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Open data
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Basic information
Entry | Database: PDB / ID: 7tr9 | ||||||||||||
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Title | Cascade complex from type I-A CRISPR-Cas system | ||||||||||||
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![]() | RNA BINDING PROTEIN / CRISPR / cascade / Cas3 / type I-A / genome editing | ||||||||||||
Function / homology | ![]() | ||||||||||||
Biological species | ![]() ![]() ![]() ![]() | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | ||||||||||||
![]() | Hu, C. / Ni, D. / Nam, K.H. / Majumdar, S. / McLean, J. / Stahlberg, H. / Terns, M. / Ke, A. | ||||||||||||
Funding support | ![]() ![]()
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![]() | ![]() Title: Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools. Authors: Chunyi Hu / Dongchun Ni / Ki Hyun Nam / Sonali Majumdar / Justin McLean / Henning Stahlberg / Michael P Terns / Ailong Ke / ![]() ![]() ![]() Abstract: Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme ...Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degradation. Contrary to this model, here, we show that type I-A Cascade and Cas3 function as an integral effector complex. We provide four cryoelectron microscopy (cryo-EM) snapshots of the Pyrococcus furiosus (Pfu) type I-A effector complex in different stages of DNA recognition and degradation. The HD nuclease of Cas3 is autoinhibited inside the effector complex. It is only allosterically activated upon full R-loop formation, when the entire targeted region has been validated by the RNA guide. The mechanistic insights inspired us to convert Pfu Cascade-Cas3 into a high-sensitivity, low-background, and temperature-activated nucleic acid detection tool. Moreover, Pfu CRISPR-Cas3 shows robust bi-directional deletion-editing activity in human cells, which could find usage in allele-specific inactivation of disease-causing mutations. | ||||||||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 739.6 KB | Display | ![]() |
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PDB format | ![]() | Display | ![]() | |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 26083MC ![]() 7tr6C ![]() 7tr8C ![]() 7traC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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1 |
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Components
-Protein , 4 types, 14 molecules CDEFGHIJKLMNOP
#1: Protein | Mass: 38800.719 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() | ||||
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#2: Protein | Mass: 12208.933 Da / Num. of mol.: 5 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() #3: Protein | Mass: 36989.148 Da / Num. of mol.: 7 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() #4: Protein | | Mass: 29147.219 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
-CRISPR-associated ... , 2 types, 2 molecules AQ
#6: Protein | Mass: 61412.555 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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#7: Protein | Mass: 26057.617 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
-DNA chain , 2 types, 2 molecules ST
#8: DNA chain | Mass: 6929.449 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
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#9: DNA chain | Mass: 2411.606 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
-RNA chain / Non-polymers , 2 types, 3 molecules R![](data/chem/img/NI.gif)
![](data/chem/img/NI.gif)
#10: Chemical | #5: RNA chain | | Mass: 14373.537 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
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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 |
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Sample preparation
Component | Name: Partial R-loop state of Cas3-Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system Type: COMPLEX / Details: Cascade alone / Entity ID: #1-#9 / Source: MULTIPLE SOURCES | ||||||||||||
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Molecular weight | Value: 0.45 MDa / Experimental value: YES | ||||||||||||
Source (natural) |
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Source (recombinant) | Organism: ![]() ![]() | ||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||
Buffer component | Conc.: 150 mM / Name: sodium chloride / Formula: NaCl | ||||||||||||
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: 6 seconds |
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Electron microscopy imaging
Microscopy | Model: TFS TALOS |
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Electron gun | Electron source: ![]() |
Electron lens | Mode: DIFFRACTION / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm |
Image recording | Average exposure time: 2.5 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 1209 |
EM imaging optics | Phase plate: VOLTA PHASE PLATE |
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Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||
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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: 13781 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: OTHER | ||||||||||||||||||||||||
Refine LS restraints |
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