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- EMDB-20730: Cryo-EM structure of p97-A232E mutant bound to cofactors UFD1L an... -

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Basic information

Entry
Database: EMDB / ID: EMD-20730
TitleCryo-EM structure of p97-A232E mutant bound to cofactors UFD1L and NPLOC4
Map dataCryo-EM structure of the p97-A232E:NPLOC4-UFD1L complex
Sample
  • Complex: Complex of p97-A232E and cofactors UFD1L and NPLOC4
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.26 Å
AuthorsBlythe EE / Gates SN / Deshaies RJ / Martin A
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM094497 United States
CitationJournal: Structure / Year: 2019
Title: Multisystem Proteinopathy Mutations in VCP/p97 Increase NPLOC4·UFD1L Binding and Substrate Processing.
Authors: Emily E Blythe / Stephanie N Gates / Raymond J Deshaies / Andreas Martin /
Abstract: Valosin-containing protein (VCP)/p97 is an essential ATP-dependent protein unfoldase. Dominant mutations in p97 cause multisystem proteinopathy (MSP), a disease affecting the brain, muscle, and bone. ...Valosin-containing protein (VCP)/p97 is an essential ATP-dependent protein unfoldase. Dominant mutations in p97 cause multisystem proteinopathy (MSP), a disease affecting the brain, muscle, and bone. Despite the identification of numerous pathways that are perturbed in MSP, the molecular-level defects of these p97 mutants are not completely understood. Here, we use biochemistry and cryoelectron microscopy to explore the effects of MSP mutations on the unfoldase activity of p97 in complex with its substrate adaptor NPLOC4⋅UFD1L (UN). We show that all seven analyzed MSP mutants unfold substrates faster. Mutant homo- and heterohexamers exhibit tighter UN binding and faster substrate processing. Our structural studies suggest that the increased UN affinity originates from a decoupling of p97's nucleotide state and the positioning of its N-terminal domains. Together, our data support a gain-of-function model for p97-UN-dependent processes in MSP and underscore the importance of N-terminal domain movements for adaptor recruitment and substrate processing by p97.
History
DepositionSep 17, 2019-
Header (metadata) releaseOct 30, 2019-
Map releaseNov 6, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20730.png

Downloads & links

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Map

FileDownload / File: emd_20730.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the p97-A232E:NPLOC4-UFD1L complex
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.016 / Movie #1: 0.014
Minimum - Maximum-0.031397544 - 0.064114965
Average (Standard dev.)0.00036890848 (±0.0025830094)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 294.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z294.400294.400294.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0310.0640.000

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Supplemental data

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Sample components

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Entire : Complex of p97-A232E and cofactors UFD1L and NPLOC4

EntireName: Complex of p97-A232E and cofactors UFD1L and NPLOC4
Components
  • Complex: Complex of p97-A232E and cofactors UFD1L and NPLOC4

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Supramolecule #1: Complex of p97-A232E and cofactors UFD1L and NPLOC4

SupramoleculeName: Complex of p97-A232E and cofactors UFD1L and NPLOC4 / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET24b
Molecular weightTheoretical: 191 KDa

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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

Concentration0.5 mg/mL
BufferpH: 7.6
Component:
ConcentrationNameFormula
60.0 mMHepes
200.0 mMSodium ChlorideNaClSodium chloride
20.0 mMMagnesium ChlorideMgCl2
1.0 mMDTT
10.0 mMATPAdenosine triphosphate
0.05 %NP-40
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: Gatan Solarus Plasma Cleaner
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: 2 second blot, 3 second wait.
DetailsIncubate complex for 5 minutes at 37 degrees Celsius before plunging.

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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 / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Number real images: 5897 / Average exposure time: 8.0 sec. / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.26 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 50032
FSC plot (resolution estimation)

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