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- EMDB-27142: Structure of Acidothermus cellulolyticus Cas9 ternary complex (Cl... -
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Open data
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Basic information
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Title | Structure of Acidothermus cellulolyticus Cas9 ternary complex (Cleavage Intermediate 1) | |||||||||
![]() | AcCas9 Cleavage intermediate 1 | |||||||||
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![]() | Cas9 / AcCas9 / Crispr / RNA BINDING PROTEIN / RNA BINDING PROTEIN-DNA-RNA complex | |||||||||
Function / homology | ![]() endonuclease activity / defense response to virus / DNA binding / RNA binding / zinc ion binding Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.21 Å | |||||||||
![]() | Rai J / Das A / Li H | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Coupled catalytic states and the role of metal coordination in Cas9. Authors: Anuska Das / Jay Rai / Mitchell O Roth / Yuerong Shu / Megan L Medina / Mackenzie R Barakat / Hong Li / ![]() Abstract: Controlling the activity of the CRISPR-Cas9 system is essential to its safe adoption for clinical and research applications. Although the conformational dynamics of Cas9 are known to control its ...Controlling the activity of the CRISPR-Cas9 system is essential to its safe adoption for clinical and research applications. Although the conformational dynamics of Cas9 are known to control its enzymatic activity, details of how Cas9 influences the catalytic processes at both nuclease domains remain elusive. Here we report five cryo-electron microscopy structures of the active Cas9 complex along the reaction path at 2.2-2.9 Å resolution. We observed that a large movement in one nuclease domain, triggered by the cognate DNA, results in noticeable changes in the active site of the other domain that is required for metal coordination and catalysis. Furthermore, the conformations synchronize the reaction intermediates, enabling coupled cutting of the two DNA strands. Consistent with the roles of conformations in organizing the active sites, adjustments to the metal-coordination residues lead to altered metal specificity of Cas9 and commonly used Cas9 in cells. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 100.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 21.6 KB 21.6 KB | Display Display | ![]() |
Images | ![]() | 50.6 KB | ||
Filedesc metadata | ![]() | 6.9 KB | ||
Others | ![]() ![]() | 84.2 MB 84.1 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 1019.2 KB | Display | ![]() |
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Full document | ![]() | 1018.8 KB | Display | |
Data in XML | ![]() | 13 KB | Display | |
Data in CIF | ![]() | 15.2 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8d2lMC ![]() 8d2kC ![]() 8d2nC ![]() 8d2oC ![]() 8d2pC ![]() 8d2qC C: citing same article ( M: atomic model generated by this map |
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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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Annotation | AcCas9 Cleavage intermediate 1 | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.825 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: Halfmap1
File | emd_27142_half_map_1.map | ||||||||||||
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Annotation | Halfmap1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Halfmap2
File | emd_27142_half_map_2.map | ||||||||||||
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Annotation | Halfmap2 | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
+Entire : CryoEM Structure of AceCas9 (Cleavage intermediate 1)
+Supramolecule #1: CryoEM Structure of AceCas9 (Cleavage intermediate 1)
+Supramolecule #2: CRISPR-associated endonuclease, Csn1 family/RNA
+Supramolecule #3: DNA
+Macromolecule #1: CRISPR-associated endonuclease, Csn1 family
+Macromolecule #2: Single guide RNA (106-MER)
+Macromolecule #3: DNA target strand (5'-D(*AP*GP*CP*TP*TP*GP*GP*TP*GP*TP*AP*TP*A)-3')
+Macromolecule #4: DNA target strand (5'-D(P*CP*CP*AP*GP*GP*AP*TP*CP*TP*TP*GP*CP*CP*...
+Macromolecule #5: DNA non-target strand (5'-D(P*TP*AP*TP*AP*CP*AP*CP*CP*AP*AP*GP*CP...
+Macromolecule #6: DNA non-target strand (5'-D(P*AP*GP*A)-3')
+Macromolecule #7: MAGNESIUM ION
+Macromolecule #8: water
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: OTHER |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 2.21 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 196600 |
Initial angle assignment | Type: OTHER |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4) |