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- PDB-7k56: Structure of VCP dodecamer purified from H1299 cells -

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Basic information

Entry
Database: PDB / ID: 7k56
TitleStructure of VCP dodecamer purified from H1299 cells
ComponentsTransitional endoplasmic reticulum ATPase
KeywordsCYTOSOLIC PROTEIN / HYDROLASE / AAA ATPase / ATP hydrolysis / segregase
Function / homology
Function and homology information


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 ...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 / 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 / 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 / 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 / nucleus / cytosol
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 / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / CDC48, domain 2 / CDC48 domain 2-like superfamily / Vps4 oligomerisation, C-terminal / Vps4 C terminal oligomerisation domain ...AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / CDC48, domain 2 / CDC48 domain 2-like superfamily / Vps4 oligomerisation, C-terminal / Vps4 C terminal oligomerisation domain / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 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

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Structure visualization

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  • Deposited structure unit
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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
G: Transitional endoplasmic reticulum ATPase
H: Transitional endoplasmic reticulum ATPase
I: Transitional endoplasmic reticulum ATPase
J: Transitional endoplasmic reticulum ATPase
K: Transitional endoplasmic reticulum ATPase
L: Transitional endoplasmic reticulum ATPase


Theoretical massNumber of molelcules
Total (without water)1,073,24212
Polymers1,073,24212
Non-polymers00
Water0
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: 89436.820 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VCP / Plasmid: pCMV / Details (production host): pCMV-Flag-VCP / Cell line (production host): H1299 / Organ (production host): Lung / Production host: Homo sapiens (human) / Tissue (production host): Lung / 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 dodecamer expressed and purified from H1299 cells
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.08 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: H1299 / Plasmid: pCMV
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 chlorideNaClSodium chloride1
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 expressed and purified from H1299 cells using anti-Flag resin and 3XFlag peptide.
Specimen supportDetails: 15mA for 40s using Emitech. / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid 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 FIELDBright-field microscopy / 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: 10 sec. / Electron dose: 46 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: 600
Details: A dataset of ~600 movies was collected using FEI Titan Krios TEM operating at 300 KV and recorded with the Gatan K2 Summit direct electron detector at super-resolution mode with 22500 ...Details: A dataset of ~600 movies was collected using FEI Titan Krios TEM operating at 300 KV and recorded with the Gatan K2 Summit direct electron detector at super-resolution mode with 22500 nominal magnification. A frame rate of 5 frames per second, a dose rate of 8 eps and a total exposure of 10 seconds were used.

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.14_3260refinement
PHENIX1.14_3260refinement
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: 40000
SymmetryPoint symmetry: D6 (2x6 fold dihedral)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27516 / Num. of class averages: 1 / Symmetry type: POINT
RefinementStereochemistry target values: CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007971256
ELECTRON MICROSCOPYf_angle_d1.05896216
ELECTRON MICROSCOPYf_chiral_restr0.062510812
ELECTRON MICROSCOPYf_plane_restr0.007912768
ELECTRON MICROSCOPYf_dihedral_angle_d8.552244436

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