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- EMDB-22675: Structure of VCP dodecamer purified from H1299 cells -

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Basic information

Entry
Database: EMDB / ID: EMD-22675
TitleStructure of VCP dodecamer purified from H1299 cells
Map datacryo-EM density map of VCP/p97 expressed and purified from H1299 cells
Sample
  • Complex: VCP/p97
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
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 / endosome to lysosome transport via multivesicular body sorting pathway / protein-DNA covalent cross-linking repair / endoplasmic reticulum stress-induced pre-emptive quality control / cellular response to arsenite ion / BAT3 complex binding / Derlin-1 retrotranslocation complex ...positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / endosome to lysosome transport via multivesicular body sorting pathway / protein-DNA covalent cross-linking repair / endoplasmic reticulum stress-induced pre-emptive quality control / cellular response to arsenite ion / BAT3 complex binding / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / deubiquitinase activator activity / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / aggresome assembly / regulation of protein localization to chromatin / NADH metabolic process / vesicle-fusing ATPase / cellular response to misfolded protein / : / stress granule disassembly / K48-linked polyubiquitin modification-dependent protein binding / positive regulation of mitochondrial membrane potential / negative regulation of protein localization to chromatin / ERAD pathway / ubiquitin-modified protein reader activity / retrograde protein transport, ER to cytosol / regulation of aerobic respiration / ATPase complex / regulation of synapse organization / ubiquitin-specific protease binding / positive regulation of ATP biosynthetic process / ubiquitin-like protein ligase binding / autophagosome maturation / RHOH GTPase cycle / polyubiquitin modification-dependent protein binding / HSF1 activation / translesion synthesis / endoplasmic reticulum to Golgi vesicle-mediated transport / MHC class I protein binding / Protein methylation / negative regulation of smoothened signaling pathway / interstrand cross-link repair / Attachment and Entry / : / ATP metabolic process / endoplasmic reticulum unfolded protein response / proteasome complex / lipid droplet / viral genome replication / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / ADP binding / proteasomal protein catabolic process / Hh mutants are degraded by ERAD / positive regulation of protein-containing complex assembly / Defective CFTR causes cystic fibrosis / Hedgehog ligand biogenesis / macroautophagy / Translesion Synthesis by POLH / ABC-family proteins mediated transport / establishment of protein localization / autophagy / Aggrephagy / cytoplasmic stress granule / positive regulation of non-canonical NF-kappaB signal transduction / positive regulation of canonical Wnt signaling pathway / positive regulation of protein catabolic process / activation of cysteine-type endopeptidase activity involved in apoptotic process / KEAP1-NFE2L2 pathway / azurophil granule lumen / double-strand break repair / Ovarian tumor domain proteases / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / E3 ubiquitin ligases ubiquitinate target proteins / site of double-strand break / cellular response to heat / Neddylation / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / protein phosphatase binding / regulation of apoptotic process / secretory granule lumen / ficolin-1-rich granule lumen / Attachment and Entry / protein ubiquitination / protein domain specific binding / DNA repair / intracellular membrane-bounded organelle / lipid binding / glutamatergic synapse / DNA damage response / 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, AAA+ lid 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 / 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: 3.9 Å
AuthorsYu G / Bai Y / Li K / Jiang W / Zhang ZY
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-
UpdateApr 6, 2022-
Current statusApr 6, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.37
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.37
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7k56
  • Surface level: 0.37
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22675.png

Downloads & links

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Map

FileDownload / File: emd_22675.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM density map of VCP/p97 expressed and purified from H1299 cells
Voxel sizeX=Y=Z: 1.4805 Å
Density
Contour LevelBy AUTHOR: 0.37 / Movie #1: 0.37
Minimum - Maximum-1.1348504 - 2.4659681
Average (Standard dev.)0.0056899553 (±0.10353951)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 379.008 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.48051.48051.4805
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z379.008379.008379.008
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-1.1352.4660.006

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Supplemental data

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Mask #1

Fileemd_22675_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_22675_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_22675_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 dodecamer expressed and purified from H1299 cells
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: H1299 / Recombinant plasmid: pCMV
Molecular weightTheoretical: 1.08 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: 89.43682 KDa
Recombinant expressionOrganism: Homo sapiens (human)
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 DDDLYG

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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 chlorideSodium chloride
1.0 mMTCEPtris(2-carboxyethyl)phosphine)

Details: 20mM Hepes pH7.4, 150mM NaCl, 1mM TCEP
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Mesh: 400 / 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 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 15mA for 40s using Emitech.
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 expressed and purified from H1299 cells using anti-Flag resin and 3XFlag peptide.

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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: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 600 / Average exposure time: 10.0 sec. / Average electron dose: 46.0 e/Å2
Details: A dataset of ~600 movies was collected using FEI Titan Krios TEM operating at 300 KV and recorded with the Gatan K2 Summit direct electron detector at super-resolution mode with 22500 ...Details: A dataset of ~600 movies was collected using FEI Titan Krios TEM operating at 300 KV and recorded with the Gatan K2 Summit direct electron detector at super-resolution mode with 22500 nominal magnification. A frame rate of 5 frames per second, a dose rate of 8 eps and a total exposure of 10 seconds were used.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 40000
CTF correctionSoftware: (Name: Gctf, cryoSPARC)
Startup modelType of model: NONE
Details: random initial models were generated in cryoSPARC from data.
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC
Details: 27,500 particles were classified to class1, 8,800 particles to class 2 and 3,800 particles to class 3. Further processing and analysis were performed with class 1 with best quality.
Final angle assignmentType: OTHER / Software - Name: cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 27516
FSC plot (resolution estimation)

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