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- PDB-8fcl: Cryo-EM structure of p97:UBXD1 closed state -

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Basic information

Entry
Database: PDB / ID: 8fcl
TitleCryo-EM structure of p97:UBXD1 closed state
Components
  • Transitional endoplasmic reticulum ATPase
  • UBX domain-containing protein 6
KeywordsCHAPERONE / HYDROLASE / AAA+
Function / homology
Function and homology information


positive regulation of Lys63-specific deubiquitinase activity / 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 / cytoplasm protein quality control / Derlin-1 retrotranslocation complex / protein-DNA covalent cross-linking repair ...positive regulation of Lys63-specific deubiquitinase activity / 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 / cytoplasm protein quality control / Derlin-1 retrotranslocation complex / protein-DNA covalent cross-linking repair / positive regulation of protein K63-linked deubiquitination / positive regulation of oxidative phosphorylation / mitotic spindle disassembly / NADH metabolic process / 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 / regulation of synapse organization / positive regulation of ATP biosynthetic process / 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 / negative regulation of hippo signaling / endoplasmic reticulum to Golgi vesicle-mediated transport / 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 / negative regulation of smoothened signaling pathway / proteasome complex / viral genome replication / : / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Hh mutants are degraded by ERAD / positive regulation of protein-containing complex assembly / macroautophagy / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / establishment of protein localization / Translesion Synthesis by POLH / positive regulation of non-canonical NF-kappaB signal transduction / ABC-family proteins mediated transport / ADP binding / autophagy / Aggrephagy / positive regulation of protein catabolic process / cytoplasmic stress granule / azurophil granule lumen / KEAP1-NFE2L2 pathway / Ovarian tumor domain proteases / late endosome membrane / 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 / Neddylation / site of double-strand break / cellular response to heat / early endosome membrane / 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 / endosome / protein ubiquitination / protein domain specific binding / lysosomal membrane / chromatin extrusion motor activity / ATP-dependent H2AZ histone chaperone activity / ATP-dependent H3-H4 histone complex chaperone activity / cohesin loader activity / DNA clamp loader activity / intracellular membrane-bounded organelle / DNA repair / centrosome / lipid binding
Similarity search - Function
UBXN6, PUB domain / Domain present in ubiquitin-regulatory proteins / UBX domain / PUB domain / PUB-like domain superfamily / UBX domain / PUB domain / UBX domain profile. / domain in protein kinases, N-glycanases and other nuclear proteins / AAA ATPase, CDC48 family ...UBXN6, PUB domain / Domain present in ubiquitin-regulatory proteins / UBX domain / PUB domain / PUB-like domain superfamily / UBX domain / PUB domain / UBX domain profile. / domain in protein kinases, N-glycanases and other nuclear proteins / 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 / Ubiquitin-like domain superfamily / 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 / UBX domain-containing protein 6
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.51 Å
AuthorsBraxton, J.R. / Tucker, M.R. / Tse, E. / Southworth, D.R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM138690 United States
Citation
Journal: Nat Struct Mol Biol / Year: 2023
Title: The p97/VCP adaptor UBXD1 drives AAA+ remodeling and ring opening through multi-domain tethered interactions.
Authors: Julian R Braxton / Chad R Altobelli / Maxwell R Tucker / Eric Tse / Aye C Thwin / Michelle R Arkin / Daniel R Southworth /
Abstract: p97, also known as valosin-containing protein, is an essential cytosolic AAA+ (ATPases associated with diverse cellular activities) hexamer that unfolds substrate polypeptides to support protein ...p97, also known as valosin-containing protein, is an essential cytosolic AAA+ (ATPases associated with diverse cellular activities) hexamer that unfolds substrate polypeptides to support protein homeostasis and macromolecular disassembly. Distinct sets of p97 adaptors guide cellular functions but their roles in direct control of the hexamer are unclear. The UBXD1 adaptor localizes with p97 in critical mitochondria and lysosome clearance pathways and contains multiple p97-interacting domains. Here we identify UBXD1 as a potent p97 ATPase inhibitor and report structures of intact human p97-UBXD1 complexes that reveal extensive UBXD1 contacts across p97 and an asymmetric remodeling of the hexamer. Conserved VIM, UBX and PUB domains tether adjacent protomers while a connecting strand forms an N-terminal domain lariat with a helix wedged at the interprotomer interface. An additional VIM-connecting helix binds along the second (D2) AAA+ domain. Together, these contacts split the hexamer into a ring-open conformation. Structures, mutagenesis and comparisons to other adaptors further reveal how adaptors containing conserved p97-remodeling motifs regulate p97 ATPase activity and structure.
#1: Journal: bioRxiv / Year: 2023
Title: The p97/VCP adapter UBXD1 drives AAA+ remodeling and ring opening through multi-domain tethered interactions.
Authors: Julian R Braxton / Chad R Altobelli / Maxwell R Tucker / Eric Tse / Aye C Thwin / Michelle R Arkin / Daniel R Southworth /
Abstract: p97/VCP is an essential cytosolic AAA+ ATPase hexamer that extracts and unfolds substrate polypeptides during protein homeostasis and degradation. Distinct sets of p97 adapters guide cellular ...p97/VCP is an essential cytosolic AAA+ ATPase hexamer that extracts and unfolds substrate polypeptides during protein homeostasis and degradation. Distinct sets of p97 adapters guide cellular functions but their roles in direct control of the hexamer are unclear. The UBXD1 adapter localizes with p97 in critical mitochondria and lysosome clearance pathways and contains multiple p97-interacting domains. We identify UBXD1 as a potent p97 ATPase inhibitor and report structures of intact p97:UBXD1 complexes that reveal extensive UBXD1 contacts across p97 and an asymmetric remodeling of the hexamer. Conserved VIM, UBX, and PUB domains tether adjacent protomers while a connecting strand forms an N-terminal domain lariat with a helix wedged at the interprotomer interface. An additional VIM-connecting helix binds along the second AAA+ domain. Together these contacts split the hexamer into a ring-open conformation. Structures, mutagenesis, and comparisons to other adapters further reveal how adapters containing conserved p97-remodeling motifs regulate p97 ATPase activity and structure.
History
DepositionDec 1, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Revision 1.2Nov 22, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Dec 20, 2023Group: 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

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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
G: UBX domain-containing protein 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)591,57119
Polymers586,4457
Non-polymers5,12612
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
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.: 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: Protein UBX domain-containing protein 6 / UBX domain-containing protein 1


Mass: 49823.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBXN6, UBXD1, UBXDC2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BZV1
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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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: Complex of p97 and UBXD1 / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 1.89 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Pelco easiGlow, 15 mA / Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Wait time 10 sec, blot time 3 sec, blot force 0

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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 magnification: 105000 X / Calibrated magnification: 59952 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.024 sec. / Electron dose: 43 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 22536
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.51 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 82334 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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