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- PDB-8i3q: Cryo-EM structure of Cas12g-sgRNA binary complex -

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Basic information

Entry
Database: PDB / ID: 8i3q
TitleCryo-EM structure of Cas12g-sgRNA binary complex
Components
  • Cas12g
  • RNA (139-MER)
KeywordsRNA BINDING PROTEIN / CRISPR-Cas
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsOuyang, S.Y. / Liu, M.X. / Li, Z.K.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82172287 China
CitationJournal: 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.
History
DepositionJan 17, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 6, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 4, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: RNA (139-MER)
A: Cas12g
hetero molecules


Theoretical massNumber of molelcules
Total (without water)134,6853
Polymers134,6192
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain RNA (139-MER)


Mass: 44771.703 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#2: Protein Cas12g


Mass: 89847.672 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Cas12g-sgRNA binary complexCOMPLEX#1-#20MULTIPLE SOURCES
2Cas12gCOMPLEX#21RECOMBINANT
3RNACOMPLEX#11SYNTHETIC
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8.5
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 88696 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037734
ELECTRON MICROSCOPYf_angle_d0.60810957
ELECTRON MICROSCOPYf_dihedral_angle_d15.7771982
ELECTRON MICROSCOPYf_chiral_restr0.0351242
ELECTRON MICROSCOPYf_plane_restr0.0051026

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