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- EMDB-31897: Human p97 single hexamer conformer I with ATPgammaS bound -

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Basic information

Entry
Database: EMDB / ID: EMD-31897
TitleHuman p97 single hexamer conformer I with ATPgammaS bound
Map datap97 SH I
Sample
  • Complex: human p97 double hexamer conformer II with ATPgammaS bound
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsAAA+ ATPase / unfoldase / CELL CYCLE / HYDROLASE
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 / ATPase complex ...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 / ATPase complex / 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 / 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 / ubiquitin-like protein ligase binding / RHOH GTPase cycle / intracellular membrane-bounded organelle / MHC class I protein binding / autophagosome maturation / negative regulation of hippo signaling / HSF1 activation / endoplasmic reticulum to Golgi vesicle-mediated transport / polyubiquitin modification-dependent protein binding / mitophagy / interstrand cross-link repair / ATP metabolic process / protein unfolding / proteasome complex / ribosome-associated ubiquitin-dependent protein catabolic process / Attachment and Entry / endoplasmic reticulum unfolded protein response / Protein methylation / ERAD pathway / negative regulation of smoothened signaling pathway / viral genome replication / translesion synthesis / negative regulation of protein localization to chromatin / rescue of stalled cytosolic ribosome / macroautophagy / lipid droplet / 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 / Hh mutants are degraded by ERAD / positive regulation of non-canonical NF-kappaB signal transduction / ADP binding / 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 / autophagy / cytoplasmic stress granule / Ribosome Quality Control (RQC) complex extracts and degrades nascent peptide / positive regulation of protein catabolic process / Aggrephagy / positive regulation of canonical Wnt signaling pathway / double-strand break repair / 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 / regulation of apoptotic process / Attachment and Entry / protein ubiquitination / protein domain specific binding / ubiquitin protein ligase binding / DNA repair / DNA damage response / Neutrophil degranulation / lipid binding / endoplasmic reticulum membrane / perinuclear region of cytoplasm
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
Transitional endoplasmic reticulum ATPase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsGao H / Li F / Shi Z / Li Y / Yu H
Funding support United States, 3 items
OrganizationGrant numberCountry
Welch FoundationI-1441 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RP160667-P2 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RP170644 United States
CitationJournal: Cell Discov / Year: 2022
Title: Cryo-EM structures of human p97 double hexamer capture potentiated ATPase-competent state.
Authors: Haishan Gao / Faxiang Li / Zhejian Ji / Zhubing Shi / Yang Li / Hongtao Yu /
Abstract: The conserved ATPase p97 (Cdc48 in yeast) and adaptors mediate diverse cellular processes through unfolding polyubiquitinated proteins and extracting them from macromolecular assemblies and membranes ...The conserved ATPase p97 (Cdc48 in yeast) and adaptors mediate diverse cellular processes through unfolding polyubiquitinated proteins and extracting them from macromolecular assemblies and membranes for disaggregation and degradation. The tandem ATPase domains (D1 and D2) of the p97/Cdc48 hexamer form stacked rings. p97/Cdc48 can unfold substrates by threading them through the central pore. The pore loops critical for substrate unfolding are, however, not well-ordered in substrate-free p97/Cdc48 conformations. How p97/Cdc48 organizes its pore loops for substrate engagement is unclear. Here we show that p97/Cdc48 can form double hexamers (DH) connected through the D2 ring. Cryo-EM structures of p97 DH reveal an ATPase-competent conformation with ordered pore loops. The C-terminal extension (CTE) links neighboring D2s in each hexamer and expands the central pore of the D2 ring. Mutations of Cdc48 CTE abolish substrate unfolding. We propose that the p97/Cdc48 DH captures a potentiated state poised for substrate engagement.
History
DepositionSep 4, 2021-
Header (metadata) releaseMar 2, 2022-
Map releaseMar 2, 2022-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7vcv
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7vcv
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31897.png

Downloads & links

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Map

FileDownload / File: emd_31897.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationp97 SH I
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 300 pix.
= 324. Å		1.08 Å/pix.
x 300 pix.
= 324. Å		1.08 Å/pix.
x 300 pix.
= 324. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.014
Minimum - Maximum-0.073167376 - 0.15733653
Average (Standard dev.)0.0000031424063 (±0.004278633)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z324.000324.000324.000
α/β/γ90.00090.00090.000
start NX/NY/NZ535455
NX/NY/NZ134138134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0730.1570.000

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Supplemental data

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Sample components

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Entire : human p97 double hexamer conformer II with ATPgammaS bound

EntireName: human p97 double hexamer conformer II with ATPgammaS bound
Components
  • Complex: human p97 double hexamer conformer II with ATPgammaS bound
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: human p97 double hexamer conformer II with ATPgammaS bound

SupramoleculeName: human p97 double hexamer conformer II with ATPgammaS bound
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.2 MDa

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Macromolecule #1: Transitional endoplasmic reticulum ATPase

MacromoleculeName: Transitional endoplasmic reticulum ATPase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: vesicle-fusing ATPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 90.265711 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MASGADSKGD DLSTAILKQK NRPNRLIVDE AINEDNSVVS LSQPKMDELQ LFRGDTVLLK GKKRREAVCI VLSDDTCSDE KIRMNRVVR NNLRVRLGDV ISIQPCPDVK YGKRIHVLPI DDTVEGITGN LFEVYLKPYF LEAYRPIRKG DIFLVRGGMR A VEFKVVET ...String:
MASGADSKGD DLSTAILKQK NRPNRLIVDE AINEDNSVVS LSQPKMDELQ LFRGDTVLLK GKKRREAVCI VLSDDTCSDE KIRMNRVVR NNLRVRLGDV ISIQPCPDVK YGKRIHVLPI DDTVEGITGN LFEVYLKPYF LEAYRPIRKG DIFLVRGGMR A VEFKVVET DPSPYCIVAP DTVIHCEGEP IKREDEEESL NEVGYDDIGG CRKQLAQIKE MVELPLRHPA LFKAIGVKPP RG ILLYGPP GTGKTLIARA VANETGAFFF LINGPEIMSK LAGESESNLR KAFEEAEKNA PAIIFIDELD AIAPKREKTH GEV ERRIVS QLLTLMDGLK QRAHVIVMAA TNRPNSIDPA LRRFGRFDRE VDIGIPDATG RLEILQIHTK NMKLADDVDL EQVA NETHG HVGADLAALC SEAALQAIRK KMDLIDLEDE TIDAEVMNSL AVTMDDFRWA LSQSNPSALR ETVVEVPQVT WEDIG GLED VKRELQELVQ YPVEHPDKFL KFGMTPSKGV LFYGPPGCGK TLLAKAIANE CQANFISIKG PELLTMWFGE SEANVR EIF DKARQAAPCV LFFDELDSIA KARGGNIGDG GGAADRVINQ ILTEMDGMST KKNVFIIGAT NRPDIIDPAI LRPGRLD QL IYIPLPDEKS RVAILKANLR KSPVAKDVDL EFLAKMTNGF SGADLTEICQ RACKLAIRES IESEIRRERE RQTNPSAM E VEEDDPVPEI RRDHFEEAMR FARRSVSDND IRKYEMFAQT LQQSRGFGSF RFPSGNQGGA GPSQGSGGGT GGSVYTEDN DDDLYGHHHH HH

UniProtKB: Transitional endoplasmic reticulum ATPase

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Macromolecule #2: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 2 / Number of copies: 12 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 12 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 7.5
Details: 25 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.5 mM TCEP, 0.01% NP40
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 3ul sample was applied and the grids were blotted for 3.0 s under 100% humidity at 277K before being plunged into liquid ethane using a Mark IV Vitrobot (FEI)..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 0.3 sec. / Average electron dose: 1.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5ftj

Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 79221
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final 3D classificationNumber classes: 6 / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3cf3


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-7vcv:
Human p97 single hexamer conformer I with ATPgammaS bound

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