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Yorodumi- EMDB-4904: Cryo-EM structure of St1Cas9-sgRNA-AcrIIA6-tDNA59-ntPAM complex. -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-4904 | |||||||||
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Title | Cryo-EM structure of St1Cas9-sgRNA-AcrIIA6-tDNA59-ntPAM complex. | |||||||||
Map data | Postprocessed 3D reconstruction of St1Cas9%u2022sgRNA%u2022AcrIIA6%u2022tDNA59-ntPAM complex. | |||||||||
Sample |
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Keywords | CRISPR-Cas9 / anti-CRISPR protein / bacteriophages / Streptococcus thermophilus Cas9 / St1Cas9 / hydrolase | |||||||||
Function / homology | Function and homology information symbiont-mediated suppression of host CRISPR-cas system / maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding Similarity search - Function | |||||||||
Biological species | Streptococcus phage D1811 (virus) / Streptococcus thermophilus DGCC 7710 (bacteria) / Streptococcus thermophilus (bacteria) / Streptococcus virus 2972 | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
Authors | Swuec P | |||||||||
Funding support | France, 1 items
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Citation | Journal: Mol Cell / Year: 2019 Title: Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6. Authors: Olivier Fuchsbauer / Paolo Swuec / Claire Zimberger / Béatrice Amigues / Sébastien Levesque / Daniel Agudelo / Alexis Duringer / Antonio Chaves-Sanjuan / Silvia Spinelli / Geneviève M ...Authors: Olivier Fuchsbauer / Paolo Swuec / Claire Zimberger / Béatrice Amigues / Sébastien Levesque / Daniel Agudelo / Alexis Duringer / Antonio Chaves-Sanjuan / Silvia Spinelli / Geneviève M Rousseau / Minja Velimirovic / Martino Bolognesi / Alain Roussel / Christian Cambillau / Sylvain Moineau / Yannick Doyon / Adeline Goulet / Abstract: In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with ...In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_4904.map.gz | 92.8 MB | EMDB map data format | |
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Header (meta data) | emd-4904-v30.xml emd-4904.xml | 20.9 KB 20.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_4904_fsc.xml | 10.6 KB | Display | FSC data file |
Images | emd_4904.png | 121.7 KB | ||
Masks | emd_4904_msk_1.map | 98.9 MB | Mask map | |
Filedesc metadata | emd-4904.cif.gz | 7.3 KB | ||
Others | emd_4904_additional.map.gz | 77.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-4904 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-4904 | HTTPS FTP |
-Validation report
Summary document | emd_4904_validation.pdf.gz | 609.7 KB | Display | EMDB validaton report |
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Full document | emd_4904_full_validation.pdf.gz | 609.2 KB | Display | |
Data in XML | emd_4904_validation.xml.gz | 11.5 KB | Display | |
Data in CIF | emd_4904_validation.cif.gz | 15.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4904 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4904 | HTTPS FTP |
-Related structure data
Related structure data | 6rjgMC 4900C 4901C 4902C 6rj9C 6rjaC 6rjdC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_4904.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Postprocessed 3D reconstruction of St1Cas9%u2022sgRNA%u2022AcrIIA6%u2022tDNA59-ntPAM complex. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.889 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_4904_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: Unsharpened 3D reconstruction of St1Cas9%u2022sgRNA%u2022AcrIIA6%u2022tDNA59-ntPAM complex....
File | emd_4904_additional.map | ||||||||||||
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Annotation | Unsharpened 3D reconstruction of St1Cas9%u2022sgRNA%u2022AcrIIA6%u2022tDNA59-ntPAM complex. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Cryo-EM structure of St1Cas9-sgRNA-AcrIIA6-tDNA59-ntPAM complex.
+Supramolecule #1: Cryo-EM structure of St1Cas9-sgRNA-AcrIIA6-tDNA59-ntPAM complex.
+Supramolecule #2: AcrIIA6
+Supramolecule #3: Cas 9
+Supramolecule #4: sgRNA
+Supramolecule #5: tDNA59
+Supramolecule #6: ntPAM
+Macromolecule #1: AcrIIA6
+Macromolecule #2: Cas 9
+Macromolecule #3: sgRNA
+Macromolecule #4: tDNA59
+Macromolecule #5: ntPAM
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.75 mg/mL |
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Buffer | pH: 7.5 |
Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: OTHER |
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Output model | PDB-6rjg: |