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- PDB-6w5c: Cryo-EM structure of Cas12i(E894A)-crRNA-dsDNA complex -

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Basic information

Entry
Database: PDB / ID: 6w5c
TitleCryo-EM structure of Cas12i(E894A)-crRNA-dsDNA complex
Components
  • Cas12i
  • NTS
  • Substrate
  • TS
  • crRNA
KeywordsHYDROLASE/DNA/RNA / CRISPR / HYDROLASE-DNA-RNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesLachnospiraceae bacterium ND2006 (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsChang, L. / Li, Z. / Zhang, H.
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Mechanisms for target recognition and cleavage by the Cas12i RNA-guided endonuclease.
Authors: Heng Zhang / Zhuang Li / Renjian Xiao / Leifu Chang /
Abstract: Cas12i is a recently identified type V CRISPR-Cas endonuclease that predominantly cleaves the non-target strand of a double-stranded DNA substrate. This nicking activity of Cas12i could potentially ...Cas12i is a recently identified type V CRISPR-Cas endonuclease that predominantly cleaves the non-target strand of a double-stranded DNA substrate. This nicking activity of Cas12i could potentially be used for genome editing with high specificity. To elucidate its mechanisms for target recognition and cleavage, we determined cryo-EM structures of Cas12i in multiple functional states. Cas12i pre-orders a seven-nucleotide seed sequence of the crRNA for target recognition and undergoes a two-step activation through crRNA-DNA hybridization. Formation of 14 base pairs activates the nickase activity, and 28-bp hybridization promotes cleavage of the target strand. The atomic structures and mechanistic insights gained should facilitate the manipulation of Cas12i for genome editing applications.
History
DepositionMar 13, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 16, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 31, 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.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Cas12i
C: TS
D: NTS
E: Substrate
B: crRNA


Theoretical massNumber of molelcules
Total (without water)161,4245
Polymers161,4245
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area19450 Å2
ΔGint-129 kcal/mol
Surface area54990 Å2

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Components

#1: Protein Cas12i


Mass: 125722.008 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium ND2006 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#2: DNA chain TS


Mass: 11083.159 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium ND2006 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#3: DNA chain NTS


Mass: 2970.954 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium ND2006 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#4: DNA chain Substrate


Mass: 2788.862 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium ND2006 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#5: RNA chain crRNA


Mass: 18859.102 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)

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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: CRISPR-Cas complexCRISPR / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 0.5 mg/ml / Embedding 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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 35 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.1_3865: / Classification: refinement
EM software
IDNameVersionCategory
2RELION3image acquisition
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 731231 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410604
ELECTRON MICROSCOPYf_angle_d0.62514780
ELECTRON MICROSCOPYf_dihedral_angle_d18.4084150
ELECTRON MICROSCOPYf_chiral_restr0.0381680
ELECTRON MICROSCOPYf_plane_restr0.0031508

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