+
Open data
-
Basic information
Entry | Database: PDB / ID: 6gtg | ||||||
---|---|---|---|---|---|---|---|
Title | Transition state structure of Cpf1(Cas12a) I4 conformation | ||||||
![]() |
| ||||||
![]() | HYDROLASE / genome editing CRISPR ribonucleoprotein complex | ||||||
Function / homology | ![]() Bacillus subtilis ribonuclease / : / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus / lyase activity / DNA binding / RNA binding Similarity search - Function | ||||||
Biological species | ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.27 Å | ||||||
![]() | Mesa, P. / Montoya, G. / Stella, S. | ||||||
Funding support | ![]()
| ||||||
![]() | ![]() 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 |
|
-
Structure visualization
Movie |
![]() |
---|---|
Structure viewer | Molecule: ![]() ![]() |
-
Downloads & links
-
Download
PDBx/mmCIF format | ![]() | 300.9 KB | Display | ![]() |
---|---|---|---|---|
PDB format | ![]() | 226.9 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
---|
-Related structure data
Related structure data | ![]() 0065MC ![]() 0061C ![]() 0062C ![]() 0063C ![]() 0064C ![]() 6gtcC ![]() 6gtdC ![]() 6gteC ![]() 6gtfC M: map data used to model this data C: citing same article ( |
---|---|
Similar structure data |
-
Links
-
Assembly
Deposited unit | ![]()
|
---|---|
1 |
|
-
Components
-DNA chain , 2 types, 2 molecules CD
#3: DNA chain | Mass: 16898.826 Da / Num. of mol.: 1 / Source method: obtained synthetically Source: (synth.) ![]() |
---|---|
#4: DNA chain | Mass: 16992.936 Da / Num. of mol.: 1 / Source method: obtained synthetically Source: (synth.) ![]() |
-Protein / RNA chain , 2 types, 2 molecules AB
#1: Protein | Mass: 155639.828 Da / Num. of mol.: 1 / Mutation: E1006Q Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: cas12a, cpf1, FTN_1397 / Production host: ![]() ![]() References: UniProt: A0Q7Q2, deoxyribonuclease I, EC: 3.1.27.2 |
---|---|
#2: RNA chain | Mass: 13749.146 Da / Num. of mol.: 1 / Source method: obtained synthetically Source: (synth.) ![]() |
-Non-polymers , 2 types, 7 molecules ![](data/chem/img/MG.gif)
![](data/chem/img/HOH.gif)
![](data/chem/img/HOH.gif)
#5: Chemical | ChemComp-MG / #6: Water | ChemComp-HOH / | |
---|
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-
Sample preparation
Component |
| ||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Molecular weight | Value: 0.180 MDa / Experimental value: YES | ||||||||||||||||||||||||||||||
Source (natural) |
| ||||||||||||||||||||||||||||||
Source (recombinant) |
| ||||||||||||||||||||||||||||||
Buffer solution | pH: 6 | ||||||||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE |
-
Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
---|---|
Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) |
-
Processing
Software |
| ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
EM software |
| ||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 358 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Stereochemistry target values: GeoStd + Monomer Library | ||||||||||||||||||||||||
Refine LS restraints |
|