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- PDB-8rsc: p97 (VCP) mutant - F539A -

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Basic information

Entry
Database: PDB / ID: 8rsc
Titlep97 (VCP) mutant - F539A
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 / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair ...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 / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / deubiquitinase activator activity / positive regulation of oxidative phosphorylation / cytoplasm protein quality control / regulation of protein localization to chromatin / ubiquitin-modified protein reader activity / 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 / NAD+ metabolic process / regulation of aerobic respiration / retrograde protein transport, ER to cytosol / stress granule disassembly / ATPase complex / ubiquitin-specific protease binding / regulation of synapse organization / polyubiquitin modification-dependent protein binding / positive regulation of ATP biosynthetic process / ubiquitin-like protein ligase binding / RHOH GTPase cycle / intracellular membrane-bounded organelle / autophagosome maturation / MHC class I protein binding / HSF1 activation / endoplasmic reticulum to Golgi vesicle-mediated transport / negative regulation of hippo signaling / mitophagy / interstrand cross-link repair / ATP metabolic process / proteasome complex / protein unfolding / endoplasmic reticulum unfolded protein response / ribosome-associated ubiquitin-dependent protein catabolic process / Attachment and Entry / ERAD pathway / Protein methylation / translesion synthesis / negative regulation of smoothened signaling pathway / viral genome replication / negative regulation of protein localization to chromatin / macroautophagy / rescue of stalled cytosolic ribosome / lipid droplet / Josephin domain DUBs / autophagy / 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 / positive regulation of non-canonical NF-kappaB signal transduction / Hh mutants are degraded by ERAD / Dengue Virus Genome Translation and Replication / Translesion Synthesis by POLH / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / AMPK-induced ERAD and lysosome mediated degradation of PD-L1(CD274) / ABC-family protein mediated transport / cytoplasmic stress granule / Ribosome Quality Control (RQC) complex extracts and degrades nascent peptide / double-strand break repair / Aggrephagy / positive regulation of canonical Wnt signaling pathway / positive regulation of protein catabolic process / azurophil granule lumen / Ovarian tumor domain proteases / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / KEAP1-NFE2L2 pathway / site of double-strand break / cellular response to heat / E3 ubiquitin ligases ubiquitinate target proteins / Neddylation / secretory granule lumen / protein phosphatase binding / ubiquitin-dependent protein catabolic process / ficolin-1-rich granule lumen / proteasome-mediated ubiquitin-dependent protein catabolic process / Attachment and Entry / regulation of apoptotic process / protein ubiquitination / protein domain specific binding / DNA repair / ubiquitin protein ligase binding / lipid binding / DNA damage response / Neutrophil degranulation / endoplasmic reticulum membrane / perinuclear region of cytoplasm
Similarity search - Function
AAA ATPase, CDC48 family / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48), N-terminal domain / 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 / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48), N-terminal domain / 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 / Transitional endoplasmic reticulum ATPase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 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
DepositionJan 24, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 29, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
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)540,43616
Polymers536,1646
Non-polymers4,27210
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, native gel electrophoresis, electron microscopy, Negative stain, isothermal titration calorimetry, Thermal Shift Assay (TSA)
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 / Production host: Escherichia coli (E. coli) / References: UniProt: P55072, vesicle-fusing ATPase
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationN

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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 / 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 secounds and blot for 3.5 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: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 3053

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4RELION3.1.2CTF correction
7RELION3.1.2model fitting
9RELION3.1.2initial Euler assignment
10RELION3.1.2final Euler assignment
11RELION3.1.2classification
12RELION3.1.23D reconstruction
13RELION3.1.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1156843
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162750 / Symmetry type: POINT
Atomic model buildingB value: 151
Atomic model buildingPDB-ID: 5FTN

5ftn


Accession code: 5FTN / Source name: PDB / Type: experimental model
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 47.05 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00332320
ELECTRON MICROSCOPYf_angle_d0.670443925
ELECTRON MICROSCOPYf_chiral_restr0.04245107
ELECTRON MICROSCOPYf_plane_restr0.00385890
ELECTRON MICROSCOPYf_dihedral_angle_d19.673911493

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