+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3297 | |||||||||
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Title | Cryo-EM structure of human p97 bound to ATPgS (Conformation I) | |||||||||
Map data | Reconstruction of human p97 bound to ATPgS (conformation I). | |||||||||
Sample |
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Keywords | cryo-electron microscopy / single-particle / p97 / AAA ATPase | |||||||||
Function / homology | Function and homology information protein binding / 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 / endoplasmic reticulum stress-induced pre-emptive quality control / cellular response to arsenite ion / Derlin-1 retrotranslocation complex / BAT3 complex binding ...protein binding / 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 / endoplasmic reticulum stress-induced pre-emptive quality control / cellular response to arsenite ion / Derlin-1 retrotranslocation complex / BAT3 complex binding / protein-DNA covalent cross-linking repair / positive regulation of protein K63-linked deubiquitination / deubiquitinase activator activity / mitotic spindle disassembly / aggresome assembly / VCP-NPL4-UFD1 AAA ATPase complex / regulation of protein localization to chromatin / ubiquitin-modified protein reader activity / vesicle-fusing ATPase / NADH metabolic process / cellular response to misfolded protein / stress granule disassembly / positive regulation of mitochondrial membrane potential / negative regulation of protein localization to chromatin / K48-linked polyubiquitin modification-dependent protein binding / retrograde protein transport, ER to cytosol / regulation of aerobic respiration / positive regulation of ATP biosynthetic process / regulation of synapse organization / ATPase complex / ubiquitin-specific protease binding / ubiquitin-like protein ligase binding / MHC class I protein binding / autophagosome maturation / RHOH GTPase cycle / polyubiquitin modification-dependent protein binding / HSF1 activation / proteasomal protein catabolic process / endoplasmic reticulum to Golgi vesicle-mediated transport / translesion synthesis / Protein methylation / interstrand cross-link repair / ERAD pathway / ATP metabolic process / negative regulation of smoothened signaling pathway / endoplasmic reticulum unfolded protein response / Attachment and Entry / proteasome complex / viral genome replication / lipid droplet / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / macroautophagy / Hh mutants are degraded by ERAD / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / Translesion Synthesis by POLH / positive regulation of protein-containing complex assembly / establishment of protein localization / positive regulation of non-canonical NF-kappaB signal transduction / ABC-family proteins mediated transport / activation of cysteine-type endopeptidase activity involved in apoptotic process / ADP binding / autophagy / Aggrephagy / cytoplasmic stress granule / positive regulation of canonical Wnt signaling pathway / positive regulation of protein catabolic process / azurophil granule lumen / KEAP1-NFE2L2 pathway / double-strand break repair / Ovarian tumor domain proteases / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / E3 ubiquitin ligases ubiquitinate target proteins / Neddylation / site of double-strand break / cellular response to heat / protein phosphatase binding / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / secretory granule lumen / regulation of apoptotic process / ficolin-1-rich granule lumen / Attachment and Entry / protein ubiquitination / protein domain specific binding / intracellular membrane-bounded organelle / DNA repair / signaling receptor binding / DNA damage response / ubiquitin protein ligase binding / lipid binding / glutamatergic synapse / endoplasmic reticulum membrane / Neutrophil degranulation / perinuclear region of cytoplasm / endoplasmic reticulum / ATP hydrolysis activity / protein-containing complex / RNA binding Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) / unidentified (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 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. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3297.map.gz | 73.4 MB | EMDB map data format | |
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Header (meta data) | emd-3297-v30.xml emd-3297.xml | 11.8 KB 11.8 KB | Display Display | EMDB header |
Images | emd_3297.png | 226.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3297 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3297 | HTTPS FTP |
-Validation report
Summary document | emd_3297_validation.pdf.gz | 336.2 KB | Display | EMDB validaton report |
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Full document | emd_3297_full_validation.pdf.gz | 335.3 KB | Display | |
Data in XML | emd_3297_validation.xml.gz | 6.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3297 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3297 | HTTPS FTP |
-Related structure data
Related structure data | 5ftlMC 3295C 3296C 3298C 3299C 5ftjC 5ftkC 5ftmC 5ftnC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_3297.map.gz / Format: CCP4 / Size: 78.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of human p97 bound to ATPgS (conformation I). | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.676 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Full-length human p97 bound to ATPgS
Entire | Name: Full-length human p97 bound to ATPgS |
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Components |
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-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 |
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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 |
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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 |
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Source (natural) | Organism: unidentified (others) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.9 mg/mL |
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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. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 79.6 K / Max: 79.8 K / Average: 79.7 K |
Specialist optics | Energy filter - Name: Gatan, Inc. / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV |
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. |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 36980 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.95 µm / Nominal magnification: 105000 |
Sample stage | Specimen holder: Liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: each particle |
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Final reconstruction | Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: OTHER / Software - Name: FREALIGN / Details: Map was corrected using a B-factor of -85 A^2. / Number images used: 33882 |