|Entry||Database: EMDB / ID: EMD-3298|
|Title||Cryo-EM structure of human p97 bound to ATPgS (Conformation II)|
|Map data||Reconstruction of human p97 bound to ATPgS (conformation II).|
|Keywords||cryo-electron microscopy / single-particle / p97 / AAA ATPase|
|Function / homology|
Function and homology information
protein binding / flavin adenine dinucleotide catabolic process / positive regulation of Lys63-specific deubiquitinase activity / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / 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 ...protein binding / flavin adenine dinucleotide catabolic process / positive regulation of Lys63-specific deubiquitinase activity / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / 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 / positive regulation of oxidative phosphorylation / Derlin-1 retrotranslocation complex / BAT3 complex binding / mitotic spindle disassembly / ERAD pathway / 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 / stress granule disassembly / K48-linked polyubiquitin modification-dependent protein binding / retrograde protein transport, ER to cytosol / regulation of aerobic respiration / positive regulation of mitochondrial membrane potential / ATPase complex / regulation of synapse organization / positive regulation of ATP biosynthetic process / ubiquitin-like protein ligase binding / ubiquitin-specific protease binding / negative regulation of smoothened signaling pathway / autophagosome maturation / ATP metabolic process / RHOH GTPase cycle / polyubiquitin modification-dependent protein binding / Attachment and Entry / endoplasmic reticulum to Golgi vesicle-mediated transport / Protein methylation / MHC class I protein binding / HSF1 activation / translesion synthesis / interstrand cross-link repair / endoplasmic reticulum unfolded protein response / lipid droplet / viral genome replication / ubiquitin-dependent ERAD pathway / Josephin domain DUBs / proteasome complex / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / macroautophagy / proteasomal protein catabolic process / ADP binding / Hh mutants are degraded by ERAD / Defective CFTR causes cystic fibrosis / Hedgehog ligand biogenesis / Translesion Synthesis by POLH / positive regulation of protein-containing complex assembly / ABC-family proteins mediated transport / positive regulation of protein catabolic process / double-strand break repair / establishment of protein localization / Aggrephagy / cytoplasmic stress granule / autophagy / azurophil granule lumen / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / activation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of canonical Wnt signaling pathway / site of double-strand break / Ovarian tumor domain proteases / E3 ubiquitin ligases ubiquitinate target proteins / proteasome-mediated ubiquitin-dependent protein catabolic process / cellular response to heat / Attachment and Entry / secretory granule lumen / protein phosphatase binding / protein ubiquitination / ficolin-1-rich granule lumen / regulation of apoptotic process / lipid binding / glutamatergic synapse / DNA repair / intracellular membrane-bounded organelle / protein domain specific binding / signaling receptor binding / cellular response to DNA damage stimulus / ubiquitin protein ligase binding / endoplasmic reticulum membrane / Neutrophil degranulation / ATP hydrolysis activity / perinuclear region of cytoplasm / endoplasmic reticulum / protein-containing complex / RNA binding / extracellular exosome / extracellular region / nucleoplasm / ATP binding / identical protein binding
Similarity search - Function
AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / CDC48, domain 2 / CDC48 domain 2-like superfamily / Vps4 C terminal oligomerisation domain / Vps4 oligomerisation, C-terminal ...AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / CDC48, domain 2 / CDC48 domain 2-like superfamily / Vps4 C terminal oligomerisation domain / Vps4 oligomerisation, C-terminal / 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 species||Homo sapiens (human) / unidentified (others)|
|Method||single particle reconstruction / cryo EM / Resolution: 3.2 Å|
|Authors||Banerjee S / Bartesaghi A / Merk A / Rao P / Bulfer SL / Yan Y / Green N / Mroczkowski B / Neitz RJ / Wipf P ...Banerjee S / Bartesaghi A / Merk A / Rao P / Bulfer SL / Yan Y / Green N / Mroczkowski B / Neitz RJ / Wipf P / Falconieri V / Deshaies RJ / Milne JLS / Huryn D / Arkin M / Subramaniam S|
|Citation||Journal: 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.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_3298.map.gz / Format: CCP4 / Size: 78.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Annotation||Reconstruction of human p97 bound to ATPgS (conformation II).|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 0.676 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire : Full-length human p97 bound to ATPgS
|Entire||Name: Full-length human p97 bound to ATPgS|
-Supramolecule #1000: Full-length human p97 bound to ATPgS
|Supramolecule||Name: Full-length human p97 bound to ATPgS / type: sample / ID: 1000 / Details: The sample was monodisperse. / Oligomeric state: hexamer / Number unique components: 2|
|Molecular weight||Theoretical: 540 KDa / Method: sequence|
-Macromolecule #1: p97/VCP Transitional endoplasmic reticulum ATPase
|Macromolecule||Name: p97/VCP Transitional endoplasmic reticulum ATPase / type: protein_or_peptide / ID: 1 / Name.synonym: p97 / Number of copies: 1 / Oligomeric state: hexamer / Recombinant expression: Yes|
|Source (natural)||Organism: Homo sapiens (human) / synonym: Human / Location in cell: cytoplasm|
|Molecular weight||Theoretical: 540 KDa|
|Recombinant expression||Organism: Bacteria (eubacteria) / Recombinant cell: E. coli / Recombinant plasmid: Rosetta2 (DE3)|
|Sequence||UniProtKB: Transitional endoplasmic reticulum ATPase|
GO: ATP binding, signaling receptor binding, protein binding, lipid binding
-Macromolecule #2: Adenosine 5 gamma-thio triphosphate
|Macromolecule||Name: Adenosine 5 gamma-thio triphosphate / type: ligand / ID: 2 / Name.synonym: ATPgS / Number of copies: 12 / Oligomeric state: monomer / Recombinant expression: No|
|Source (natural)||Organism: unidentified (others)|
|Processing||single particle reconstruction|
|Buffer||pH: 8 / Details: 25 mM Tris, 150 mM NaCl, 1 mM MgCl2, 1.0 mM TCEP|
|Grid||Details: 200 mesh Quantifoil R1.2/1/3 grids|
|Vitrification||Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90.15 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 2.5 seconds before plunging.|
|Microscope||FEI TITAN KRIOS|
|Electron beam||Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN|
|Electron optics||Calibrated magnification: 36980 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.95 µm / Nominal magnification: 105000|
|Specialist optics||Energy filter - Name: Gatan, Inc. / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV|
|Sample stage||Specimen holder: Liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Temperature||Min: 79.6 K / Max: 79.8 K / Average: 79.7 K|
|Details||Parallel beam illumination|
|Date||Jan 17, 2015|
|Image recording||Category: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 628 / Average electron dose: 40 e/Å2|
Details: Every image is the average of 40 frames recorded by the direct electron detector.
Model: Titan Krios / Image courtesy: FEI Company
|CTF correction||Details: each particle|
|Final reconstruction||Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: OTHER / Software - Name: FREALIGN / Details: Map was corrected using a B-factor of -85 A^2. / Number images used: 32406|
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