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- PDB-8gaf: Exploiting Activation and Inactivation Mechanisms in Type I-C CRI... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8gaf | ||||||
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Title | Exploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications | ||||||
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![]() | HYDROLASE/RNA / CRISPR / type I-C / cascade / anti-CRISPR / HYDROLASE-RNA complex | ||||||
Function / homology | ![]() maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding Similarity search - Function | ||||||
Biological species | ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.64 Å | ||||||
![]() | Hu, C. / Nam, K.H. / Ke, A. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications. Authors: Chunyi Hu / Mason T Myers / Xufei Zhou / Zhonggang Hou / Macy L Lozen / Ki Hyun Nam / Yan Zhang / Ailong Ke / ![]() ![]() ![]() Abstract: Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size ...Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here, we use four cryoelectron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9 that strongly inhibit Neisseria lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through allosteric inhibition, whereas AcrIC9 achieves so through direct competition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for type I CRISPR eukaryotic genome engineering. | ||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 544.2 KB | Display | ![]() |
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PDB format | ![]() | 442.7 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 29896MC ![]() 8g9sC ![]() 8g9tC ![]() 8g9uC ![]() 8gamC ![]() 8ganC 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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Components
#1: Protein | Mass: 32208.111 Da / Num. of mol.: 7 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #2: Protein | Mass: 64715.652 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #3: Protein | Mass: 14245.184 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #4: RNA chain | | Mass: 13816.202 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() #5: Protein | | Mass: 23870.451 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: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-loop formation Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES |
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Molecular weight | Value: 0.4 MDa / Experimental value: YES |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.5 / Details: 25mM Tris pH 7.5, 150mM NaCl |
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: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: ![]() |
Electron lens | Mode: DIFFRACTION / Nominal magnification: 67000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 2500 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 70 K |
Image recording | Average exposure time: 2.5 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1798 / Num. of real images: 1798 |
EM imaging optics | Energyfilter name: GIF Bioquantum |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||
3D reconstruction | Resolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 103405 / Symmetry type: POINT | ||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT |