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- EMDB-23445: Cryo-EM structure of human p97 in complex with Npl4/Ufd1 and Ub6 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-23445
TitleCryo-EM structure of human p97 in complex with Npl4/Ufd1 and Ub6 (Class 3)
Map data
SampleHuman p97 in complex with Npl4/Ufd1 and K48-linked Ub6 in the presence of ATP
  • Transitional endoplasmic reticulum ATPase
  • Hexa-ubiquitin
  • (ligand) x 3
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 / ATPase complex / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / deubiquitinase activator activity / endosome to lysosome transport via multivesicular body sorting pathway / cellular response to arsenite ion ...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 / ATPase complex / positive regulation of oxidative phosphorylation / 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 / Derlin-1 retrotranslocation complex / BAT3 complex binding / 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 / K48-linked polyubiquitin modification-dependent protein binding / stress granule disassembly / positive regulation of mitochondrial membrane potential / retrograde protein transport, ER to cytosol / autophagosome maturation / regulation of aerobic respiration / MHC class I protein binding / regulation of synapse organization / positive regulation of ATP biosynthetic process / negative regulation of smoothened signaling pathway / ubiquitin-specific protease binding / ubiquitin-like protein ligase binding / ATP metabolic process / RHOH GTPase cycle / Attachment and Entry / polyubiquitin modification-dependent protein binding / proteasomal protein catabolic process / endoplasmic reticulum to Golgi vesicle-mediated transport / Protein methylation / translesion synthesis / HSF1 activation / proteasome complex / interstrand cross-link repair / endoplasmic reticulum unfolded protein response / lipid droplet / ubiquitin-dependent ERAD pathway / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / macroautophagy / ADP binding / Hh mutants are degraded by ERAD / Defective CFTR causes cystic fibrosis / Hedgehog ligand biogenesis / Translesion Synthesis by POLH / ABC-family proteins mediated transport / positive regulation of protein catabolic process / positive regulation of protein-containing complex assembly / double-strand break repair / establishment of protein localization / Aggrephagy / cytoplasmic stress granule / autophagy / positive regulation of canonical Wnt signaling pathway / azurophil granule lumen / activation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / site of double-strand break / viral genome replication / Ovarian tumor domain proteases / Attachment and Entry / E3 ubiquitin ligases ubiquitinate target proteins / proteasome-mediated ubiquitin-dependent protein catabolic process / cellular response to heat / secretory granule lumen / protein phosphatase binding / regulation of apoptotic process / protein ubiquitination / ficolin-1-rich granule lumen / lipid binding / : / glutamatergic synapse / DNA repair / protein domain specific binding / intracellular membrane-bounded organelle / cellular response to DNA damage stimulus / ubiquitin protein ligase binding / Neutrophil degranulation / endoplasmic reticulum membrane / 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 / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (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 / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (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: 3.4 Å
AuthorsPan M / Yu Y / Liu L / Zhao M
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: Mechanistic insight into substrate processing and allosteric inhibition of human p97.
Authors: Man Pan / Yuanyuan Yu / Huasong Ai / Qingyun Zheng / Yuan Xie / Lei Liu / Minglei Zhao /
Abstract: p97 processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Here, we report a series of cryo-EM structures of the substrate-engaged human p97 complex with ...p97 processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Here, we report a series of cryo-EM structures of the substrate-engaged human p97 complex with resolutions ranging from 2.9 to 3.8 Å that captured 'power-stroke'-like motions of both the D1 and D2 ATPase rings of p97. A key feature of these structures is the critical conformational changes of the intersubunit signaling (ISS) motifs, which tighten the binding of nucleotides and neighboring subunits and contribute to the spiral staircase conformation of the D1 and D2 rings. In addition, we determined the cryo-EM structure of human p97 in complex with NMS-873, a potent p97 inhibitor, at a resolution of 2.4 Å. The structures showed that NMS-873 binds at a cryptic groove in the D2 domain and interacts with the ISS motif, preventing its conformational change and thus blocking substrate translocation allosterically.
History
DepositionFeb 6, 2021-
Header (metadata) releaseSep 15, 2021-
Map releaseSep 15, 2021-
UpdateSep 15, 2021-
Current statusSep 15, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ln1
  • Surface level: 0.015
  • 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_23445.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 320 pix.
= 340.16 Å
1.06 Å/pix.
x 320 pix.
= 340.16 Å
1.06 Å/pix.
x 320 pix.
= 340.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.063 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.0185894 - 0.07590106
Average (Standard dev.)-6.175131e-05 (±0.0023001279)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 340.15997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0631.0631.063
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z340.160340.160340.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0190.076-0.000

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Supplemental data

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Segmentation: #1

Fileemd_23445_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: #1

Fileemd_23445_additional_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_23445_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_23445_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

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Sample components

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Entire Human p97 in complex with Npl4/Ufd1 and K48-linked Ub6 in the pre...

EntireName: Human p97 in complex with Npl4/Ufd1 and K48-linked Ub6 in the presence of ATP
Number of Components: 6

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Component #1: protein, Human p97 in complex with Npl4/Ufd1 and K48-linked Ub6 i...

ProteinName: Human p97 in complex with Npl4/Ufd1 and K48-linked Ub6 in the presence of ATP
Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli K-12 (bacteria)

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Component #2: protein, Transitional endoplasmic reticulum ATPase

ProteinName: Transitional endoplasmic reticulum ATPase / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 89.493852 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, Hexa-ubiquitin

ProteinName: Hexa-ubiquitin / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.783958 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Component #5: ligand, ADENOSINE-5'-TRIPHOSPHATE

LigandName: ADENOSINE-5'-TRIPHOSPHATE / Number of Copies: 7 / Recombinant expression: No
MassTheoretical: 0.507181 kDa

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Component #6: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 7 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2.5 mg/mL / pH: 8
VitrificationCryogen Name: ETHANE / Temperature: 281 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 50 e/Å2 / Illumination Mode: FLOOD BEAM
LensImaging Mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: OTHER

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Image processing

ProcessingMethod: single particle reconstruction / Applied Symmetry: C1 (asymmetric) / Number of Projections: 98051
3D reconstructionSoftware: RELION / Resolution: 3.4 Å / Resolution Method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Output model

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