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- PDB-7r7s: p47-bound p97-R155H mutant with ATPgammaS -

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Basic information

Entry
Database: PDB / ID: 7r7s
Titlep47-bound p97-R155H mutant with ATPgammaS
Components
  • NSFL1 cofactor p47
  • Transitional endoplasmic reticulum ATPase
KeywordsHYDROLASE/Lipid Binding Protein / AAA+ ATPase / MOTOR PROTEIN / HYDROLASE / HYDROLASE-Lipid Binding Protein complex
Function / homology
Function and homology information


positive regulation of mitotic centrosome separation / negative regulation of protein localization to centrosome / RHOH GTPase cycle / nuclear membrane reassembly / Golgi stack / positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / endosome to lysosome transport via multivesicular body sorting pathway ...positive regulation of mitotic centrosome separation / negative regulation of protein localization to centrosome / RHOH GTPase cycle / nuclear membrane reassembly / Golgi stack / positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / endosome to lysosome transport via multivesicular body sorting pathway / protein-DNA covalent cross-linking repair / endoplasmic reticulum stress-induced pre-emptive quality control / cellular response to arsenite ion / BAT3 complex binding / Derlin-1 retrotranslocation complex / spindle pole centrosome / positive regulation of protein K63-linked deubiquitination / deubiquitinase activator activity / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / aggresome assembly / NADH metabolic process / regulation of protein localization to chromatin / vesicle-fusing ATPase / cellular response to misfolded protein / : / stress granule disassembly / K48-linked polyubiquitin modification-dependent protein binding / positive regulation of mitochondrial membrane potential / negative regulation of protein localization to chromatin / ERAD pathway / ubiquitin-modified protein reader activity / retrograde protein transport, ER to cytosol / regulation of aerobic respiration / ATPase complex / regulation of synapse organization / ubiquitin-specific protease binding / positive regulation of ATP biosynthetic process / Golgi organization / ubiquitin-like protein ligase binding / autophagosome maturation / establishment of mitotic spindle orientation / RHOH GTPase cycle / autophagosome assembly / polyubiquitin modification-dependent protein binding / HSF1 activation / translesion synthesis / endoplasmic reticulum to Golgi vesicle-mediated transport / MHC class I protein binding / Protein methylation / negative regulation of smoothened signaling pathway / interstrand cross-link repair / Attachment and Entry / : / ATP metabolic process / endoplasmic reticulum unfolded protein response / proteasome complex / lipid droplet / viral genome replication / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / ubiquitin binding / ADP binding / proteasomal protein catabolic process / Hh mutants are degraded by ERAD / positive regulation of protein-containing complex assembly / Defective CFTR causes cystic fibrosis / Hedgehog ligand biogenesis / macroautophagy / Translesion Synthesis by POLH / ABC-family proteins mediated transport / establishment of protein localization / autophagy / Aggrephagy / cytoplasmic stress granule / positive regulation of non-canonical NF-kappaB signal transduction / positive regulation of canonical Wnt signaling pathway / 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 / 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 / chromosome / Neddylation / ATPase binding / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / protein phosphatase binding / regulation of apoptotic process / secretory granule lumen / ficolin-1-rich granule lumen / Attachment and Entry / membrane fusion / protein ubiquitination / protein domain specific 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. / UBA-like domain ...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. / UBA-like 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 / UBA-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 / 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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / NSFL1 cofactor p47 / Transitional endoplasmic reticulum ATPase
Similarity search - Component
Biological speciesHomo sapiens (human)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.23 Å
AuthorsNandi, P. / Li, S. / Coulmbres, R.C.A. / Wang, F. / Williams, D.R. / Malyutin, A.G. / Poh, Y.-P. / Chou, T.-F. / Chiu, P.-L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS102279 United States
CitationJournal: Int J Mol Sci / Year: 2021
Title: Structural and Functional Analysis of Disease-Linked p97 ATPase Mutant Complexes.
Authors: Purbasha Nandi / Shan Li / Rod Carlo A Columbres / Feng Wang / Dewight R Williams / Yu-Ping Poh / Tsui-Fen Chou / Po-Lin Chiu /
Abstract: IBMPFD/ALS is a genetic disorder caused by a single amino acid mutation on the p97 ATPase, promoting ATPase activity and cofactor dysregulation. The disease mechanism underlying p97 ATPase ...IBMPFD/ALS is a genetic disorder caused by a single amino acid mutation on the p97 ATPase, promoting ATPase activity and cofactor dysregulation. The disease mechanism underlying p97 ATPase malfunction remains unclear. To understand how the mutation alters the ATPase regulation, we assembled a full-length p97 with its p47 cofactor and first visualized their structures using single-particle cryo-EM. More than one-third of the population was the dodecameric form. Nucleotide presence dissociates the dodecamer into two hexamers for its highly elevated function. The N-domains of the p97 mutant all show up configurations in ADP- or ATPS-bound states. Our functional and structural analyses showed that the p47 binding is likely to impact the p97 ATPase activities via changing the conformations of arginine fingers. These functional and structural analyses underline the ATPase dysregulation with the miscommunication between the functional modules of the p97.
History
DepositionJun 25, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 4, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 25, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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: NSFL1 cofactor p47
J: NSFL1 cofactor p47
hetero molecules


Theoretical massNumber of molelcules
Total (without water)624,24920
Polymers617,9708
Non-polymers6,27912
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 89417.773 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VCP / Production host: Escherichia coli (E. coli) / References: UniProt: P55072, vesicle-fusing ATPase
#2: Protein NSFL1 cofactor p47 / XY body-associated protein XY40 / p97 cofactor p47


Mass: 40731.855 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Nsfl1c / Production host: Escherichia coli (E. coli) / References: UniProt: O35987
#3: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
Has ligand of interestY

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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: p47-bound p97-R155H mutant with ATPgammaS / Type: COMPLEX / Details: p47-bound p97-R155H mutant with ATPgammaS / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.723 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia virus LL11 / Strain: BL21
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESHEPES1
2150 mMpotassium chlorideKCl1
31 mMTCEPTCEP1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The protein sample was monodisperse.
Specimen supportGrid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 292 K / Details: The grid was blotted for 6 seconds.

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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 FIELDBright-field microscopy / Nominal magnification: 48077 X / Nominal defocus max: -2500 nm / Nominal defocus min: -800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Residual tilt: 0.001 mradians
Image recordingElectron dose: 44.4 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2796
Image scansSampling size: 5 µm

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1Topaz0.2.3particle selection
2SerialEM3.9image acquisition
4cryoSPARC2.15CTF correction
7UCSF Chimera1.14model fitting
9cryoSPARC2.15initial Euler assignment
10cryoSPARC2.15final Euler assignment
11cryoSPARC2.15classification
12cryoSPARC2.153D reconstruction
13PHENIX1.18.2-3874model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 401303
3D reconstructionResolution: 4.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63353 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 124.8 / Protocol: OTHER / Space: REAL / Target criteria: CC
Atomic model buildingPDB-ID: 5FTN

5ftn

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00433789
ELECTRON MICROSCOPYf_angle_d0.9445698
ELECTRON MICROSCOPYf_dihedral_angle_d21.3474666
ELECTRON MICROSCOPYf_chiral_restr0.0585167
ELECTRON MICROSCOPYf_plane_restr0.0075980

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