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Yorodumi- PDB-8g9s: Exploiting Activation and Inactivation Mechanisms in Type I-C CRI... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8g9s | ||||||
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Title | Exploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications | ||||||
Components |
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Keywords | HYDROLASE/RNA / CRISPR / type I-C / cascade / anti-CRISPR / HYDROLASE-RNA complex | ||||||
Function / homology | Function and homology information maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding Similarity search - Function | ||||||
Biological species | Neisseria lactamica (bacteria) Rhodobacter phage RcNL1 (virus) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å | ||||||
Authors | Hu, C. / Nam, K.H. / Ke, A. | ||||||
Funding support | United States, 1items
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Citation | Journal: Mol Cell / Year: 2024 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: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8g9s.cif.gz | 560.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8g9s.ent.gz | 463.9 KB | Display | PDB format |
PDBx/mmJSON format | 8g9s.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8g9s_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 8g9s_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 8g9s_validation.xml.gz | 94.5 KB | Display | |
Data in CIF | 8g9s_validation.cif.gz | 147.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/g9/8g9s ftp://data.pdbj.org/pub/pdb/validation_reports/g9/8g9s | HTTPS FTP |
-Related structure data
Related structure data | 29877MC 8g9tC 8g9uC 8gafC 8gamC 8ganC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 5 types, 14 molecules BCDEFGMHIJLNKA
#1: Protein | Mass: 32208.111 Da / Num. of mol.: 7 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Neisseria lactamica (bacteria) / Gene: NCTC10618_01084 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A378VEU0 #2: Protein | Mass: 14245.184 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Neisseria lactamica (bacteria) / Gene: NCTC10618_01085 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A378VF47 #3: Protein | | Mass: 23854.451 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Neisseria lactamica (bacteria) / Gene: cas5d / Production host: Escherichia coli (E. coli) / References: UniProt: D0W8X4 #5: Protein | | Mass: 45864.527 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Neisseria lactamica (bacteria) / Gene: NCTC10618_01085 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A378VF47 #6: Protein | | Mass: 8190.161 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Rhodobacter phage RcNL1 (virus) / Production host: Escherichia coli (E. coli) |
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-RNA chain , 1 types, 1 molecules O
#4: RNA chain | Mass: 13495.021 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Neisseria lactamica (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: Binary complex of AcrIC8 with crRNA bound type I-C Cascade Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES | ||||||||||||
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Molecular weight | Value: 0.4 MDa / Experimental value: YES | ||||||||||||
Source (natural) |
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Source (recombinant) | Organism: Escherichia coli (E. coli) / Strain: BL21 | ||||||||||||
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 |
-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: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
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: 1200 / Num. of real images: 1200 |
EM imaging optics | Energyfilter name: GIF Bioquantum |
-Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 200000 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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