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- EMDB-22678: Structure of apo VCP hexamer generated from bacterially recombina... -

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Basic information

Entry
Database: EMDB / ID: EMD-22678
TitleStructure of apo VCP hexamer generated from bacterially recombinant VCP/p97
Map dataCryo-EM density map of apo VCP hexamer generated from bacterially recombinant VCP/p97.
Sample
  • Complex: VCP/p97
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
KeywordsAAA ATPase / ATP hydrolysis / segregase / CYTOSOLIC PROTEIN / HYDROLASE
Function / homology
Function and homology information


: / 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 / BAT3 complex binding / cellular response to arsenite ion / protein-DNA covalent cross-linking repair / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination ...: / 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 / BAT3 complex binding / cellular response to arsenite ion / protein-DNA covalent cross-linking repair / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / cytoplasm protein quality control / positive regulation of oxidative phosphorylation / : / aggresome assembly / deubiquitinase activator activity / mitotic spindle disassembly / ubiquitin-modified protein reader activity / regulation of protein localization to chromatin / VCP-NPL4-UFD1 AAA ATPase complex / cellular response to misfolded protein / negative regulation of protein localization to chromatin / positive regulation of mitochondrial membrane potential / vesicle-fusing ATPase / K48-linked polyubiquitin modification-dependent protein binding / regulation of aerobic respiration / retrograde protein transport, ER to cytosol / stress granule disassembly / ATPase complex / regulation of synapse organization / ubiquitin-specific protease binding / positive regulation of ATP biosynthetic process / 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 / ATP metabolic process / endoplasmic reticulum unfolded protein response / proteasomal protein catabolic process / Protein methylation / Attachment and Entry / ERAD pathway / lipid droplet / proteasome complex / viral genome replication / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / negative regulation of smoothened signaling pathway / macroautophagy / positive regulation of protein-containing complex assembly / Hh mutants are degraded by ERAD / establishment of protein localization / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / positive regulation of non-canonical NF-kappaB signal transduction / Translesion Synthesis by POLH / ADP binding / ABC-family proteins mediated transport / autophagy / cytoplasmic stress granule / Aggrephagy / positive regulation of protein catabolic process / azurophil granule lumen / KEAP1-NFE2L2 pathway / Ovarian tumor domain proteases / 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 / site of double-strand break / cellular response to heat / Neddylation / ubiquitin-dependent protein catabolic process / secretory granule lumen / protein phosphatase binding / regulation of apoptotic process / ficolin-1-rich granule lumen / proteasome-mediated ubiquitin-dependent protein catabolic process / Attachment and Entry / protein ubiquitination / ciliary basal body / protein domain specific binding / DNA repair / intracellular membrane-bounded organelle / lipid binding / ubiquitin protein ligase binding / DNA damage response / Neutrophil degranulation / endoplasmic reticulum membrane / perinuclear region of cytoplasm / glutamatergic synapse / endoplasmic reticulum / protein-containing complex / ATP hydrolysis activity / 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: 4.2 Å
AuthorsYu G / Bai Y
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)RO1 CA69202 United States
CitationJournal: iScience / Year: 2021
Title: Cryo-electron microscopy structures of VCP/p97 reveal a new mechanism of oligomerization regulation.
Authors: Guimei Yu / Yunpeng Bai / Kunpeng Li / Ovini Amarasinghe / Wen Jiang / Zhong-Yin Zhang /
Abstract: VCP/p97 is an evolutionarily conserved AAA+ ATPase important for cellular homeostasis. Previous studies suggest that VCP predominantly exists as a homohexamer. Here, we performed structural and ...VCP/p97 is an evolutionarily conserved AAA+ ATPase important for cellular homeostasis. Previous studies suggest that VCP predominantly exists as a homohexamer. Here, we performed structural and biochemical characterization of VCP dodecamer, an understudied state of VCP. The structure revealed an apo nucleotide status that has rarely been captured, a tail-to-tail assembly of two hexamers, and the up-elevated N-terminal domains akin to that seen in the ATP-bound hexamer. Further analyses elucidated a nucleotide status-dependent dodecamerization mechanism, where nucleotide dissociation from the D2 AAA domains induces and promotes VCP dodecamerization. In contrast, nucleotide-free D1 AAA domains are associated with the up-rotation of N-terminal domains, which may prime D1 for ATP binding. These results therefore reveal new nucleotide status-dictated intra- and interhexamer conformational changes and suggest that modulation of D2 domain nucleotide occupancy may serve as a mechanism in controlling VCP oligomeric states.
History
DepositionSep 16, 2020-
Header (metadata) releaseOct 13, 2021-
Map releaseOct 13, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.45
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7k59
  • Surface level: 0.45
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22678.png

Downloads & links

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Map

FileDownload / File: emd_22678.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM density map of apo VCP hexamer generated from bacterially recombinant VCP/p97.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.55 Å/pix.
x 256 pix.
= 397.44 Å		1.55 Å/pix.
x 256 pix.
= 397.44 Å		1.55 Å/pix.
x 256 pix.
= 397.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5525 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.45 / Movie #1: 0.45
Minimum - Maximum-0.8458452 - 2.608808
Average (Standard dev.)0.0038240724 (±0.090986595)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 397.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.55251.55251.5525
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z397.440397.440397.440
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.8462.6090.004

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Supplemental data

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Mask #1

Fileemd_22678_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_22678_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_22678_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : VCP/p97

EntireName: VCP/p97
Components
  • Complex: VCP/p97
    • Protein or peptide: Transitional endoplasmic reticulum ATPase

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Supramolecule #1: VCP/p97

SupramoleculeName: VCP/p97 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: VCP/p97 was overexpressed and purified from E.coli BL21(DE3). Purified protein was treated with apyrase to remove prebound ADP and generate apo VCP/p97.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 540 KDa

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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: 89.464828 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 LQQSRGFGDF RFPSGNQGGA GPSQGSGGGT GGSVYTEDN DDDLYG

UniProtKB: Transitional endoplasmic reticulum ATPase

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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 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMHepes4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
150.0 mMNaClSodium chloride
1.0 mMTCEPtris(2-carboxyethyl)phosphine)

Details: 20mM Hepes pH7.4, 150mM NaCl, 1mM TCEP
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: LACEY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: GRAPHENE OXIDE / Support film - #1 - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 293 K / Instrument: GATAN CRYOPLUNGE 3
Details: 3.5ul sample was applied to a lacy carbon grid coated with graphene oxide. 7 seconds blot with filter paper was performed using Gatan Cp3..
DetailsVCP/p97 was overexpressed and purified with E.coli BL21(DE3). Purified proteins were treated with apyrase to remove prebound ADP and generate apo VCP/p97.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 807 / Average exposure time: 8.0 sec. / Average electron dose: 40.0 e/Å2
Details: A frame rate of 5 frames per second, a dose rate of 7.6 eps and a total exposure of 8 seconds were used.
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

Particle selectionNumber selected: 71600
Startup modelType of model: NONE
Details: random initial models were generated in cryoSPARC from data.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 22172
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC
Details: Two major classes including a dodecamer and a hexamer class. This submission is reporting reconstruction result for the hexamer class.
FSC plot (resolution estimation)

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