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- PDB-7k59: Structure of apo VCP hexamer generated from bacterially recombina... -

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Basic information

Entry
Database: PDB / ID: 7k59
TitleStructure of apo VCP hexamer generated from bacterially recombinant VCP/p97
ComponentsTransitional endoplasmic reticulum ATPase
KeywordsCYTOSOLIC PROTEIN / HYDROLASE / AAA ATPase / ATP hydrolysis / segregase
Function / homology
Function and homology information


: / 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 ...: / 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 / endoplasmic reticulum unfolded protein response / Protein methylation / proteasomal protein catabolic process / Attachment and Entry / ERAD pathway / lipid droplet / proteasome complex / viral genome replication / Josephin domain DUBs / negative regulation of smoothened signaling pathway / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / 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 / Ovarian tumor domain proteases / KEAP1-NFE2L2 pathway / 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 / cellular response to heat / site of double-strand break / 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 / protein domain specific binding / DNA repair / intracellular membrane-bounded organelle / DNA damage response / ubiquitin protein ligase binding / lipid binding / Neutrophil degranulation / endoplasmic reticulum membrane / perinuclear region of cytoplasm / glutamatergic synapse / endoplasmic reticulum / protein-containing complex / ATP hydrolysis activity / RNA 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 / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsYu, G. / Bai, Y. / Li, K. / Jiang, W. / Zhang, Z.Y.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)RO1 CA69202 United States
CitationJournal: iScience / Year: 2021
Title: Cryo-electron microscopy structures of VCP/p97 reveal a new mechanism of oligomerization regulation.
Authors: Guimei Yu / Yunpeng Bai / Kunpeng Li / Ovini Amarasinghe / Wen Jiang / Zhong-Yin Zhang /
Abstract: VCP/p97 is an evolutionarily conserved AAA+ ATPase important for cellular homeostasis. Previous studies suggest that VCP predominantly exists as a homohexamer. Here, we performed structural and ...VCP/p97 is an evolutionarily conserved AAA+ ATPase important for cellular homeostasis. Previous studies suggest that VCP predominantly exists as a homohexamer. Here, we performed structural and biochemical characterization of VCP dodecamer, an understudied state of VCP. The structure revealed an apo nucleotide status that has rarely been captured, a tail-to-tail assembly of two hexamers, and the up-elevated N-terminal domains akin to that seen in the ATP-bound hexamer. Further analyses elucidated a nucleotide status-dependent dodecamerization mechanism, where nucleotide dissociation from the D2 AAA domains induces and promotes VCP dodecamerization. In contrast, nucleotide-free D1 AAA domains are associated with the up-rotation of N-terminal domains, which may prime D1 for ATP binding. These results therefore reveal new nucleotide status-dictated intra- and interhexamer conformational changes and suggest that modulation of D2 domain nucleotide occupancy may serve as a mechanism in controlling VCP oligomeric states.
History
DepositionSep 16, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 13, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 6, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: Transitional endoplasmic reticulum ATPase
B: Transitional endoplasmic reticulum ATPase
C: Transitional endoplasmic reticulum ATPase
D: Transitional endoplasmic reticulum ATPase
E: Transitional endoplasmic reticulum ATPase
F: Transitional endoplasmic reticulum ATPase


Theoretical massNumber of molelcules
Total (without water)536,7896
Polymers536,7896
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Transitional endoplasmic reticulum ATPase / TER ATPase / 15S Mg(2+)-ATPase p97 subunit / Valosin-containing protein / VCP


Mass: 89464.828 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VCP / Plasmid: PET28a / Details (production host): PET28a-VCP / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P55072, vesicle-fusing ATPase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: VCP/p97 / Type: COMPLEX
Details: VCP/p97 was overexpressed and purified from E.coli BL21(DE3). Purified protein was treated with apyrase to remove prebound ADP and generate apo VCP/p97.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.54 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3) / Plasmid: PET28a
Buffer solutionpH: 7.4 / Details: 20mM Hepes pH7.4, 150mM NaCl, 1mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)Hepes1
2150 mMSodium chlorideNaCl1
31 mMtris(2-carboxyethyl)phosphine)TCEP1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: VCP/p97 was overexpressed and purified with E.coli BL21(DE3). Purified proteins were treated with apyrase to remove prebound ADP and generate apo VCP/p97.
Specimen supportGrid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 70 % / Chamber temperature: 293 K
Details: 3.5ul sample was applied to a lacy carbon grid coated with graphene oxide. 7 seconds blot with filter paper was performed using Gatan Cp3.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 807
Details: A frame rate of 5 frames per second, a dose rate of 7.6 eps and a total exposure of 8 seconds were used.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
1Gautomatchparticle selection
2Leginonimage acquisition
4GctfCTF correction
5cryoSPARCCTF correction
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
14cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 71600
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 22172 / Num. of class averages: 1 / Symmetry type: POINT

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