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- EMDB-31894: Human p97 double hexamer conformer II with ATPgammaS bound -

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Basic information

Entry
Database: EMDB / ID: EMD-31894
TitleHuman p97 double hexamer conformer II with ATPgammaS bound
Map datahuman p97 double hexamer conformer II
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
Function / homology
Function and homology information


flavin adenine dinucleotide catabolic process / positive regulation of Lys63-specific deubiquitinase activity / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / VCP-NSFL1C complex / deubiquitinase activator activity / endosome to lysosome transport via multivesicular body sorting pathway / cellular response to arsenite ion / endoplasmic reticulum stress-induced pre-emptive quality control / BAT3 complex binding ...flavin adenine dinucleotide catabolic process / positive regulation of Lys63-specific deubiquitinase activity / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / VCP-NSFL1C complex / deubiquitinase activator activity / endosome to lysosome transport via multivesicular body sorting pathway / cellular response to arsenite ion / endoplasmic reticulum stress-induced pre-emptive quality control / BAT3 complex binding / Derlin-1 retrotranslocation complex / positive regulation of oxidative phosphorylation / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / NADH metabolic process / ERAD pathway / aggresome assembly / regulation of protein localization to chromatin / vesicle-fusing ATPase / ER-associated misfolded protein catabolic process / stress granule disassembly / K48-linked polyubiquitin modification-dependent protein binding / retrograde protein transport, ER to cytosol / regulation of aerobic respiration / positive regulation of mitochondrial membrane potential / ATPase complex / regulation of synapse organization / positive regulation of ATP biosynthetic process / ubiquitin-like protein ligase binding / ubiquitin-specific protease binding / negative regulation of smoothened signaling pathway / autophagosome maturation / RHOH GTPase cycle / polyubiquitin modification-dependent protein binding / Attachment and Entry / endoplasmic reticulum to Golgi vesicle-mediated transport / Protein methylation / ATP metabolic process / MHC class I protein binding / HSF1 activation / translesion synthesis / endoplasmic reticulum unfolded protein response / interstrand cross-link repair / lipid droplet / viral genome replication / ubiquitin-dependent ERAD pathway / Josephin domain DUBs / proteasome complex / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / macroautophagy / proteasomal protein catabolic process / ADP binding / Hh mutants are degraded by ERAD / Defective CFTR causes cystic fibrosis / Hedgehog ligand biogenesis / Translesion Synthesis by POLH / positive regulation of protein-containing complex assembly / ABC-family proteins mediated transport / positive regulation of protein catabolic process / double-strand break repair / establishment of protein localization / Aggrephagy / cytoplasmic stress granule / autophagy / azurophil granule lumen / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / activation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of canonical Wnt signaling pathway / site of double-strand break / Ovarian tumor domain proteases / E3 ubiquitin ligases ubiquitinate target proteins / cellular response to heat / proteasome-mediated ubiquitin-dependent protein catabolic process / Attachment and Entry / secretory granule lumen / protein phosphatase binding / protein ubiquitination / ficolin-1-rich granule lumen / regulation of apoptotic process / lipid binding / glutamatergic synapse / DNA repair / intracellular membrane-bounded organelle / protein domain specific binding / cellular response to DNA damage stimulus / ubiquitin protein ligase binding / Neutrophil degranulation / endoplasmic reticulum membrane / ATP hydrolysis activity / perinuclear region of cytoplasm / endoplasmic reticulum / protein-containing complex / RNA binding / extracellular exosome / extracellular region / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol
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 / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / CDC48, domain 2 / CDC48 domain 2-like superfamily / Vps4 oligomerisation, C-terminal / Vps4 C terminal oligomerisation domain ...AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / CDC48, domain 2 / CDC48 domain 2-like superfamily / Vps4 oligomerisation, C-terminal / Vps4 C terminal oligomerisation domain / Aspartate decarboxylase-like domain superfamily / AAA+ lid domain / AAA ATPase, AAA+ lid domain / AAA-protein family signature. / ATPase, AAA-type, conserved site / 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: 3.32 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 3, 2021-
Header (metadata) releaseMar 2, 2022-
Map releaseMar 2, 2022-
UpdateMar 9, 2022-
Current statusMar 9, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
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  • Surface view with fitted model
  • Atomic models: PDB-7vcs
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7vcs
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

FileDownload / File: emd_31894.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman p97 double hexamer conformer II
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 280 pix.
= 302.4 Å
1.08 Å/pix.
x 280 pix.
= 302.4 Å
1.08 Å/pix.
x 280 pix.
= 302.4 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
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.02
Minimum - Maximum-0.041696727 - 0.12146865
Average (Standard dev.)0.0017219124 (±0.006915755)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 302.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z302.400302.400302.400
α/β/γ90.00090.00090.000
start NX/NY/NZ535455
NX/NY/NZ134138134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0420.1210.002

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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)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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: 12 / 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

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Macromolecule #2: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 2 / Number of copies: 24 / 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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final 3D classificationNumber classes: 6 / Software - Name: RELION
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 50746
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Chain ID: A
RefinementProtocol: RIGID BODY FIT
Output model

PDB-7vcs:
Human p97 double hexamer conformer II with ATPgammaS bound

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