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- PDB-6xmg: Cryo-EM structure of Cas12g ternary complex -

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Basic information

Entry
Database: PDB / ID: 6xmg
TitleCryo-EM structure of Cas12g ternary complex
Components
  • CRISPR-CasCRISPR
  • RNA (130-MER)
  • RNA (5'-R(P*UP*UP*AP*AP*UP*GP*CP*GP*GP*UP*AP*GP*UP*UP*UP*AP*UP*CP*AP*CP*AP*GP*UP*U)-3')
KeywordsHYDROLASE/RNA / CRISPR / HYDROLASE-RNA complex
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesmetagenome (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsChang, L. / Li, Z. / Zhang, H.
CitationJournal: Nat Chem Biol / Year: 2021
Title: Cryo-EM structure of the RNA-guided ribonuclease Cas12g.
Authors: Zhuang Li / Heng Zhang / Renjian Xiao / Ruijie Han / Leifu Chang /
Abstract: Cas12g, the type V-G CRISPR-Cas effector, is an RNA-guided ribonuclease that targets single-stranded RNA substrate. The CRISPR-Cas12g system offers a potential platform for transcriptome engineering ...Cas12g, the type V-G CRISPR-Cas effector, is an RNA-guided ribonuclease that targets single-stranded RNA substrate. The CRISPR-Cas12g system offers a potential platform for transcriptome engineering and diagnostic applications. We determined the structures of Cas12g-guide RNA complexes in the absence and presence of target RNA by cryo-EM to a resolution of 3.1 Å and 4.8 Å, respectively. Cas12g adopts a bilobed structure with miniature REC2 and Nuc domains, whereas the guide RNAs fold into a flipped 'F' shape, which is primarily recognized by the REC lobe. Target RNA and the CRISPR RNA (crRNA) guide form a duplex that inserts into the central cavity between the REC and NUC lobes, inducing conformational changes in both lobes to activate Cas12g. The structural insights would facilitate the development of Cas12g-based applications.
History
DepositionJun 30, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 13, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 3, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Feb 10, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Apr 7, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4May 10, 2023Group: Database references / Source and taxonomy
Category: database_2 / em_entity_assembly_naturalsource / entity_src_gen
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_entity_assembly_naturalsource.ncbi_tax_id / _em_entity_assembly_naturalsource.organism / _entity_src_gen.gene_src_details / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name
Revision 1.5Jun 7, 2023Group: Source and taxonomy / Structure summary / Category: em_entity_assembly / pdbx_entity_src_syn
Item: _em_entity_assembly.source / _pdbx_entity_src_syn.details ..._em_entity_assembly.source / _pdbx_entity_src_syn.details / _pdbx_entity_src_syn.ncbi_taxonomy_id / _pdbx_entity_src_syn.organism_scientific

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Structure visualization

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Assembly

Deposited unit
A: CRISPR-Cas
C: RNA (130-MER)
E: RNA (5'-R(P*UP*UP*AP*AP*UP*GP*CP*GP*GP*UP*AP*GP*UP*UP*UP*AP*UP*CP*AP*CP*AP*GP*UP*U)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)141,3684
Polymers141,3023
Non-polymers651
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13960 Å2
ΔGint-99 kcal/mol
Surface area50550 Å2

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Components

#1: Protein CRISPR-Cas / CRISPR


Mass: 89891.680 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: hot spring metagenome / Source: (gene. exp.) metagenome (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: RNA chain RNA (130-MER)


Mass: 43777.043 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: hot spring metagenome / Source: (synth.) metagenome (others)
#3: RNA chain RNA (5'-R(P*UP*UP*AP*AP*UP*GP*CP*GP*GP*UP*AP*GP*UP*UP*UP*AP*UP*CP*AP*CP*AP*GP*UP*U)-3')


Mass: 7633.511 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: hot spring metagenome / Source: (synth.) metagenome (others)
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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

ComponentName: CRISP-Cas complex / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: metagenome (others)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 1.35 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.1_3865: / Classification: refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 38238 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028863
ELECTRON MICROSCOPYf_angle_d0.42812718
ELECTRON MICROSCOPYf_dihedral_angle_d19.8732551
ELECTRON MICROSCOPYf_chiral_restr0.0281500
ELECTRON MICROSCOPYf_plane_restr0.0041088

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