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- EMDB-23928: Cryo-EM structure of affinity captured human p97 double-hexamer -

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Basic information

Entry
Database: EMDB / ID: EMD-23928
TitleCryo-EM structure of affinity captured human p97 double-hexamer
Map data
Sample
  • Complex: Full-length human p97
Function / homology
Function and homology information


flavin adenine dinucleotide catabolic process / positive regulation of Lys63-specific deubiquitinase activity / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / VCP-NSFL1C complex / deubiquitinase activator activity / endosome to lysosome transport via multivesicular body sorting pathway / cellular response to arsenite ion / endoplasmic reticulum stress-induced pre-emptive quality control / positive regulation of oxidative phosphorylation ...flavin adenine dinucleotide catabolic process / positive regulation of Lys63-specific deubiquitinase activity / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / VCP-NSFL1C complex / deubiquitinase activator activity / endosome to lysosome transport via multivesicular body sorting pathway / cellular response to arsenite ion / endoplasmic reticulum stress-induced pre-emptive quality control / positive regulation of oxidative phosphorylation / BAT3 complex binding / Derlin-1 retrotranslocation complex / ERAD pathway / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / NADH metabolic process / aggresome assembly / regulation of protein localization to chromatin / vesicle-fusing ATPase / ER-associated misfolded protein catabolic process / stress granule disassembly / K48-linked polyubiquitin modification-dependent protein binding / retrograde protein transport, ER to cytosol / regulation of aerobic respiration / positive regulation of mitochondrial membrane potential / ATPase complex / regulation of synapse organization / positive regulation of ATP biosynthetic process / ubiquitin-like protein ligase binding / ubiquitin-specific protease binding / negative regulation of smoothened signaling pathway / autophagosome maturation / ATP metabolic process / RHOH GTPase cycle / polyubiquitin modification-dependent protein binding / Attachment and Entry / endoplasmic reticulum to Golgi vesicle-mediated transport / Protein methylation / MHC class I protein binding / HSF1 activation / translesion synthesis / interstrand cross-link repair / endoplasmic reticulum unfolded protein response / lipid droplet / viral genome replication / ubiquitin-dependent ERAD pathway / Josephin domain DUBs / proteasome complex / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / macroautophagy / proteasomal protein catabolic process / ADP binding / Hh mutants are degraded by ERAD / Defective CFTR causes cystic fibrosis / Hedgehog ligand biogenesis / Translesion Synthesis by POLH / positive regulation of protein-containing complex assembly / ABC-family proteins mediated transport / positive regulation of protein catabolic process / double-strand break repair / establishment of protein localization / Aggrephagy / cytoplasmic stress granule / autophagy / azurophil granule lumen / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / activation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of canonical Wnt signaling pathway / site of double-strand break / Ovarian tumor domain proteases / E3 ubiquitin ligases ubiquitinate target proteins / proteasome-mediated ubiquitin-dependent protein catabolic process / cellular response to heat / Attachment and Entry / secretory granule lumen / protein phosphatase binding / protein ubiquitination / ficolin-1-rich granule lumen / regulation of apoptotic process / lipid binding / glutamatergic synapse / DNA repair / intracellular membrane-bounded organelle / protein domain specific binding / cellular response to DNA damage stimulus / ubiquitin protein ligase binding / endoplasmic reticulum membrane / Neutrophil degranulation / ATP hydrolysis activity / perinuclear region of cytoplasm / endoplasmic reticulum / protein-containing complex / RNA binding / extracellular exosome / extracellular region / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol
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 / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / CDC48, domain 2 / CDC48 domain 2-like superfamily / Vps4 oligomerisation, C-terminal / Vps4 C terminal oligomerisation 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 / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / CDC48, domain 2 / CDC48 domain 2-like superfamily / Vps4 oligomerisation, C-terminal / Vps4 C terminal oligomerisation domain / Aspartate decarboxylase-like domain superfamily / AAA+ lid domain / AAA ATPase, AAA+ lid domain / AAA-protein family signature. / ATPase, AAA-type, conserved site / 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: 3.57 Å
AuthorsHoq MR / Vago FS / Li K / Kovaliov M / Nicholas RJ / Huryn DM / Wipf P / Jiang W / Thompson DH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: ACS Nano / Year: 2021
Title: Affinity Capture of p97 with Small-Molecule Ligand Bait Reveals a 3.6 Å Double-Hexamer Cryoelectron Microscopy Structure.
Authors: Md Rejaul Hoq / Frank S Vago / Kunpeng Li / Marina Kovaliov / Robert J Nicholas / Donna M Huryn / Peter Wipf / Wen Jiang / David H Thompson /
Abstract: Recent progress in the development of affinity grids for cryoelectron microscopy (cryo-EM) typically employs genetic engineering of the protein sample such as histidine or Spy tagging, immobilized ...Recent progress in the development of affinity grids for cryoelectron microscopy (cryo-EM) typically employs genetic engineering of the protein sample such as histidine or Spy tagging, immobilized antibody capture, or nonselective immobilization via electrostatic interactions or Schiff base formation. We report a powerful and flexible method for the affinity capture of target proteins for cryo-EM analysis that utilizes small-molecule ligands as bait for concentrating human target proteins directly onto the grid surface for single-particle reconstruction. This approach is demonstrated for human p97, captured using two different small-molecule high-affinity ligands of this AAA+ ATPase. Four electron density maps are revealed, each representing a p97 conformational state captured from solution, including a double-hexamer structure resolved to 3.6 Å. These results demonstrate that the noncovalent capture of protein targets on EM grids modified with high-affinity ligands can enable the structure elucidation of multiple configurational states of the target and potentially inform structure-based drug design campaigns.
History
DepositionMay 3, 2021-
Header (metadata) releaseJun 2, 2021-
Map releaseJun 2, 2021-
UpdateJun 9, 2021-
Current statusJun 9, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 5.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_23928.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.36 Å/pix.
x 240 pix.
= 326.4 Å
1.36 Å/pix.
x 240 pix.
= 326.4 Å
1.36 Å/pix.
x 240 pix.
= 326.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.36 Å
Density
Contour LevelBy AUTHOR: 5.5 / Movie #1: 5.5
Minimum - Maximum-11.566196 - 18.911308
Average (Standard dev.)-1.5263049e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-120-120-120
Dimensions240240240
Spacing240240240
CellA=B=C: 326.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z326.400326.400326.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ192192192
MAP C/R/S123
start NC/NR/NS-120-120-120
NC/NR/NS240240240
D min/max/mean-11.56618.911-0.000

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Supplemental data

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Mask #1

Fileemd_23928_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: #1

Fileemd_23928_additional_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_23928_half_map_1.map
Projections & Slices
AxesZYX

Projections

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Density Histograms

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Half map: #2

Fileemd_23928_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

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Sample components

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Entire : Full-length human p97

EntireName: Full-length human p97
Components
  • Complex: Full-length human p97

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Supramolecule #1: Full-length human p97

SupramoleculeName: Full-length human p97 / type: complex / ID: 1 / Parent: 0 / Details: p97/VCP Transitional endoplasmic reticulum ATPase
Source (natural)Organism: Homo sapiens (human) / Location in cell: Cytoplasm
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 1.080 MDa

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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

Concentration0.015 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
250.0 mMKClpotasium chloride
1.0 mMMgCl2magnesium chloride
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 3.0 nm
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Instrument: GATAN CRYOPLUNGE 3 / Details: Blot for 8 seconds 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: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
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 / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Sampling interval: 2.5 µm / Number real images: 1232 / Average exposure time: 10.0 sec. / Average electron dose: 43.25 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 77783
CTF correctionSoftware:
Namedetails
Gctf (ver. 1.06)Gctf was used to determine CTF correction parameters.
cryoSPARC (ver. 3.1.0)cryoSPARC was used to apply CTF correction parameters.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0)
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1.0) / Number images used: 12921
FSC plot (resolution estimation)

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