic information

Entry
Database: EMDB / ID: EMD-3298
TitleCryo-EM structure of human p97 bound to ATPgS (Conformation II)
Map dataReconstruction of human p97 bound to ATPgS (conformation II).
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 / positive regulation of Lys63-specific deubiquitinase activity / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / 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 ...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 / 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 / positive regulation of oxidative phosphorylation / Derlin-1 retrotranslocation complex / BAT3 complex binding / mitotic spindle disassembly / ERAD pathway / VCP-NPL4-UFD1 AAA ATPase complex / NADH metabolic process / aggresome assembly / vesicle-fusing ATPase / regulation of protein localization to chromatin / ER-associated misfolded protein catabolic process / stress granule disassembly / K48-linked polyubiquitin modification-dependent protein binding / retrograde protein transport, ER to cytosol / regulation of aerobic respiration / positive regulation of mitochondrial membrane potential / ATPase complex / regulation of synapse organization / positive regulation of ATP biosynthetic process / ubiquitin-like protein ligase binding / ubiquitin-specific protease binding / negative regulation of smoothened signaling pathway / autophagosome maturation / ATP metabolic process / RHOH GTPase cycle / polyubiquitin modification-dependent protein binding / Attachment and Entry / endoplasmic reticulum to Golgi vesicle-mediated transport / Protein methylation / MHC class I protein binding / HSF1 activation / translesion synthesis / interstrand cross-link repair / endoplasmic reticulum unfolded protein response / lipid droplet / viral genome replication / ubiquitin-dependent ERAD pathway / Josephin domain DUBs / proteasome complex / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / macroautophagy / proteasomal protein catabolic process / ADP binding / Hh mutants are degraded by ERAD / Defective CFTR causes cystic fibrosis / Hedgehog ligand biogenesis / Translesion Synthesis by POLH / positive regulation of protein-containing complex assembly / ABC-family proteins mediated transport / positive regulation of protein catabolic process / double-strand break repair / establishment of protein localization / Aggrephagy / cytoplasmic stress granule / autophagy / azurophil granule lumen / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / activation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of canonical Wnt signaling pathway / site of double-strand break / Ovarian tumor domain proteases / E3 ubiquitin ligases ubiquitinate target proteins / proteasome-mediated ubiquitin-dependent protein catabolic process / cellular response to heat / Attachment and Entry / secretory granule lumen / protein phosphatase binding / protein ubiquitination / ficolin-1-rich granule lumen / regulation of apoptotic process / lipid binding / glutamatergic synapse / DNA repair / intracellular membrane-bounded organelle / protein domain specific binding / signaling receptor binding / cellular response to DNA damage stimulus / ubiquitin protein ligase binding / endoplasmic reticulum membrane / Neutrophil degranulation / ATP hydrolysis activity / 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 / Cell division protein 48 (CDC48) N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / 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 / Cell division protein 48 (CDC48) N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / CDC48, domain 2 / CDC48 domain 2-like superfamily / Vps4 C terminal oligomerisation domain / Vps4 oligomerisation, C-terminal / 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: 3.2 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-5ftm
  • 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_3298.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 II).
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: 0.0183 / Movie #1: 0.016
Minimum - Maximum-0.02500867 - 0.07105248
Average (Standard dev.)0.0003613 (±0.00482665)
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-0.0250.0710.000

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 36980 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.95 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: Gatan, Inc. / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 79.6 K / Max: 79.8 K / Average: 79.7 K
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.
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: 3.2 Å / Resolution method: OTHER / Software - Name: FREALIGN / Details: Map was corrected using a B-factor of -85 A^2. / Number images used: 32406

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