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Yorodumi- EMDB-20865: Cryo-EM structure of the asymmetric AAA+ domain hexamer from Ther... -
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-Basic information
Entry | Database: EMDB / ID: EMD-20865 | |||||||||
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Title | Cryo-EM structure of the asymmetric AAA+ domain hexamer from Thermococcus gammatolerans McrB | |||||||||
Map data | TgMcrB-AAA hexamer | |||||||||
Sample |
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Keywords | AAA protein / GTPase / Methylation-dependent restriction / DNA BINDING PROTEIN | |||||||||
Function / homology | Function and homology information endonuclease activity / ATP hydrolysis activity / ATP binding / metal ion binding Similarity search - Function | |||||||||
Biological species | Thermococcus gammatolerans (archaea) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
Authors | Niu Y / Suzuki H | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Nat Commun / Year: 2020 Title: Structural asymmetry governs the assembly and GTPase activity of McrBC restriction complexes. Authors: Yiming Niu / Hiroshi Suzuki / Christopher J Hosford / Thomas Walz / Joshua S Chappie / Abstract: McrBC complexes are motor-driven nucleases functioning in bacterial self-defense by cleaving foreign DNA. The GTP-specific AAA + protein McrB powers translocation along DNA and its hydrolysis ...McrBC complexes are motor-driven nucleases functioning in bacterial self-defense by cleaving foreign DNA. The GTP-specific AAA + protein McrB powers translocation along DNA and its hydrolysis activity is stimulated by its partner nuclease McrC. Here, we report cryo-EM structures of Thermococcus gammatolerans McrB and McrBC, and E. coli McrBC. The McrB hexamers, containing the necessary catalytic machinery for basal GTP hydrolysis, are intrinsically asymmetric. This asymmetry directs McrC binding so that it engages a single active site, where it then uses an arginine/lysine-mediated hydrogen-bonding network to reposition the asparagine in the McrB signature motif for optimal catalytic function. While the two McrBC complexes use different DNA-binding domains, these contribute to the same general GTP-recognition mechanism employed by all G proteins. Asymmetry also induces distinct inter-subunit interactions around the ring, suggesting a coordinated and directional GTP-hydrolysis cycle. Our data provide insights into the conserved molecular mechanisms governing McrB family AAA + motors. | |||||||||
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_20865.map.gz | 60 MB | EMDB map data format | |
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Header (meta data) | emd-20865-v30.xml emd-20865.xml | 17.3 KB 17.3 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_20865_fsc.xml | 8.9 KB | Display | FSC data file |
Images | emd_20865.png | 60 KB | ||
Masks | emd_20865_msk_1.map | 64 MB | Mask map | |
Filedesc metadata | emd-20865.cif.gz | 6 KB | ||
Others | emd_20865_half_map_1.map.gz emd_20865_half_map_2.map.gz | 49.6 MB 49.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-20865 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-20865 | HTTPS FTP |
-Validation report
Summary document | emd_20865_validation.pdf.gz | 1002.6 KB | Display | EMDB validaton report |
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Full document | emd_20865_full_validation.pdf.gz | 1002.2 KB | Display | |
Data in XML | emd_20865_validation.xml.gz | 13.4 KB | Display | |
Data in CIF | emd_20865_validation.cif.gz | 19.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20865 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20865 | HTTPS FTP |
-Related structure data
Related structure data | 6ut4MC 6ut3C 6ut5C 6ut6C 6ut7C 6ut8C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10582 (Title: Single-particle Cryo-EM of the Thermococcus gammatolerans McrB AAA+ hexamer Data size: 644.6 Data #1: Unaligned multi-frame micrographs of the Thermococcus gammatolerans McrB AAA+ hexamer [micrographs - multiframe]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_20865.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | TgMcrB-AAA hexamer | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.25 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_20865_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: Half map 1
File | emd_20865_half_map_1.map | ||||||||||||
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Annotation | Half map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 2
File | emd_20865_half_map_2.map | ||||||||||||
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Annotation | Half map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Homo-hexameric assembly of the AAA domain of Thermococcus gammato...
Entire | Name: Homo-hexameric assembly of the AAA domain of Thermococcus gammatolerans McrB |
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Components |
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-Supramolecule #1: Homo-hexameric assembly of the AAA domain of Thermococcus gammato...
Supramolecule | Name: Homo-hexameric assembly of the AAA domain of Thermococcus gammatolerans McrB type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Thermococcus gammatolerans (archaea) |
-Macromolecule #1: GTPase subunit of restriction endonuclease
Macromolecule | Name: GTPase subunit of restriction endonuclease / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO |
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Source (natural) | Organism: Thermococcus gammatolerans (archaea) |
Molecular weight | Theoretical: 50.137219 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MNESAGHNIK EDYFRVDMLL NKKGQVILYG PPGTGKTWIA RKYVVEETNE KTPGNKWEFI TFHQSYSYEE FIEGFRPRTD NEEKIRYVV EDGIFKKIAL RALVKGLFEL EDATIGKDKI HRLYILLTKK EPLSPTEYEE YLRLKRYLWE LVGGLPKDKL K NLTPKFYL ...String: MNESAGHNIK EDYFRVDMLL NKKGQVILYG PPGTGKTWIA RKYVVEETNE KTPGNKWEFI TFHQSYSYEE FIEGFRPRTD NEEKIRYVV EDGIFKKIAL RALVKGLFEL EDATIGKDKI HRLYILLTKK EPLSPTEYEE YLRLKRYLWE LVGGLPKDKL K NLTPKFYL IIDEINRGNI SKIFGELITL LEKDKRLGGE NQLIVRLPYS GEPFAVPPNL YIIGTMNTAD RSIALLDVAL RR RFAFIEV EPRPEFLEKE NLKKIREKKL KTEDRKRLNE KLNELFSKLG NDNYFLKTLL EKINVRITVV KDRDHRIGHS YFL NVETVE DLHHVWYYEV LPLLMEYFYN DWETIKWVLN EKGKEHGNVF FEKLRLTGPN GEEAYQLKVL EGDAFIGALK RIIS KNTPS QEGGATTNEE NSPENTQSQT EGD UniProtKB: GTPase subunit of restriction endonuclease |
-Macromolecule #2: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE
Macromolecule | Name: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / type: ligand / ID: 2 / Number of copies: 6 / Formula: GSP |
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Molecular weight | Theoretical: 539.246 Da |
Chemical component information | ChemComp-GSP: |
-Macromolecule #3: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 6 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #4: water
Macromolecule | Name: water / type: ligand / ID: 4 / Number of copies: 12 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 10.0 mg/mL |
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Buffer | pH: 7.5 |
Grid | Model: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 1599 / Average exposure time: 10.0 sec. / Average electron dose: 8.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |