+Open data
-Basic information
Entry | Database: PDB / ID: 7kfu | ||||||
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Title | Cas6-RT-Cas1--Cas2 complex | ||||||
Components |
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Keywords | HYDROLASE / Complex / CRISPR Cas Protein / Reverse Transcriptase | ||||||
Biological species | Thiomicrospira sp. (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | ||||||
Authors | Hoel, C.M. / Wang, J.Y. / Doudna, J.A. / Brohawn, S.G. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nat Commun / Year: 2021 Title: Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex. Authors: Joy Y Wang / Christopher M Hoel / Basem Al-Shayeb / Jillian F Banfield / Stephen G Brohawn / Jennifer A Doudna / Abstract: CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation ...CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1-Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference. | ||||||
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 | 7kfu.cif.gz | 510.4 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7kfu.ent.gz | 397.1 KB | Display | PDB format |
PDBx/mmJSON format | 7kfu.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7kfu_validation.pdf.gz | 750.6 KB | Display | wwPDB validaton report |
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Full document | 7kfu_full_validation.pdf.gz | 771.2 KB | Display | |
Data in XML | 7kfu_validation.xml.gz | 69.8 KB | Display | |
Data in CIF | 7kfu_validation.cif.gz | 107.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/kf/7kfu ftp://data.pdbj.org/pub/pdb/validation_reports/kf/7kfu | HTTPS FTP |
-Related structure data
Related structure data | 22856MC 7kftC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | |
EM raw data | EMPIAR-10642 (Title: Cas6-reverse transcriptase-Cas1—Cas2 CRISPR integrase complex Data size: 2.2 TB Data #1: Unaligned multi-frame movies of a Cas6-reverse transcriptase-Cas1—Cas2 CRISPR integrase complex [micrographs - multiframe]) |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 11583.263 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Details: N-terminal residues SNA are from the expression tag. Protein sequence corresponds to GenBank NCN43132.1. Source: (gene. exp.) Thiomicrospira sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta #2: Protein | Mass: 112869.414 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Details: Protein sequence corresponds to GenBank NCN66177.1. Source: (gene. exp.) Thiomicrospira sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta |
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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: Cas6-RT-Cas1--Cas2 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Value: 0.47 MDa / Experimental value: NO |
Source (natural) | Organism: Thiomicrospira sp. (bacteria) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Rosetta |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Blot force 1, 3 second blot |
-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 FIELD |
Image recording | Electron dose: 49.9 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 129175 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 183.8 Å2 | ||||||||||||||||||||||||
Refine LS restraints |
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