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Open data
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Basic information
Entry | Database: PDB / ID: 6c66 | |||||||||
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Title | CRISPR RNA-guided surveillance complex, pre-nicking | |||||||||
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![]() | DNA BINDING PROTEIN/DNA/RNA / CRISPR-Cas / Cascade / Cas3 / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex | |||||||||
Function / homology | ![]() maintenance of CRISPR repeat elements / defense response to virus / RNA helicase activity / hydrolase activity / RNA binding / ATP binding / identical protein binding / metal ion binding / cytosol Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.66 Å | |||||||||
![]() | Xiao, Y. / Luo, M. / Liao, M. / Ke, A. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Structure basis for RNA-guided DNA degradation by Cascade and Cas3. Authors: Yibei Xiao / Min Luo / Adam E Dolan / Maofu Liao / Ailong Ke / ![]() Abstract: Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA by the RNA-guided Cascade (CRISPR associated complex for antiviral defense) complex and ...Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA by the RNA-guided Cascade (CRISPR associated complex for antiviral defense) complex and the nuclease-helicase fusion enzyme Cas3, respectively. Here, we present a 3.7-angstrom-resolution cryo-electron microscopy (cryo-EM) structure of the Type I-E Cascade/R-loop/Cas3 complex, poised to initiate DNA degradation. Cas3 distinguishes Cascade conformations and only captures the R-loop-forming Cascade, to avoid cleaving partially complementary targets. Its nuclease domain recruits the nontarget strand (NTS) DNA at a bulged region for the nicking of single-stranded DNA. An additional 4.7-angstrom-resolution cryo-EM structure captures the postnicking state, in which the severed NTS retracts to the helicase entrance, to be threaded for adenosine 5'-triphosphate-dependent processive degradation. These snapshots form the basis for understanding RNA-guided DNA degradation in Type I-E CRISPR-Cas systems. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 805.6 KB | Display | ![]() |
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PDB format | ![]() | 650.5 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 7347MC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-Protein , 2 types, 3 molecules GIK
#1: Protein | Mass: 104039.219 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: YX / Gene: Tfu_1593 / Production host: ![]() ![]() |
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#4: Protein | Mass: 27446.613 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: YX / Gene: Tfu_1591 / Production host: ![]() ![]() |
-CRISPR-associated protein, ... , 4 types, 9 molecules ABCDEFHMO
#2: Protein | Mass: 61433.297 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: YX / Gene: Tfu_1592 / Production host: ![]() ![]() | ||||
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#3: Protein | Mass: 41043.043 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: YX / Gene: Tfu_1590 / Production host: ![]() ![]() #7: Protein | | Mass: 28279.260 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: YX / Gene: Tfu_1589 / Production host: ![]() ![]() #9: Protein | | Mass: 26327.938 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: YX / Gene: Tfu_1588 / Production host: ![]() ![]() |
-DNA chain , 2 types, 2 molecules LN
#6: DNA chain | Mass: 16801.725 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
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#8: DNA chain | Mass: 17099.920 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
-RNA chain / Non-polymers , 2 types, 3 molecules J![](data/chem/img/FE.gif)
![](data/chem/img/FE.gif)
#10: Chemical | #5: RNA chain | | Mass: 19790.793 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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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 |
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Sample preparation
Component | Name: CRISPR RNA-guided surveillance complex with Cas3 bound in its pre-nicking state Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT |
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Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.5 / Details: 10 mM HEPES pH 7.5, 150 mM NaCl, 5 mM DTT |
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 85 % |
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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: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 31000 X / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2700 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: 1428 |
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Processing
Software | Name: PHENIX / Version: 1.11.1_2575: / 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: 322268 | ||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51889 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER | ||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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