+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-0065 | |||||||||
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Title | Transition state structure of Cpf1(Cas12a)I4 conformation | |||||||||
Map data | ||||||||||
Sample |
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Keywords | genome editing CRISPR ribonucleoprotein complex / HYDROLASE | |||||||||
Function / homology | Function and homology information Bacillus subtilis ribonuclease / : / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus / lyase activity / DNA binding / RNA binding Similarity search - Function | |||||||||
Biological species | Francisella tularensis subsp. novicida U112 (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.27 Å | |||||||||
Authors | Mesa P / Montoya G | |||||||||
Funding support | Denmark, 1 items
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Citation | Journal: Cell / Year: 2018 Title: Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity. Authors: Stefano Stella / Pablo Mesa / Johannes Thomsen / Bijoya Paul / Pablo Alcón / Simon B Jensen / Bhargav Saligram / Matias E Moses / Nikos S Hatzakis / Guillermo Montoya / Abstract: Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures ...Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs. | |||||||||
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_0065.map.gz | 35.1 MB | EMDB map data format | |
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Header (meta data) | emd-0065-v30.xml emd-0065.xml | 15.6 KB 15.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_0065_fsc.xml | 8.6 KB | Display | FSC data file |
Images | emd_0065.png | 134.2 KB | ||
Filedesc metadata | emd-0065.cif.gz | 6.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-0065 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-0065 | HTTPS FTP |
-Validation report
Summary document | emd_0065_validation.pdf.gz | 268.6 KB | Display | EMDB validaton report |
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Full document | emd_0065_full_validation.pdf.gz | 267.7 KB | Display | |
Data in XML | emd_0065_validation.xml.gz | 10.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0065 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0065 | HTTPS FTP |
-Related structure data
Related structure data | 6gtgMC 0061C 0062C 0063C 0064C 6gtcC 6gtdC 6gteC 6gtfC M: atomic model generated by this map C: citing same article (ref.) |
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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_0065.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 0.832 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
+Entire : Transition state complex I4 conformation
+Supramolecule #1: Transition state complex I4 conformation
+Supramolecule #2: CRISPR-associated endonuclease Cas12a
+Supramolecule #3: RNA (28-mer)
+Supramolecule #4: DNA
+Macromolecule #1: CRISPR-associated endonuclease Cas12a
+Macromolecule #2: RNA (40-MER)
+Macromolecule #3: DNA (32-MER)
+Macromolecule #4: DNA (5'-D(P*CP*GP*AP*GP*CP*TP*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')
+Macromolecule #5: MAGNESIUM ION
+Macromolecule #6: water
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 6 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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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: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |