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- PDB-8pqx: p97 (VCP) mutant - F539A state III -

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Basic information

Entry
Database: PDB / ID: 8pqx
Titlep97 (VCP) mutant - F539A state III
ComponentsTransitional endoplasmic reticulum ATPase
KeywordsCHAPERONE / Hexameric complex / ATPase / Unfoldase / Protein Quality Control / Segregase
Function / homology
Function and homology information


flavin adenine dinucleotide catabolic process / VCP-NSFL1C complex / endoplasmic reticulum stress-induced pre-emptive quality control / endosome to lysosome transport via multivesicular body sorting pathway / BAT3 complex binding / cellular response to arsenite ion / cytoplasmic ubiquitin ligase complex / 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 / endoplasmic reticulum stress-induced pre-emptive quality control / endosome to lysosome transport via multivesicular body sorting pathway / BAT3 complex binding / cellular response to arsenite ion / cytoplasmic ubiquitin ligase complex / protein-DNA covalent cross-linking repair / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / ATPase complex / deubiquitinase activator activity / positive regulation of oxidative phosphorylation / cytoplasm protein quality control / aggresome assembly / ubiquitin-modified protein reader activity / regulation of protein localization to chromatin / cellular response to misfolded protein / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / positive regulation of mitochondrial membrane potential / vesicle-fusing ATPase / K48-linked polyubiquitin modification-dependent protein binding / ciliary transition zone / regulation of aerobic respiration / NAD+ metabolic process / retrograde protein transport, ER to cytosol / stress granule disassembly / ubiquitin-specific protease binding / regulation of synapse organization / positive regulation of ATP biosynthetic process / intracellular membrane-bounded organelle / ubiquitin-like protein ligase binding / RHOH GTPase cycle / MHC class I protein binding / autophagosome maturation / negative regulation of hippo signaling / endoplasmic reticulum to Golgi vesicle-mediated transport / HSF1 activation / polyubiquitin modification-dependent protein binding / interstrand cross-link repair / ATP metabolic process / Attachment and Entry / endoplasmic reticulum unfolded protein response / Protein methylation / ERAD pathway / ciliary tip / translesion synthesis / negative regulation of protein localization to chromatin / lipid droplet / viral genome replication / proteasome complex / macroautophagy / negative regulation of smoothened signaling pathway / Josephin domain DUBs / establishment of protein localization / proteasomal protein catabolic process / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / positive regulation of protein-containing complex assembly / ADP binding / Hh mutants are degraded by ERAD / positive regulation of non-canonical NF-kappaB signal transduction / Translesion Synthesis by POLH / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / ABC-family protein mediated transport / autophagy / cytoplasmic stress granule / positive regulation of protein catabolic process / positive regulation of canonical Wnt signaling pathway / Aggrephagy / azurophil granule lumen / Ovarian tumor domain proteases / KEAP1-NFE2L2 pathway / double-strand break repair / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / cellular response to heat / E3 ubiquitin ligases ubiquitinate target proteins / site of double-strand break / Neddylation / secretory granule lumen / protein phosphatase binding / regulation of apoptotic process / ficolin-1-rich granule lumen / ciliary basal body / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / Attachment and Entry / protein ubiquitination / protein domain specific binding / DNA repair / ubiquitin protein ligase binding / Neutrophil degranulation / DNA damage response / lipid binding / endoplasmic reticulum membrane / perinuclear region of cytoplasm / glutamatergic synapse / endoplasmic reticulum / ATP hydrolysis activity
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 / Transitional endoplasmic reticulum ATPase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsArie, M. / Matzov, D. / Karmona, R. / Szenkier, N. / Stanhill, A. / Navon, A.
Funding support Israel, 1items
OrganizationGrant numberCountry
Israel Science Foundation2038/17 Israel
CitationJournal: iScience / Year: 2024
Title: A non-symmetrical p97 conformation initiates a multistep recruitment of Ufd1/Npl4.
Authors: Michal Arie / Donna Matzov / Rotem Karmona / Natalia Szenkier / Ariel Stanhill / Ami Navon /
Abstract: experiments and cryo-EM structures of p97 and its cofactor, Ufd1/Npl4 (UN), elucidated substrate processing. Yet, the structural transitions and the related ATPase cycle upon UN binding remain ... experiments and cryo-EM structures of p97 and its cofactor, Ufd1/Npl4 (UN), elucidated substrate processing. Yet, the structural transitions and the related ATPase cycle upon UN binding remain unresolved. We captured two discrete conformations: One in which D1 protomers are ATP bound, while the D2 subunits are in the ADP state, presumably required for substrate engagement with the D2 pore; and a heterologous nucleotide state within the D1 ring in which only two NTDs are in the "up" ATP state that favors UN binding. Further analysis suggests that initially, UN binds p97's non-symmetrical conformation, this association promotes a structural transition upon which five NTDs shift to an "up" state and are poised to bind ATP. The UBXL domain of Npl4 was captured bound to an NTD in the ADP state, demonstrating a conformation that may provide directionality to incoming substrate and introduce the flexibility needed for substrate processing.
History
DepositionJul 12, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 29, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2024Group: Advisory / Data collection ...Advisory / Data collection / Database references / Derived calculations / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details / pdbx_modification_feature / pdbx_validate_close_contact / struct_conn / struct_conn_type
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 / _citation_author.identifier_ORCID / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)541,37118
Polymers536,1646
Non-polymers5,20612
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, assay for oligomerization, Thermal Shift Assay (TSA), gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 89360.727 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VCP / Plasmid: pQE9 / Production host: Escherichia coli (E. coli) / References: UniProt: P55072, vesicle-fusing ATPase
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
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: Tertiary complex of the hexameric p97 F539A mutant (state III)
Type: COMPLEX
Details: The mutant generated by the replacement of phenylalanine to alanine at position 539
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.53 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pQE9
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
230 mMpotassium chlorideKCl1
31 mMDTTC4H10O2S21
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: pre-blotting incubation time of 20 seconds and blot for 2.0 seconds with -1 blot force

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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: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 42 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 22245

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5686568
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 163728 / Symmetry type: POINT
Atomic model buildingB value: 102
Atomic model buildingPDB-ID: 8AJF

8ajf
PDB Unreleased entry


Accession code: 8AJF / Source name: PDB / Type: experimental model
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 122.67 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00327178
ELECTRON MICROSCOPYf_angle_d0.56636769
ELECTRON MICROSCOPYf_dihedral_angle_d6.4133796
ELECTRON MICROSCOPYf_chiral_restr0.0414116
ELECTRON MICROSCOPYf_plane_restr0.0044842

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