National Natural Science Foundation of China (NSFC)
31770948
China
Citation
Journal: PLoS Genet / Year: 2023 Title: Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage. Authors: Mengxi Liu / Zekai Li / Jing Chen / Jinying Lin / Qiuhua Lu / Yangmiao Ye / Hongmin Zhang / Bo Zhang / Songying Ouyang / Abstract: Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC ...Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. The conserved zinc finger motifs in Cas12g undergo an ordered-to-disordered transition from the apo to the sgRNA-bound state and their mutations negatively impact on target RNA cleavage. Moreover, we identified a lid motif in the RuvC domain that undergoes transformation from a helix to loop to regulate the access to the RuvC active site and subsequent cleavage of the RNA substrate. Overall, our study provides valuable insights into the mechanisms by which Cas12g recognizes sgRNA and the conformational changes it undergoes from sgRNA binding to the activation of the RNase active site, thereby laying a foundation for the potential repurposing of Cas12g as a tool for RNA-editing.
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
Wavelength: 0.9792 Å / Relative weight: 1
Reflection
Resolution: 2.24→62.86 Å / Num. obs: 49819 / % possible obs: 99.4 % / Redundancy: 13 % / CC1/2: 0.997 / Rmerge(I) obs: 0.091 / Net I/σ(I): 17
Reflection shell
Resolution: 2.24→2.3 Å / Mean I/σ(I) obs: 2.3 / Num. unique obs: 3596 / CC1/2: 0.786 / % possible all: 98.2
-
Processing
Software
Name
Version
Classification
REFMAC
5.8.0267
refinement
xia2
datareduction
xia2
datascaling
PHENIX
1.13-2998
phasing
Refinement
Method to determine structure: SAD / Resolution: 2.24→62.86 Å / Cor.coef. Fo:Fc: 0.948 / Cor.coef. Fo:Fc free: 0.926 / SU B: 7.013 / SU ML: 0.17 / Cross valid method: THROUGHOUT / ESU R: 0.233 / ESU R Free: 0.207 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.26849
2416
4.9 %
RANDOM
Rwork
0.22398
-
-
-
obs
0.2262
47341
99.32 %
-
Solvent computation
Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK