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

Basic information

• Entry: Database: EMDB / ID: EMD-22675
• Title: Structure of VCP dodecamer purified from H1299 cells
• Map data: cryo-EM density map of VCP/p97 expressed and purified from H1299 cells

Sample

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

• Keywords: AAA ATPase / ATP hydrolysis / segregase / CYTOSOLIC PROTEIN / HYDROLASE

Function / homology

Function:

: / 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 / cellular response to arsenite ion / BAT3 complex binding / cytoplasm protein quality control / Derlin-1 retrotranslocation complex / protein-DNA covalent cross-linking repair / positive regulation of protein K63-linked deubiquitination / positive regulation of oxidative phosphorylation / mitotic spindle disassembly / NADH metabolic process / aggresome assembly / deubiquitinase activator activity / regulation of protein localization to chromatin / ubiquitin-modified protein reader activity / VCP-NPL4-UFD1 AAA ATPase complex / vesicle-fusing ATPase / cellular response to misfolded protein / negative regulation of protein localization to chromatin / positive regulation of mitochondrial membrane potential / K48-linked polyubiquitin modification-dependent protein binding / retrograde protein transport, ER to cytosol / regulation of aerobic respiration / stress granule disassembly / 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 / negative regulation of hippo signaling / endoplasmic reticulum to Golgi vesicle-mediated transport / HSF1 activation / translesion synthesis / interstrand cross-link repair / proteasomal protein catabolic process / ATP metabolic process / Protein methylation / endoplasmic reticulum unfolded protein response / ERAD pathway / Attachment and Entry / lipid droplet / negative regulation of smoothened signaling pathway / proteasome complex / viral genome replication / : / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / positive regulation of protein-containing complex assembly / Hh mutants are degraded by ERAD / macroautophagy / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / establishment of protein localization / Translesion Synthesis by POLH / positive regulation of non-canonical NF-kappaB signal transduction / ADP binding / ABC-family proteins mediated transport / autophagy / positive regulation of protein catabolic process / Aggrephagy / cytoplasmic stress granule / KEAP1-NFE2L2 pathway / azurophil granule lumen / 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 / Neddylation / site of double-strand break / 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 / chromatin extrusion motor activity / ATP-dependent H2AZ histone chaperone activity / ATP-dependent H3-H4 histone complex chaperone activity / cohesin loader activity / intracellular membrane-bounded organelle / DNA clamp loader activity / DNA repair / lipid binding / ubiquitin protein ligase binding / DNA damage response / Neutrophil degranulation / endoplasmic reticulum membrane / perinuclear region of cytoplasm

Domain/homology:

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

Component:

Transitional endoplasmic reticulum ATPase

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 3.9 Å
• Authors: Yu G / Bai Y

Funding support

United States, 1 items

Organization	Grant number	Country
National Institutes of Health/National Cancer Institute (NIH/NCI)	RO1 CA69202	United States

• Citation: Journal: 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 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

Deposition	Sep 16, 2020	-
Header (metadata) release	Oct 13, 2021	-
Map release	Oct 13, 2021	-
Update	May 29, 2024	-
Current status	May 29, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_22675.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: cryo-EM density map of VCP/p97 expressed and purified from H1299 cells
• Voxel size: X=Y=Z: 1.4805 Å

Density

Contour Level	By AUTHOR: 0.37 / Movie #1: 0.37
Minimum - Maximum	-1.1348504 - 2.4659681
Average (Standard dev.)	0.0056899553 (±0.10353951)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 256 256 256 Spacing 256 256 256
Cell	A=B=C: 379.008 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.4805	1.4805	1.4805
M x/y/z	256	256	256
origin x/y/z	0.000	0.000	0.000
length x/y/z	379.008	379.008	379.008
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	256	256	256
D min/max/mean	-1.135	2.466	0.006

Supplemental data

Mask #1

• File: emd_22675_msk_1.map

Half map: half map 1

• File: emd_22675_half_map_1.map
• Annotation: half map 1

Half map: half map 2

• File: emd_22675_half_map_2.map
• Annotation: half map 2

Sample components

Entire : VCP/p97

• Entire: Name: VCP/p97

Components

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

Supramolecule #1: VCP/p97

• Supramolecule: Name: 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)
• Molecular weight: Theoretical: 1.08 MDa

Macromolecule #1: Transitional endoplasmic reticulum ATPase

• Macromolecule: Name: 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 weight: Theoretical: 89.43682 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

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

UniProtKB: Transitional endoplasmic reticulum ATPase

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 1 mg/mL

Buffer

pH: 7.4
Component:

Concentration	Formula	Name
20.0 mM	Hepes	4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
150.0 mM	NaCl	Sodium chloride
1.0 mM	TCEP	tris(2-carboxyethyl)phosphine)

Details: 20mM Hepes pH7.4, 150mM NaCl, 1mM TCEP

• Grid: Model: 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 - Time: 40 sec. / Pretreatment - Atmosphere: AIR / Details: 15mA for 40s using Emitech.
• Vitrification: Cryogen 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..
• Details: VCP/p97 was expressed and purified from H1299 cells using anti-Flag resin and 3XFlag peptide.

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Image recording: Film 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/Ų; 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.
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 40000
• Startup model: Type of model: NONE; Details: random initial models were generated in cryoSPARC from data.
• Final reconstruction: Number 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
• Initial angle assignment: Type: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
• Final angle assignment: Type: OTHER / Software - Name: cryoSPARC
• Final 3D classification: Number 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.