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- PDB-7n3o: Cryo-EM structure of the Cas12k-sgRNA complex -

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Basic information

Entry
Database: PDB / ID: 7n3o
TitleCryo-EM structure of the Cas12k-sgRNA complex
Components
  • Cas12k
  • Single guide RNA
KeywordsRNA BINDING PROTEIN/RNA / CRISPR Cas / RNA binding protein / protein-RNA complex / RNA BINDING PROTEIN-RNA complex
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesScytonema hofmannii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsChang, L. / Li, Z. / Xiao, R. / Wang, S. / Han, R.
CitationJournal: Mol Cell / Year: 2021
Title: Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition.
Authors: Renjian Xiao / Shukun Wang / Ruijie Han / Zhuang Li / Clinton Gabel / Indranil Arun Mukherjee / Leifu Chang /
Abstract: The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for ...The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for precise DNA insertion. To reveal the mechanism underlying target DNA recognition, we determined a cryoelectron microscopy (cryo-EM) structure of Cas12k from cyanobacteria Scytonema hofmanni in complex with a single guide RNA (sgRNA) and a double-stranded target DNA. Coupled with mutagenesis and in vitro DNA transposition assay, our results revealed mechanisms for the recognition of the GGTT protospacer adjacent motif (PAM) sequence and the structural elements of Cas12k critical for RNA-guided DNA transposition. These structural and mechanistic insights should aid in the development of type V-K CRISPR-transposon systems as tools for genome editing.
History
DepositionJun 1, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 1, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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

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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: Cas12k
B: Single guide RNA


Theoretical massNumber of molelcules
Total (without water)158,5422
Polymers158,5422
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6700 Å2
ΔGint-46 kcal/mol
Surface area57260 Å2

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Components

#1: Protein Cas12k


Mass: 73280.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
#2: RNA chain Single guide RNA


Mass: 85261.250 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (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

Component
IDNameTypeEntity IDParent-IDSource
1Cryo-EM structure of the Cas12k-sgRNA-dsDNA complexCOMPLEXall0MULTIPLE SOURCES
2Cas12kCOMPLEX#11RECOMBINANT
3Single-guide RNACOMPLEX#21SYNTHETIC
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Scytonema hofmannii (bacteria)34078
23Scytonema hofmannii (bacteria)34078
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.6
SpecimenEmbedding 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: OTHER
Electron lensMode: OTHER
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 183870 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028945
ELECTRON MICROSCOPYf_angle_d0.58913190
ELECTRON MICROSCOPYf_dihedral_angle_d16.3633157
ELECTRON MICROSCOPYf_chiral_restr0.0311669
ELECTRON MICROSCOPYf_plane_restr0.007851

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