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Yorodumi- PDB-6ut5: Cryo-EM structure of the Thermococcus gammatolerans McrBC complex -
+Open data
-Basic information
Entry | Database: PDB / ID: 6ut5 | ||||||
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Title | Cryo-EM structure of the Thermococcus gammatolerans McrBC complex | ||||||
Components |
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Keywords | DNA BINDING PROTEIN / Endonuclease / AAA protein / GTPase / Methylation-dependent restriction | ||||||
Function / homology | Function and homology information | ||||||
Biological species | Thermococcus gammatolerans (archaea) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.44 Å | ||||||
Authors | Niu, Y. / Suzuki, H. / Hosford, C.J. / Chappie, J.S. / Walz, T. | ||||||
Funding support | United States, 1items
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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 | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6ut5.cif.gz | 505.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6ut5.ent.gz | 405.8 KB | Display | PDB format |
PDBx/mmJSON format | 6ut5.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ut/6ut5 ftp://data.pdbj.org/pub/pdb/validation_reports/ut/6ut5 | HTTPS FTP |
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-Related structure data
Related structure data | 20866MC 6ut3C 6ut4C 6ut6C 6ut7C 6ut8C C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | |
EM raw data | EMPIAR-10583 (Title: Single-particle Cryo-EM of the Thermococcus gammatolerans McrBC complex Data size: 883.3 Data #1: Unaligned multi-frame micrographs of the Thermococcus gammatolerans McrBC complex [micrographs - multiframe]) |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 2 types, 7 molecules ABCDEFG
#1: Protein | Mass: 71668.867 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermococcus gammatolerans (archaea) / Production host: Escherichia coli (E. coli) / References: UniProt: C5A3Z3 #2: Protein | | Mass: 54339.406 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermococcus gammatolerans (archaea) / Production host: Escherichia coli (E. coli) / References: UniProt: C5A3Z2 |
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-Non-polymers , 4 types, 40 molecules
#3: Chemical | ChemComp-GSP / #4: Chemical | ChemComp-MG / #5: Chemical | ChemComp-GDP / | #6: Water | ChemComp-HOH / | |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Complex of the full-length Thermococcus gammatolerans McrBC Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1-#2 / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Thermococcus gammatolerans (archaea) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 16 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 10 sec. / Electron dose: 8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2078 |
Image scans | Movie frames/image: 40 / Used frames/image: 1-40 |
-Processing
Software | Name: PHENIX / Version: 1.15.2_3472: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.44 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 226846 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||
Refinement | Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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