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- PDB-5ftk: Cryo-EM structure of human p97 bound to ADP -

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Basic information

Entry
Database: PDB / ID: 5ftk
TitleCryo-EM structure of human p97 bound to ADP
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 protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / positive regulation of oxidative phosphorylation / ATPase complex / VCP-NSFL1C complex / deubiquitinase activator activity / endosome to lysosome transport via multivesicular body sorting pathway / cellular response to arsenite ion ...positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / positive regulation of oxidative phosphorylation / ATPase complex / 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 / vesicle-fusing ATPase / regulation of protein localization to chromatin / 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
Vcp-like ATPase; Chain A, domain 2 - #10 / Vcp-like ATPase; Chain A, domain 2 / AAA ATPase, CDC48 family / Barwin-like endoglucanases - #20 / 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 ...Vcp-like ATPase; Chain A, domain 2 - #10 / Vcp-like ATPase; Chain A, domain 2 / AAA ATPase, CDC48 family / Barwin-like endoglucanases - #20 / 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 / Helicase, Ruva Protein; domain 3 - #60 / Vps4 C terminal oligomerisation domain / Vps4 oligomerisation, C-terminal / Barwin-like endoglucanases / Aspartate decarboxylase-like domain superfamily / AAA+ lid domain / AAA ATPase, AAA+ lid domain / AAA-protein family signature. / ATPase, AAA-type, conserved site / Helicase, Ruva Protein; domain 3 / 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 nucleotide triphosphate hydrolases / 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
ADENOSINE-5'-DIPHOSPHATE / Transitional endoplasmic reticulum ATPase
Similarity search - Component
Biological speciesHOMO SAPIENS (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 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)541,74718
Polymers536,6216
Non-polymers5,12612
Water1,72996
1


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

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Components

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


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-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 96 / Source method: isolated from a natural source / Formula: H2O

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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 ADP / 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: Sep 19, 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: 215000 X / Calibrated magnification: 78490 X / Nominal defocus max: 2500 nm / Nominal defocus min: 700 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: 1925

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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: 2.4 Å / Num. of particles: 30616
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-3296. (DEPOSITION ID: 14171).
Symmetry type: POINT
RefinementHighest resolution: 2.4 Å
Refinement stepCycle: LAST / Highest resolution: 2.4 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms33954 0 324 96 34374

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