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- PDB-7l48: Cryo-EM structure of a CRISPR-Cas12f Binary Complex -

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Basic information

Entry
Database: PDB / ID: 7l48
TitleCryo-EM structure of a CRISPR-Cas12f Binary Complex
Components
  • Cas12f
  • sgRNA
KeywordsRNA BINDING PROTEIN/RNA / CRISPR CAS / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesunidentified (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsChang, L. / Li, Z.
CitationJournal: Nucleic Acids Res / Year: 2021
Title: Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR-Cas12f nuclease.
Authors: Renjian Xiao / Zhuang Li / Shukun Wang / Ruijie Han / Leifu Chang /
Abstract: Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR-Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate ...Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR-Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR-Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.
History
DepositionDec 18, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 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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Assembly

Deposited unit
A: Cas12f
B: Cas12f
E: sgRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)196,0597
Polymers195,7983
Non-polymers2624
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14700 Å2
ΔGint-120 kcal/mol
Surface area70980 Å2

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Components

#1: Protein Cas12f


Mass: 61677.434 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: RNA chain sgRNA


Mass: 72442.969 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) unidentified (others)
#3: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / 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
1Cryo-EM structure of a CRISPR-Cas Ternary ComplexCOMPLEX#1-#20MULTIPLE SOURCES
2Cas12fCOMPLEX#11RECOMBINANT
3sgRNACOMPLEX#21NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12unidentified (others)32644
23unidentified (others)32644
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.5
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: 1.18.2_3874: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 154090 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412021
ELECTRON MICROSCOPYf_angle_d0.97516875
ELECTRON MICROSCOPYf_dihedral_angle_d21.6762858
ELECTRON MICROSCOPYf_chiral_restr0.0531944
ELECTRON MICROSCOPYf_plane_restr0.0071620

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