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Open data
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Basic information
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Title | Cascade complex from type I-A CRISPR-Cas system | ||||||||||||
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Function / homology | ![]() | ||||||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.9 Å | ||||||||||||
![]() | Hu C / Ni D / Nam KH / 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
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 97.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 22.2 KB 22.2 KB | Display Display | ![]() |
Images | ![]() | 139.7 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7tr9MC ![]() 7tr6C ![]() 7tr8C ![]() 7traC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 1.24 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
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Sample components
+Entire : Partial R-loop state of Cas3-Cascade complex from Pyrococcus furi...
+Supramolecule #1: Partial R-loop state of Cas3-Cascade complex from Pyrococcus furi...
+Macromolecule #1: Cas8a
+Macromolecule #2: Cas11a
+Macromolecule #3: Cas7a
+Macromolecule #4: Cas5a
+Macromolecule #6: CRISPR-associated helicase Cas3
+Macromolecule #7: CRISPR-associated endonuclease Cas3-HD
+Macromolecule #5: crRNA (45-MER)
+Macromolecule #8: Target strand DNA
+Macromolecule #9: Non-Target strand DNA
+Macromolecule #10: NICKEL (II) ION
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 7.5 / Component - Concentration: 150.0 mM / Component - Formula: NaCl / Component - Name: sodium chloride |
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 6 seconds. |
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Electron microscopy
Microscope | TFS TALOS |
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Specialist optics | Phase plate: VOLTA PHASE PLATE |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 1209 / Average exposure time: 2.5 sec. / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 100.0 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm |
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Image processing
-Atomic model buiding 1
Refinement | Protocol: OTHER |
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Output model | ![]() PDB-7tr9: |