+Open data
-Basic information
Entry | Database: PDB / ID: 5u07 | ||||||
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Title | CRISPR RNA-guided surveillance complex | ||||||
Components |
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Keywords | IMMUNE SYSTEM / CRISPR-Cas / Cascade | ||||||
Function / homology | Function and homology information maintenance of CRISPR repeat elements / defense response to virus / RNA binding / identical protein binding Similarity search - Function | ||||||
Biological species | Thermobifida fusca YX (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å | ||||||
Authors | Xiao, Y. / Luo, M. / Hayes, R.P. / Kim, J. / Ng, S. / Ding, F. / Liao, M. / Ke, A. | ||||||
Citation | Journal: Cell / Year: 2017 Title: Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System. Authors: Yibei Xiao / Min Luo / Robert P Hayes / Jonathan Kim / Sherwin Ng / Fang Ding / Maofu Liao / Ailong Ke / Abstract: Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo- ...Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems. | ||||||
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 | 5u07.cif.gz | 614.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb5u07.ent.gz | 497.3 KB | Display | PDB format |
PDBx/mmJSON format | 5u07.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5u07_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 5u07_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 5u07_validation.xml.gz | 104 KB | Display | |
Data in CIF | 5u07_validation.cif.gz | 159.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/u0/5u07 ftp://data.pdbj.org/pub/pdb/validation_reports/u0/5u07 | HTTPS FTP |
-Related structure data
Related structure data | 8477MC 8478C 5u0aC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-CRISPR-associated protein, ... , 4 types, 9 molecules ACDEFGHIN
#1: Protein | Mass: 26327.938 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermobifida fusca YX (bacteria) / Strain: YX / Gene: Tfu_1588 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q47PJ5 | ||
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#3: Protein | Mass: 61433.297 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermobifida fusca YX (bacteria) / Strain: YX / Gene: Tfu_1592 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q47PJ1 | ||
#4: Protein | Mass: 41043.043 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermobifida fusca YX (bacteria) / Strain: YX / Gene: Tfu_1590 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q47PJ3 #7: Protein | | Mass: 28279.260 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermobifida fusca YX (bacteria) / Strain: YX / Gene: Tfu_1589 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q47PJ4 |
-DNA chain , 2 types, 2 molecules MO
#6: DNA chain | Mass: 6381.106 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermobifida fusca YX (bacteria) |
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#8: DNA chain | Mass: 3986.600 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermobifida fusca YX (bacteria) |
-Protein / RNA chain , 2 types, 3 molecules BJK
#2: Protein | Mass: 27446.613 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermobifida fusca YX (bacteria) / Strain: YX / Gene: Tfu_1591 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q47PJ2 #5: RNA chain | | Mass: 19790.793 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: RNA was prepared by in vitro transcription / Source: (synth.) Thermobifida fusca YX (bacteria) |
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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: CRISPR RNA-guided surveillance complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Source (natural) | Organism: Thermobifida fusca (strain YX) (bacteria) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pcdf |
Buffer solution | pH: 7.5 / Details: 10 mM Hepes, 150 mM NaCl, 5 mM DTT, pH 7.5 |
Specimen | Conc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid type: 400 mesh Quantifoil holey carbon grid |
Vitrification | Cryogen name: ETHANE / Humidity: 85 % |
-Electron microscopy imaging
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
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Microscopy | Model: FEI POLARA 300 |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 31000 X / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2300 nm / Cs: 2 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: GATAN LIQUID NITROGEN / Temperature (max): 105 K / Temperature (min): 80 K |
Image recording | Electron dose: 8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1305 |
-Processing
Software | Name: PHENIX / Version: 1.11.1-2575_1692: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 582497 | ||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.8 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 70222 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||
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