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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
Sample
  • Complex: Human p97 in complex with Npl4/Ufd1 and K48-linked Ub6 in the presence of ATP
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
    • Protein or peptide: Hexa-ubiquitin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / protein-DNA covalent cross-linking repair / 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 / Derlin-1 retrotranslocation complex ...positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / protein-DNA covalent cross-linking repair / 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 / Derlin-1 retrotranslocation complex / ERAD pathway / positive regulation of protein K63-linked deubiquitination / deubiquitinase activator activity / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / aggresome assembly / regulation of protein localization to chromatin / NADH metabolic process / vesicle-fusing ATPase / : / stress granule disassembly / K48-linked polyubiquitin modification-dependent protein binding / negative regulation of protein localization to chromatin / ubiquitin-dependent protein binding / retrograde protein transport, ER to cytosol / positive regulation of mitochondrial membrane potential / regulation of aerobic respiration / ATPase complex / regulation of synapse organization / positive regulation of ATP biosynthetic process / ubiquitin-specific protease binding / ubiquitin-like protein ligase binding / autophagosome maturation / RHOH GTPase cycle / polyubiquitin modification-dependent protein binding / HSF1 activation / translesion synthesis / endoplasmic reticulum to Golgi vesicle-mediated transport / MHC class I protein binding / Protein methylation / interstrand cross-link repair / Attachment and Entry / ATP metabolic process / endoplasmic reticulum unfolded protein response / : / negative regulation of smoothened signaling pathway / proteasome complex / viral genome replication / lipid droplet / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / proteasomal protein catabolic process / ADP binding / Hh mutants are degraded by ERAD / Defective CFTR causes cystic fibrosis / macroautophagy / Hedgehog ligand biogenesis / Translesion Synthesis by POLH / ABC-family proteins mediated transport / positive regulation of protein-containing complex assembly / establishment of protein localization / autophagy / Aggrephagy / cytoplasmic stress granule / positive regulation of protein catabolic process / activation of cysteine-type endopeptidase activity involved in apoptotic process / KEAP1-NFE2L2 pathway / azurophil granule lumen / double-strand break repair / positive regulation of canonical Wnt signaling pathway / Ovarian tumor domain proteases / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / E3 ubiquitin ligases ubiquitinate target proteins / site of double-strand break / cellular response to heat / Neddylation / proteasome-mediated ubiquitin-dependent protein catabolic process / regulation of apoptotic process / secretory granule lumen / protein phosphatase binding / ficolin-1-rich granule lumen / Attachment and Entry / protein ubiquitination / protein domain specific binding / intracellular membrane-bounded organelle / DNA repair / lipid binding / glutamatergic synapse / DNA damage response / ubiquitin protein ligase binding / Neutrophil degranulation / endoplasmic reticulum membrane / perinuclear region of cytoplasm / endoplasmic reticulum / ATP hydrolysis activity / protein-containing complex / RNA binding / extracellular exosome / extracellular region / nucleoplasm
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 / Aspartate decarboxylase-like domain superfamily / AAA ATPase, AAA+ lid 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 / 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
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)
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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Mask #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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Density Histograms

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Half map: #2

Fileemd_23445_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

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Half map: #1

Fileemd_23445_half_map_2.map
Projections & Slices
AxesZYX

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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
Components
  • Complex: Human p97 in complex with Npl4/Ufd1 and K48-linked Ub6 in the presence of ATP
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
    • Protein or peptide: Hexa-ubiquitin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Human p97 in complex with Npl4/Ufd1 and K48-linked Ub6 in the pre...

SupramoleculeName: Human p97 in complex with Npl4/Ufd1 and K48-linked Ub6 in the presence of ATP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)

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Macromolecule #1: Transitional endoplasmic reticulum ATPase

MacromoleculeName: Transitional endoplasmic reticulum ATPase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: vesicle-fusing ATPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 89.493852 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASGADSKGD DLSTAILKQK NRPNRLIVDE AINEDNSVVS LSQPKMDELQ LFRGDTVLLK GKKRREAVCI VLSDDTCSDE KIRMNRVVR NNLRVRLGDV ISIQPCPDVK YGKRIHVLPI DDTVEGITGN LFEVYLKPYF LEAYRPIRKG DIFLVRGGMR A VEFKVVET ...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 LFKEIGVKPP 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 LFFDQLDSIA 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

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Macromolecule #2: Hexa-ubiquitin

MacromoleculeName: Hexa-ubiquitin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 783.958 Da
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 7 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 7 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration2.5 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 98051
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7ln1:
Cryo-EM structure of human p97 in complex with Npl4/Ufd1 and Ub6 (Class 3)

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