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- EMDB-23158: Cryo-EM structure of a CRISPR-Cas Ternary Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-23158
TitleCryo-EM structure of a CRISPR-Cas Ternary Complex
Map data
Sample
  • Complex: Cryo-EM structure of a CRISPR-Cas Ternary Complex
    • Complex: Cas12f1
      • Protein or peptide: Cas12f1
    • Complex: target DNA, substrate DNA, and sgRNA
      • DNA: TS
      • DNA: NTS
      • RNA: sgRNA
      • DNA: Substrate
  • Ligand: ZINC ION
KeywordsCRISPR CAS / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex
Biological speciesunidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 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, 2020-
Header (metadata) releaseJun 2, 2021-
Map releaseJun 2, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7l49
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23158.png

Downloads & links

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Map

FileDownload / File: emd_23158.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.1262963 - 0.23435614
Average (Standard dev.)0.00019907801 (±0.004227985)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 268.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z268.800268.800268.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1260.2340.000

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of a CRISPR-Cas Ternary Complex

EntireName: Cryo-EM structure of a CRISPR-Cas Ternary Complex
Components
  • Complex: Cryo-EM structure of a CRISPR-Cas Ternary Complex
    • Complex: Cas12f1
      • Protein or peptide: Cas12f1
    • Complex: target DNA, substrate DNA, and sgRNA
      • DNA: TS
      • DNA: NTS
      • RNA: sgRNA
      • DNA: Substrate
  • Ligand: ZINC ION

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Supramolecule #1: Cryo-EM structure of a CRISPR-Cas Ternary Complex

SupramoleculeName: Cryo-EM structure of a CRISPR-Cas Ternary Complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5

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Supramolecule #2: Cas12f1

SupramoleculeName: Cas12f1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: unidentified (others)

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Supramolecule #3: target DNA, substrate DNA, and sgRNA

SupramoleculeName: target DNA, substrate DNA, and sgRNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#5
Source (natural)Organism: unidentified (others)

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Macromolecule #1: Cas12f1

MacromoleculeName: Cas12f1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 61.677434 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAKNTITKTL KLRIVRPYNS AEVEKIVADE KNNREKIALE KNKDKVKEAC SKHLKVAAYC TTQVERNACL FCKARKLDDK FYQKLRGQF PDAVFWQEIS EIFRQLQKQA AEIYNQSLIE LYYEIFIKGK GIANASSVEH YLSDVCYTRA AELFKNAAIA S GLRSKIKS ...String:
MAKNTITKTL KLRIVRPYNS AEVEKIVADE KNNREKIALE KNKDKVKEAC SKHLKVAAYC TTQVERNACL FCKARKLDDK FYQKLRGQF PDAVFWQEIS EIFRQLQKQA AEIYNQSLIE LYYEIFIKGK GIANASSVEH YLSDVCYTRA AELFKNAAIA S GLRSKIKS NFRLKELKNM KSGLPTTKSD NFPIPLVKQK GGQYTGFEIS NHNSDFIIKI PFGRWQVKKE IDKYRPWEKF DF EQVQKSP KPISLLLSTQ RRKRNKGWSK DEGTEAEIKK VMNGDYQTSY IEVKRGSKIC EKSAWMLNLS IDVPKIDKGV DPS IIGGID VGVKSPLVCA INNAFSRYSI SDNDLFHFNK KMFARRRILL KKNRHKRAGH GAKNKLKPIT ILTEKSERFR KKLI ERWAC EIADFFIKNK VGTVQMENLE SMKRKEDSYF NIRLRGFWPY AEMQNKIEFK LKQYGIEIRK VAPNNTSKTC SKCGH LNNY FNFEYRKKNK FPHFKCEKCN FKENADYNAA LNISNPKLKS TKEEP

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Macromolecule #2: TS

MacromoleculeName: TS / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 18.587895 KDa
SequenceString: (DG)(DC)(DT)(DG)(DA)(DT)(DG)(DC)(DA)(DT) (DC)(DT)(DA)(DG)(DA)(DT)(DT)(DG)(DT)(DG) (DC)(DA)(DC)(DG)(DC)(DC)(DG)(DG)(DC) (DG)(DA)(DC)(DG)(DT)(DT)(DG)(DG)(DG)(DT) (DC) (DA)(DA)(DC)(DT)(DT)(DA) ...String:
(DG)(DC)(DT)(DG)(DA)(DT)(DG)(DC)(DA)(DT) (DC)(DT)(DA)(DG)(DA)(DT)(DT)(DG)(DT)(DG) (DC)(DA)(DC)(DG)(DC)(DC)(DG)(DG)(DC) (DG)(DA)(DC)(DG)(DT)(DT)(DG)(DG)(DG)(DT) (DC) (DA)(DA)(DC)(DT)(DT)(DA)(DA)(DA) (DT)(DA)(DC)(DG)(DT)(DA)(DA)(DG)(DG)(DT) (DG)(DC)

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Macromolecule #3: NTS

MacromoleculeName: NTS / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 18.396789 KDa
SequenceString: (DG)(DC)(DA)(DC)(DC)(DT)(DT)(DA)(DC)(DG) (DT)(DA)(DT)(DT)(DT)(DA)(DA)(DG)(DT)(DT) (DG)(DA)(DC)(DC)(DC)(DA)(DA)(DC)(DG) (DT)(DC)(DG)(DC)(DC)(DG)(DG)(DC)(DG)(DT) (DG) (DC)(DA)(DC)(DA)(DA)(DT) ...String:
(DG)(DC)(DA)(DC)(DC)(DT)(DT)(DA)(DC)(DG) (DT)(DA)(DT)(DT)(DT)(DA)(DA)(DG)(DT)(DT) (DG)(DA)(DC)(DC)(DC)(DA)(DA)(DC)(DG) (DT)(DC)(DG)(DC)(DC)(DG)(DG)(DC)(DG)(DT) (DG) (DC)(DA)(DC)(DA)(DA)(DT)(DC)(DT) (DA)(DG)(DA)(DT)(DG)(DC)(DA)(DT)(DC)(DA) (DG)(DC)

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Macromolecule #5: Substrate

MacromoleculeName: Substrate / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 1.400952 KDa
SequenceString:
(DC)(DC)(DC)(DC)(DC)

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Macromolecule #4: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 72.442969 KDa
SequenceString: GGGCUUCACU GAUAAAGUGG AGAACCGCUU CACCAAAAGC UGUCCCUUAG GGGAUUAGAA CUUGAGUGAA GGUGGGCUGC UUGCAUCAG CCUAAUGUCG AGAAGUGCUU UCUUCGGAAA GUAACCCUCG AAACAAAUUC AUUUUUCCUC UCCAAUUCUG C ACAAGAAA ...String:
GGGCUUCACU GAUAAAGUGG AGAACCGCUU CACCAAAAGC UGUCCCUUAG GGGAUUAGAA CUUGAGUGAA GGUGGGCUGC UUGCAUCAG CCUAAUGUCG AGAAGUGCUU UCUUCGGAAA GUAACCCUCG AAACAAAUUC AUUUUUCCUC UCCAAUUCUG C ACAAGAAA GUUGCAGAAC CCGAAUAGAC GAAUGAAGGA AUGCAACAGU UGACCCAACG UCGCCGG

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 384132
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

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