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- EMDB-31896: Human p97 double hexamer conformer I with D1-ATPgammaS and D2-ADP... -

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Basic information

Entry
Database: EMDB / ID: EMD-31896
TitleHuman p97 double hexamer conformer I with D1-ATPgammaS and D2-ADP bound
Map data
Sample
  • Complex: human p97 double hexamer conformer I with D1-ATPgammaS and D2-ADP bound
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsAAA+ ATPase / unfoldase / CELL CYCLE / HYDROLASE
Function / homology
Function and homology information


positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / cytoplasm protein quality control / endosome to lysosome transport via multivesicular body sorting pathway / endoplasmic reticulum stress-induced pre-emptive quality control / cellular response to arsenite ion / Derlin-1 retrotranslocation complex / BAT3 complex binding ...positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / cytoplasm protein quality control / endosome to lysosome transport via multivesicular body sorting pathway / endoplasmic reticulum stress-induced pre-emptive quality control / cellular response to arsenite ion / Derlin-1 retrotranslocation complex / BAT3 complex binding / protein-DNA covalent cross-linking repair / positive regulation of protein K63-linked deubiquitination / deubiquitinase activator activity / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / ubiquitin-modified protein reader activity / regulation of protein localization to chromatin / aggresome assembly / NADH metabolic process / vesicle-fusing ATPase / cellular response to misfolded protein / stress granule disassembly / negative regulation of protein localization to chromatin / positive regulation of mitochondrial membrane potential / retrograde protein transport, ER to cytosol / K48-linked polyubiquitin modification-dependent protein binding / regulation of aerobic respiration / 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 / HSF1 activation / negative regulation of hippo signaling / endoplasmic reticulum to Golgi vesicle-mediated transport / translesion synthesis / proteasomal protein catabolic process / Protein methylation / interstrand cross-link repair / ATP metabolic process / negative regulation of smoothened signaling pathway / endoplasmic reticulum unfolded protein response / ERAD pathway / Attachment and Entry / proteasome complex / viral genome replication / lipid droplet / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / macroautophagy / Hh mutants are degraded by ERAD / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / positive regulation of protein-containing complex assembly / ADP binding / Translesion Synthesis by POLH / establishment of protein localization / ABC-family proteins mediated transport / : / autophagy / Aggrephagy / cytoplasmic stress granule / positive regulation of non-canonical NF-kappaB signal transduction / positive regulation of protein catabolic process / azurophil granule lumen / KEAP1-NFE2L2 pathway / positive regulation of canonical Wnt signaling pathway / Ovarian tumor domain proteases / double-strand break repair / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / E3 ubiquitin ligases ubiquitinate target proteins / site of double-strand break / Neddylation / cellular response to heat / 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 / protein ubiquitination / protein domain specific binding / intracellular membrane-bounded organelle / DNA repair / lipid binding / DNA damage response / glutamatergic synapse / ubiquitin protein ligase binding / Neutrophil degranulation / endoplasmic reticulum membrane / perinuclear region of cytoplasm / endoplasmic reticulum / ATP hydrolysis activity / protein-containing complex / RNA binding
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: 3.15 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: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7vcu
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31896.png

Downloads & links

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Map

FileDownload / File: emd_31896.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.013 / Movie #1: 0.02
Minimum - Maximum-0.046606693 - 0.1299706
Average (Standard dev.)0.0014204686 (±0.0069071096)
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-0.0470.1300.001

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Supplemental data

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Sample components

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Entire : human p97 double hexamer conformer I with D1-ATPgammaS and D2-ADP...

EntireName: human p97 double hexamer conformer I with D1-ATPgammaS and D2-ADP bound
Components
  • Complex: human p97 double hexamer conformer I with D1-ATPgammaS and D2-ADP bound
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: human p97 double hexamer conformer I with D1-ATPgammaS and D2-ADP...

SupramoleculeName: human p97 double hexamer conformer I with D1-ATPgammaS and D2-ADP 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: 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

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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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 12 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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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 reconstructionResolution.type: BY AUTHOR / Resolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 20139
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-7vcu:
Human p97 double hexamer conformer I with D1-ATPgammaS and D2-ADP bound

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