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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-23158 | |||||||||
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Title | Cryo-EM structure of a CRISPR-Cas Ternary Complex | |||||||||
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![]() | CRISPR CAS / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex | |||||||||
Biological species | unidentified (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
![]() | Chang L / Li Z | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 13.8 KB 13.8 KB | Display Display | ![]() |
Images | ![]() | 61.5 KB | ||
Filedesc metadata | ![]() | 5.7 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7l49MC ![]() 7l48C M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 1.05 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Cryo-EM structure of a CRISPR-Cas Ternary Complex
Entire | Name: Cryo-EM structure of a CRISPR-Cas Ternary Complex |
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Components |
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-Supramolecule #1: Cryo-EM structure of a CRISPR-Cas Ternary Complex
Supramolecule | Name: 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
Supramolecule | Name: Cas12f1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: unidentified (others) |
-Supramolecule #3: target DNA, substrate DNA, and sgRNA
Supramolecule | Name: target DNA, substrate DNA, and sgRNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#5 |
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Source (natural) | Organism: unidentified (others) |
-Macromolecule #1: Cas12f1
Macromolecule | Name: Cas12f1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Theoretical: 61.677434 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #2: TS
Macromolecule | Name: TS / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Theoretical: 18.587895 KDa |
Sequence | 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) ...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) |
-Macromolecule #3: NTS
Macromolecule | Name: NTS / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Theoretical: 18.396789 KDa |
Sequence | 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) ...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) |
-Macromolecule #5: Substrate
Macromolecule | Name: Substrate / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Theoretical: 1.400952 KDa |
Sequence | String: (DC)(DC)(DC)(DC)(DC) |
-Macromolecule #4: sgRNA
Macromolecule | Name: sgRNA / type: rna / ID: 4 / Number of copies: 1 |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Theoretical: 72.442969 KDa |
Sequence | String: 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 |
-Macromolecule #6: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 6 / Number of copies: 4 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: OTHER / Imaging mode: OTHER |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: INSILICO MODEL |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 384132 |
Initial angle assignment | Type: RANDOM ASSIGNMENT |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |