+Open data
-Basic information
Entry | Database: PDB / ID: 8csz | ||||||
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Title | IscB and wRNA bound to Target DNA | ||||||
Components |
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Keywords | RNA BINDING PROTEIN/RNA/DNA / CRISPR / IscB / HEARO RNA / omega RNA / RNA BINDING PROTEIN-RNA-DNA complex | ||||||
Function / homology | DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) Function and homology information | ||||||
Biological species | synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | ||||||
Authors | Schuler, G.A. / Hu, C. / Ke, A. | ||||||
Funding support | United States, 1items
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Citation | Journal: Science / Year: 2022 Title: Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9. Authors: Gabriel Schuler / Chunyi Hu / Ailong Ke / Abstract: Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ...Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ωRNA plays the combined role of CRISPR RNA (crRNA) and trans-activating CRISPR RNA tracrRNA) to guide double-stranded DNA (dsDNA) cleavage. Here we report a 2.78-angstrom cryo-electron microscopy structure of IscB-ωRNA bound to a dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 ribonucleoproteins. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9 and contacts the RNA-DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8csz.cif.gz | 216.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8csz.ent.gz | 158.9 KB | Display | PDB format |
PDBx/mmJSON format | 8csz.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8csz_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
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Full document | 8csz_full_validation.pdf.gz | 1.5 MB | Display | |
Data in XML | 8csz_validation.xml.gz | 31.5 KB | Display | |
Data in CIF | 8csz_validation.cif.gz | 46.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/cs/8csz ftp://data.pdbj.org/pub/pdb/validation_reports/cs/8csz | HTTPS FTP |
-Related structure data
Related structure data | 26976MC 7utnC 8ctlC C: citing same article (ref.) M: map data used to model this data |
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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
-DNA chain , 2 types, 2 molecules BE
#3: DNA chain | Mass: 18305.646 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#4: DNA chain | Mass: 18684.965 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Protein / RNA chain , 2 types, 2 molecules DC
#1: Protein | Mass: 56819.680 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: Locked R-loop state / Source: (gene. exp.) synthetic construct (others) Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) Strain (production host): T7 Express (NEB) |
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#2: RNA chain | Mass: 71877.797 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) synthetic construct (others) Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) Strain (production host): T7 Express (NEB) |
-Non-polymers , 2 types, 3 molecules
#5: Chemical | #6: Chemical | ChemComp-ZN / | |
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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 |
-Sample preparation
Component | Name: Cryo-EM structure of IscB in complex with RNA and target DNA Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES | |||||||||||||||||||||||||
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Molecular weight | Value: 190 kDa/nm / Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: synthetic construct (others) | |||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) Strain: T7 Express (NEB) | |||||||||||||||||||||||||
Buffer solution | pH: 7.25 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||
Specimen support | Grid type: Quantifoil R1.2/1.3 | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 6.5 seconds before plunging |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33304 / Symmetry type: POINT | ||||||||||||||||||||||||
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