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- PDB-8ooi: Full composite cryo-EM map of p97/VCP in ADP.Pi state -

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Basic information

Entry
Database: PDB / ID: 8ooi
TitleFull composite cryo-EM map of p97/VCP in ADP.Pi state
ComponentsTransitional endoplasmic reticulum ATPase
KeywordsHYDROLASE / AAA+ ATPase / complex
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 / cellular response to arsenite ion / BAT3 complex binding / Derlin-1 retrotranslocation complex / protein-DNA covalent cross-linking repair / cytoplasm protein quality control ...: / 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 / cellular response to arsenite ion / BAT3 complex binding / Derlin-1 retrotranslocation complex / protein-DNA covalent cross-linking repair / cytoplasm protein quality control / positive regulation of protein K63-linked deubiquitination / positive regulation of oxidative phosphorylation / : / mitotic spindle disassembly / aggresome assembly / deubiquitinase activator activity / regulation of protein localization to chromatin / ubiquitin-modified protein reader activity / VCP-NPL4-UFD1 AAA ATPase complex / vesicle-fusing ATPase / cellular response to misfolded protein / negative regulation of protein localization to chromatin / positive regulation of mitochondrial membrane potential / K48-linked polyubiquitin modification-dependent protein binding / retrograde protein transport, ER to cytosol / regulation of aerobic respiration / stress granule disassembly / positive regulation of ATP biosynthetic process / regulation of synapse organization / ATPase complex / ubiquitin-specific protease binding / MHC class I protein binding / ubiquitin-like protein ligase binding / RHOH GTPase cycle / polyubiquitin modification-dependent protein binding / autophagosome maturation / endoplasmic reticulum to Golgi vesicle-mediated transport / negative regulation of hippo signaling / HSF1 activation / translesion synthesis / interstrand cross-link repair / proteasomal protein catabolic process / ATP metabolic process / Protein methylation / endoplasmic reticulum unfolded protein response / ERAD pathway / Attachment and Entry / 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 / Hedgehog ligand biogenesis / establishment of protein localization / Defective CFTR causes cystic fibrosis / Translesion Synthesis by POLH / positive regulation of non-canonical NF-kappaB signal transduction / ADP binding / ABC-family proteins mediated transport / autophagy / positive regulation of protein catabolic process / cytoplasmic stress granule / Aggrephagy / 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 / Neddylation / cellular response to heat / ubiquitin-dependent protein catabolic process / protein phosphatase binding / secretory granule lumen / regulation of apoptotic process / proteasome-mediated ubiquitin-dependent protein catabolic process / ficolin-1-rich granule lumen / Attachment and Entry / protein ubiquitination / ciliary basal body / protein domain specific binding / DNA repair / intracellular membrane-bounded organelle / lipid binding / ubiquitin protein ligase binding / DNA damage response / 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
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / : / PHOSPHATE ION / Transitional endoplasmic reticulum ATPase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsCheng, T.C. / Sakata, E. / Schuetz, A.K.
Funding support Germany, 4items
OrganizationGrant numberCountry
German Research Foundation (DFG)394455587 Germany
German Research Foundation (DFG)201302640 Germany
German Research Foundation (DFG)390729940 Germany
German Research Foundation (DFG) Germany
CitationJournal: Nat Chem / Year: 2024
Title: Characterizing ATP processing by the AAA+ protein p97 at the atomic level.
Authors: Mikhail Shein / Manuel Hitzenberger / Tat Cheung Cheng / Smruti R Rout / Kira D Leitl / Yusuke Sato / Martin Zacharias / Eri Sakata / Anne K Schütz /
Abstract: The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of protein homeostasis and an ...The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of protein homeostasis and an impactful pharmacological target. The hexameric complex undergoes substantial conformational changes throughout its catalytic cycle. Here we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and molecular dynamics simulations. p97 populates a metastable reaction intermediate, the ADP·P state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes. Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.
History
DepositionApr 5, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Mar 13, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Oct 16, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)543,32342
Polymers536,6216
Non-polymers6,70236
Water2,594144
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 6 molecules AFBDCE

#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.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VCP / Production host: Escherichia coli (E. coli) / References: UniProt: P55072, vesicle-fusing ATPase

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Non-polymers , 6 types, 180 molecules

#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 144 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: p97/VCP / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 / Nominal defocus max: 2200 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategory
2EPUimage acquisition
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 198991 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00435700
ELECTRON MICROSCOPYf_angle_d0.64448282
ELECTRON MICROSCOPYf_dihedral_angle_d5.5854908
ELECTRON MICROSCOPYf_chiral_restr0.0465382
ELECTRON MICROSCOPYf_plane_restr0.0056324

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