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- PDB-5ftn: Cryo-EM structure of human p97 bound to ATPgS (Conformation III) -

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Basic information

Entry
Database: PDB / ID: 5ftn
TitleCryo-EM structure of human p97 bound to ATPgS (Conformation III)
ComponentsTRANSITIONAL ENDOPLASMIC RETICULUM ATPASE
KeywordsHYDROLASE / SINGLE-PARTICLE / P97 / AAA ATPASE
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
Vcp-like ATPase; Chain A, domain 2 - #10 / Vcp-like ATPase; Chain A, domain 2 / Vps4 C terminal oligomerisation domain / Barwin-like endoglucanases - #20 / Helicase, Ruva Protein; domain 3 - #60 / AAA ATPase, CDC48 family / Barwin-like endoglucanases / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain ...Vcp-like ATPase; Chain A, domain 2 - #10 / Vcp-like ATPase; Chain A, domain 2 / Vps4 C terminal oligomerisation domain / Barwin-like endoglucanases - #20 / Helicase, Ruva Protein; domain 3 - #60 / AAA ATPase, CDC48 family / Barwin-like endoglucanases / 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. / Helicase, Ruva Protein; domain 3 / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Roll / Beta Barrel / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Transitional endoplasmic reticulum ATPase
Similarity search - Component
Biological speciesHOMO SAPIENS (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsBanerjee, S. / Bartesaghi, A. / Merk, A. / Rao, P. / Bulfer, S.L. / Yan, Y. / Green, N. / Mroczkowski, B. / Neitz, R.J. / Wipf, P. ...Banerjee, S. / Bartesaghi, A. / Merk, A. / Rao, P. / Bulfer, S.L. / Yan, Y. / Green, N. / Mroczkowski, B. / Neitz, R.J. / Wipf, P. / Falconieri, V. / Deshaies, R.J. / Milne, J.L.S. / Huryn, D. / Arkin, M. / Subramaniam, S.
CitationJournal: Science / Year: 2016
Title: 2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition.
Authors: Soojay Banerjee / Alberto Bartesaghi / Alan Merk / Prashant Rao / Stacie L Bulfer / Yongzhao Yan / Neal Green / Barbara Mroczkowski / R Jeffrey Neitz / Peter Wipf / Veronica Falconieri / ...Authors: Soojay Banerjee / Alberto Bartesaghi / Alan Merk / Prashant Rao / Stacie L Bulfer / Yongzhao Yan / Neal Green / Barbara Mroczkowski / R Jeffrey Neitz / Peter Wipf / Veronica Falconieri / Raymond J Deshaies / Jacqueline L S Milne / Donna Huryn / Michelle Arkin / Sriram Subramaniam /
Abstract: p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo-electron microscopy (cryo-EM) structures for adenosine diphosphate ...p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo-electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)-bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5'-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.
History
DepositionJan 14, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 27, 2016Provider: repository / Type: Initial release
Revision 1.1Feb 10, 2016Group: Database references / Other
Revision 1.2Mar 9, 2016Group: Database references
Revision 1.3Oct 3, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name

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Structure visualization

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)543,19230
Polymers536,6216
Non-polymers6,57124
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: Protein
TRANSITIONAL ENDOPLASMIC RETICULUM ATPASE / TER ATPASE / 15S MG(2+)-ATPASE P97 SUBUNIT / VALOSIN-CONTAINING PROTEIN / VCP


Mass: 89436.820 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HOMO SAPIENS (human) / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): ROSETTA2 / References: UniProt: P55072, vesicle-fusing ATPase
#2: 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 / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg

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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: FULL-LENGTH HUMAN P97 BOUND TO ATPGS / Type: COMPLEX
Buffer solutionName: 25 MM TRIS, 150 MM NACL, 1 MM MGCL2, 1.0 MM TCEP / pH: 8 / Details: 25 MM TRIS, 150 MM NACL, 1 MM MGCL2, 1.0 MM TCEP
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 100, TEMPERATURE- 90.15, INSTRUMENT- FEI VITROBOT MARK IV, METHOD- BLOT FOR 2.5 SECONDS BEFORE PLUNGING.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Jan 17, 2015 / Details: PARALLEL BEAM ILLUMINATION
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Calibrated magnification: 36980 X / Nominal defocus max: 2700 nm / Nominal defocus min: 950 nm / Cs: 2.7 mm
Specimen holderTemperature: 79.7 K
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansNum. digital images: 628

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Processing

EM softwareName: FREALIGN / Category: 3D reconstruction
CTF correctionDetails: EACH PARTICLE
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionMethod: SCORE MINIMIZATION / Resolution: 3.3 Å / Num. of particles: 23306
Details: N-TERMINAL RESIDUES 21-200 DISORDERED. LINKER CONNECTING RESIDUES 707 TO 728 IS NOT IN THE MODEL. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3299. (DEPOSITION ID: 14179).
Symmetry type: POINT
RefinementHighest resolution: 3.3 Å
Refinement stepCycle: LAST / Highest resolution: 3.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms34572 0 384 0 34956

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