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- EMDB-30154: Human AAA+ ATPase VCP mutant - T76E, AMP-PNP-bound form, Conforma... -

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Basic information

Entry
Database: EMDB / ID: EMD-30154
TitleHuman AAA+ ATPase VCP mutant - T76E, AMP-PNP-bound form, Conformation II
Map dataHuman AAA ATPase VCP, T76E mutant with AMP-PNP-bound, Conformation II
Sample
  • Complex: Transitional endoplasmic reticulum ATPase, VCP.
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsYang C / Zhang H
CitationJournal: Cell Death Differ / Year: 2022
Title: The phosphorylation and dephosphorylation switch of VCP/p97 regulates the architecture of centrosome and spindle.
Authors: Kaiyuan Zhu / Yang Cai / Xiaotong Si / Zuodong Ye / Yuanzhu Gao / Chuang Liu / Rui Wang / Zhibin Ma / Huazhang Zhu / Liang Zhang / Shengjin Li / Hongmin Zhang / Jianbo Yue /
Abstract: The proper orientation of centrosome and spindle is essential for genome stability; however, the mechanism that governs these processes remains elusive. Here, we demonstrated that polo-like kinase 1 ...The proper orientation of centrosome and spindle is essential for genome stability; however, the mechanism that governs these processes remains elusive. Here, we demonstrated that polo-like kinase 1 (Plk1), a key mitotic kinase, phosphorylates residue Thr76 in VCP/p97 (an AAA-ATPase), at the centrosome from prophase to anaphase. This phosphorylation process recruits VCP to the centrosome and in this way, it regulates centrosome orientation. VCP exhibits strong co-localization with Eg5 (a mitotic kinesin motor), at the mitotic spindle, and the dephosphorylation of Thr76 in VCP is required for the enrichment of both VCP and Eg5 at the spindle, thus ensuring proper spindle architecture and chromosome segregation. We also showed that the phosphatase, PTEN, is responsible for the dephosphorylation of Thr76 in VCP; when PTEN was knocked down, the normal spread of VCP from the centrosome to the spindle was abolished. Cryo-EM structures of VCP and VCP, which represent dephosphorylated and phosphorylated states of VCP, respectively, revealed that the Thr76 phosphorylation modulates VCP by altering the inter-domain and inter-subunit interactions, and ultimately the nucleotide-binding pocket conformation. Interestingly, the tumor growth in nude mice implanted with VCP-reconstituted cancer cells was significantly slower when compared with those implanted with VCP-reconstituted cancer cells. Collectively, our findings demonstrate that the phosphorylation and dephosphorylation switch of VCP regulates the architecture of centrosome and spindle for faithful chromosome segregation.
History
DepositionMar 23, 2020-
Header (metadata) releaseMar 31, 2021-
Map releaseMar 31, 2021-
UpdateApr 27, 2022-
Current statusApr 27, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.302
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.302
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30154.png

Downloads & links

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Map

FileDownload / File: emd_30154.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman AAA ATPase VCP, T76E mutant with AMP-PNP-bound, Conformation II
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 360 pix.
= 386.28 Å		1.07 Å/pix.
x 360 pix.
= 386.28 Å		1.07 Å/pix.
x 360 pix.
= 386.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.073 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.302 / Movie #1: 0.302
Minimum - Maximum-0.53 - 1.49
Average (Standard dev.)-1.9524956e-05 (±0.0499997)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 386.27997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0731.0731.073
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z386.280386.280386.280
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ336336336
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.5301.490-0.000

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Supplemental data

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Sample components

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Entire : Transitional endoplasmic reticulum ATPase, VCP.

EntireName: Transitional endoplasmic reticulum ATPase, VCP.
Components
  • Complex: Transitional endoplasmic reticulum ATPase, VCP.

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Supramolecule #1: Transitional endoplasmic reticulum ATPase, VCP.

SupramoleculeName: Transitional endoplasmic reticulum ATPase, VCP. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: VCP T76E mutant of AMP-PNP-bound form,Conformation II
Source (natural)Organism: Homo sapiens (human) / Location in cell: cytoplasm nucleus ER
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: T7 SHuffle (NEB C3026) / Recombinant plasmid: pET
Molecular weightExperimental: 97 kDa/nm

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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

Concentration2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPESHEPES
50.0 mMNaClSodium Chloride
5.0 mMMgCl2Magnesium Chloride
0.001 %2-ME2-Mercaptoethanol
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: RELION (ver. 3.0.6)
Startup modelType of model: EMDB MAP
EMDB ID:

3298

Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. Beta -1.0.0) / Number images used: 533495
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cisTEM (ver. Beta -1.0.0)
Final angle assignmentType: NOT APPLICABLE / Software - Name: cisTEM (ver. Beta -1.0.0)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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