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- PDB-8b5r: p97-p37-SPI substrate complex -

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Basic information

Entry
Database: PDB / ID: 8b5r
Titlep97-p37-SPI substrate complex
Components
  • (UBX domain-containing protein ...) x 2
  • I3 sequence being threaded through the p97 channel
  • Protein phosphatase 1 regulatory subunit 7
  • Serine/threonine-protein phosphatase PP1-gamma catalytic subunit
  • Transitional endoplasmic reticulum ATPase
KeywordsCHAPERONE / AAA+ ATPase / p97 / VCP / Cdc48 / unfoldase / protein phosphatase-1 / protein maturation
Function / homology
Function and homology information


negative regulation of protein localization to centrosome / positive regulation of mitotic centrosome separation / PTW/PP1 phosphatase complex / regulation of nucleocytoplasmic transport / protein phosphatase 1 binding / nuclear membrane reassembly / lamin binding / protein phosphatase regulator activity / spindle pole centrosome / : ...negative regulation of protein localization to centrosome / positive regulation of mitotic centrosome separation / PTW/PP1 phosphatase complex / regulation of nucleocytoplasmic transport / protein phosphatase 1 binding / nuclear membrane reassembly / lamin binding / protein phosphatase regulator activity / spindle pole centrosome / : / flavin adenine dinucleotide catabolic process / VCP-NSFL1C complex / endosome to lysosome transport via multivesicular body sorting pathway / endoplasmic reticulum stress-induced pre-emptive quality control / BAT3 complex binding / cellular response to arsenite ion / protein-DNA covalent cross-linking repair / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / cytoplasm protein quality control / positive regulation of oxidative phosphorylation / : / aggresome assembly / deubiquitinase activator activity / mitotic spindle disassembly / ubiquitin-modified protein reader activity / regulation of protein localization to chromatin / VCP-NPL4-UFD1 AAA ATPase complex / cellular response to misfolded protein / negative regulation of protein localization to chromatin / positive regulation of mitochondrial membrane potential / vesicle-fusing ATPase / K48-linked polyubiquitin modification-dependent protein binding / regulation of aerobic respiration / retrograde protein transport, ER to cytosol / stress granule disassembly / glycogen metabolic process / entrainment of circadian clock by photoperiod / protein-serine/threonine phosphatase / ATPase complex / Triglyceride catabolism / regulation of synapse organization / ubiquitin-specific protease binding / Golgi organization / protein serine/threonine phosphatase activity / phosphatase activity / positive regulation of ATP biosynthetic process / MHC class I protein binding / Maturation of hRSV A proteins / microtubule organizing center / cleavage furrow / ubiquitin-like protein ligase binding / establishment of mitotic spindle orientation / phosphoprotein phosphatase activity / mitotic sister chromatid segregation / RHOH GTPase cycle / positive regulation of glial cell proliferation / polyubiquitin modification-dependent protein binding / autophagosome maturation / autophagosome assembly / blastocyst development / endoplasmic reticulum to Golgi vesicle-mediated transport / negative regulation of hippo signaling / HSF1 activation / translesion synthesis / interstrand cross-link repair / protein dephosphorylation / ATP metabolic process / endoplasmic reticulum unfolded protein response / proteasomal protein catabolic process / Protein methylation / enzyme regulator activity / Attachment and Entry / ERAD pathway / lipid droplet / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / proteasome complex / viral genome replication / Resolution of Sister Chromatid Cohesion / ubiquitin binding / Josephin domain DUBs / Downregulation of TGF-beta receptor signaling / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / negative regulation of smoothened signaling pathway / macroautophagy / circadian regulation of gene expression / establishment of protein localization / Hh mutants are degraded by ERAD / positive regulation of protein-containing complex assembly / RAF activation / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / RHO GTPases Activate Formins / positive regulation of non-canonical NF-kappaB signal transduction / Translesion Synthesis by POLH / ADP binding
Similarity search - Function
SEP domain / NSFL1 cofactor p47, SEP domain superfamily / SEP domain / SEP domain profile. / Domain present in Saccharomyces cerevisiae Shp1, Drosophila melanogaster eyes closed gene (eyc), and vertebrate p47. / : / Domain present in ubiquitin-regulatory proteins / UBX domain / UBX domain / UBX domain profile. ...SEP domain / NSFL1 cofactor p47, SEP domain superfamily / SEP domain / SEP domain profile. / Domain present in Saccharomyces cerevisiae Shp1, Drosophila melanogaster eyes closed gene (eyc), and vertebrate p47. / : / Domain present in ubiquitin-regulatory proteins / UBX domain / UBX domain / UBX domain profile. / Serine-threonine protein phosphatase, N-terminal / : / Serine-threonine protein phosphatase N-terminal domain / Leucine rich repeat 4 / Leucine Rich repeats (2 copies) / U2A'/phosphoprotein 32 family A, C-terminal / occurring C-terminal to leucine-rich repeats / Leucine-rich repeat / Serine/threonine specific protein phosphatases signature. / Protein phosphatase 2A homologues, catalytic domain. / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / 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 / Leucine-rich repeat, SDS22-like subfamily / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / Aspartate decarboxylase-like domain superfamily / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / Leucine-rich repeat profile. / Leucine-rich repeat / Leucine-rich repeat domain superfamily / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Ubiquitin-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Serine/threonine-protein phosphatase PP1-gamma catalytic subunit / Transitional endoplasmic reticulum ATPase / UBX domain-containing protein 2B / Protein phosphatase 1 regulatory subunit 7
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.1 Å
Authorsvan den Boom, J. / Marini, G. / Meyer, H. / Saibil, H.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust106249/Z/14/Z United Kingdom
Wellcome Trust202679/Z/16/Z United Kingdom
Wellcome Trust206166/Z/17/Z United Kingdom
CitationJournal: EMBO J / Year: 2023
Title: Structural basis of ubiquitin-independent PP1 complex disassembly by p97.
Authors: Johannes van den Boom / Guendalina Marini / Hemmo Meyer / Helen R Saibil /
Abstract: The AAA+-ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is ...The AAA+-ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is unclear. Here, we present cryo-EM structures of p97 in the process of disassembling a protein phosphatase-1 (PP1) complex by extracting an inhibitory subunit from PP1. We show that PP1 and its partners SDS22 and inhibitor-3 (I3) are loaded tightly onto p97, surprisingly via a direct contact of SDS22 with the p97 N-domain. Loading is assisted by the p37 adapter that bridges two adjacent p97 N-domains underneath the substrate complex. A stretch of I3 is threaded into the central channel of the spiral-shaped p97 hexamer, while other elements of I3 are still attached to PP1. Thus, our data show how p97 arranges a protein complex between the p97 N-domain and central channel, suggesting a hold-and-extract mechanism for p97-mediated disassembly.
History
DepositionSep 24, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 7, 2023Provider: repository / Type: Initial release
Revision 2.0Jun 14, 2023Group: Atomic model / Database references / Category: atom_site / citation / citation_author
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_atom_id / _atom_site.label_atom_id / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 2.1Jul 26, 2023Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 2.2Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transitional endoplasmic reticulum ATPase
B: Transitional endoplasmic reticulum ATPase
C: Transitional endoplasmic reticulum ATPase
D: Transitional endoplasmic reticulum ATPase
E: Transitional endoplasmic reticulum ATPase
F: Transitional endoplasmic reticulum ATPase
I: I3 sequence being threaded through the p97 channel
P: Serine/threonine-protein phosphatase PP1-gamma catalytic subunit
S: Protein phosphatase 1 regulatory subunit 7
X: UBX domain-containing protein 2B
Y: UBX domain-containing protein 2B


Theoretical massNumber of molelcules
Total (without water)633,01411
Polymers633,01411
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 8 molecules ABCDEFPS

#1: Protein
Transitional endoplasmic reticulum ATPase / TER ATPase / 15S Mg(2+)-ATPase p97 subunit / Valosin-containing protein / VCP


Mass: 90265.695 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VCP / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P55072, vesicle-fusing ATPase
#3: Protein Serine/threonine-protein phosphatase PP1-gamma catalytic subunit / PP-1G / Protein phosphatase 1C catalytic subunit


Mass: 37030.777 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PPP1CC / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P36873, protein-serine/threonine phosphatase
#4: Protein Protein phosphatase 1 regulatory subunit 7 / Protein phosphatase 1 regulatory subunit 22


Mass: 41616.129 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PPP1R7, SDS22 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q15435

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Protein/peptide , 1 types, 1 molecules I

#2: Protein/peptide I3 sequence being threaded through the p97 channel


Mass: 1890.321 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper)

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UBX domain-containing protein ... , 2 types, 2 molecules XY

#5: Protein/peptide UBX domain-containing protein 2B / NSFL1 cofactor p37 / p97 cofactor p37


Mass: 1123.238 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBXN2B
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q14CS0
#6: Protein UBX domain-containing protein 2B / NSFL1 cofactor p37 / p97 cofactor p37


Mass: 9759.220 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBXN2B
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q14CS0

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: p97 complex with p37 adaptor and SPI substrate / Type: COMPLEX / Entity ID: #1-#4, #6, #5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3300 nm / Nominal defocus min: 1800 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 48 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 191477 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00226454
ELECTRON MICROSCOPYf_angle_d0.50737038
ELECTRON MICROSCOPYf_dihedral_angle_d4.8555196
ELECTRON MICROSCOPYf_chiral_restr0.0444868
ELECTRON MICROSCOPYf_plane_restr0.0055462

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