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Yorodumi- EMDB-13486: Cryo-EM structure of ShCas12k in complex with a sgRNA and a dsDNA... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13486 | |||||||||
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Title | Cryo-EM structure of ShCas12k in complex with a sgRNA and a dsDNA target | |||||||||
Map data | ||||||||||
Sample |
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Keywords | Cas12k / sgRNA / target DNA / Protein-RNA-DNA complex / Tn7 / DNA Binding Protein / Transposition / CRISPR | |||||||||
Function / homology | : / ShCas12k Function and homology information | |||||||||
Biological species | Scytonema hofmannii (bacteria) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.04 Å | |||||||||
Authors | Schmitz M / Jinek M | |||||||||
Funding support | European Union, 2 items
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Citation | Journal: Nature / Year: 2021 Title: Target site selection and remodelling by type V CRISPR-transposon systems. Authors: Irma Querques / Michael Schmitz / Seraina Oberli / Christelle Chanez / Martin Jinek / Abstract: Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided ...Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_13486.map.gz | 49 MB | EMDB map data format | |
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Header (meta data) | emd-13486-v30.xml emd-13486.xml | 12.7 KB 12.7 KB | Display Display | EMDB header |
Images | emd_13486.png | 56.2 KB | ||
Filedesc metadata | emd-13486.cif.gz | 5.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13486 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13486 | HTTPS FTP |
-Validation report
Summary document | emd_13486_validation.pdf.gz | 445.3 KB | Display | EMDB validaton report |
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Full document | emd_13486_full_validation.pdf.gz | 444.9 KB | Display | |
Data in XML | emd_13486_validation.xml.gz | 6.4 KB | Display | |
Data in CIF | emd_13486_validation.cif.gz | 7.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13486 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13486 | HTTPS FTP |
-Related structure data
Related structure data | 7plaMC 7oxdC 7plhC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_13486.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.64 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Ternary complex of ShCas12k with sgRNA and target dsDNA
Entire | Name: Ternary complex of ShCas12k with sgRNA and target dsDNA |
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Components |
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-Supramolecule #1: Ternary complex of ShCas12k with sgRNA and target dsDNA
Supramolecule | Name: Ternary complex of ShCas12k with sgRNA and target dsDNA type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Scytonema hofmannii (bacteria) |
Molecular weight | Theoretical: 187 KDa |
-Macromolecule #1: ShCas12k
Macromolecule | Name: ShCas12k / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Scytonema hofmannii (bacteria) |
Molecular weight | Theoretical: 73.421773 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: SNASQITIQA RLISFESNRQ QLWKLMADLN TPLINELLCQ LGQHPDFEKW QQKGKLPSTV VSQLCQPLKT DPRFAGQPSR LYMSAIHIV DYIYKSWLAI QKRLQQQLDG KTRWLEMLNS DAELVELSGD TLEAIRVKAA EILAIAMPAS ESDSASPKGK K GKKEKKPS ...String: SNASQITIQA RLISFESNRQ QLWKLMADLN TPLINELLCQ LGQHPDFEKW QQKGKLPSTV VSQLCQPLKT DPRFAGQPSR LYMSAIHIV DYIYKSWLAI QKRLQQQLDG KTRWLEMLNS DAELVELSGD TLEAIRVKAA EILAIAMPAS ESDSASPKGK K GKKEKKPS SSSPKRSLSK TLFDAYQETE DIKSRSAISY LLKNGCKLTD KEEDSEKFAK RRRQVEIQIQ RLTEKLISRM PK GRDLTNA KWLETLLTAT TTVAEDNAQA KRWQDILLTR SSSLPFPLVF ETNEDMVWSK NQKGRLCVHF NGLSDLIFEV YCG NRQLHW FQRFLEDQQT KRKSKNQHSS GLFTLRNGHL VWLEGEGKGE PWNLHHLTLY CCVDNRLWTE EGTEIVRQEK ADEI TKFIT NMKKKSDLSD TQQALIQRKQ STLTRINNSF ERPSQPLYQG QSHILVGVSL GLEKPATVAV VDAIANKVLA YRSIK QLLG DNYELLNRQR RQQQYLSHER HKAQKNFSPN QFGASELGQH IDRLLAKAIV ALARTYKAGS IVLPKLGDMR EVVQSE IQA IAEQKFPGYI EGQQKYAKQY RVNVHRWSYG RLIQSIQSKA AQTGIVIEEG KQPIRGSPHD KAKELALSAY NLRLTRR S UniProtKB: ShCas12k |
-Macromolecule #2: sgRNA
Macromolecule | Name: sgRNA / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: Scytonema hofmannii (bacteria) |
Molecular weight | Theoretical: 82.376547 KDa |
Sequence | String: GGAUAUUAAU AGCGCCGCAA UUCAUGCUGC UUGCAGCCUC UGAAUUUUGU UAAAUGAGGG UUAGUUUGAC UGUAUAAAUA CAGUCUUGC UUUCUGACCC UGGUAGCUGC UCACCCUGAU GCUGCUGUCA AUAGACAGGA UAGGUGCGCU CCCAGCAAUA A GGGCGCGG ...String: GGAUAUUAAU AGCGCCGCAA UUCAUGCUGC UUGCAGCCUC UGAAUUUUGU UAAAUGAGGG UUAGUUUGAC UGUAUAAAUA CAGUCUUGC UUUCUGACCC UGGUAGCUGC UCACCCUGAU GCUGCUGUCA AUAGACAGGA UAGGUGCGCU CCCAGCAAUA A GGGCGCGG AUGUACUGCU GUAGUGGCUA CUGAAUCACC CCCGAUCAAG GGGGAACCCU AAAUGGGUUG AAAGGAGAAG UC AUUUAAU AAGGCCACU |
-Macromolecule #3: DNA target strand
Macromolecule | Name: DNA target strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 15.301837 KDa |
Sequence | String: (DG)(DT)(DT)(DT)(DA)(DA)(DC)(DA)(DG)(DT) (DG)(DG)(DC)(DC)(DT)(DT)(DA)(DT)(DT)(DA) (DA)(DA)(DT)(DG)(DA)(DC)(DT)(DT)(DC) (DT)(DC)(DA)(DA)(DC)(DC)(DT)(DC)(DC)(DT) (DA) (DC)(DG)(DT)(DA)(DT)(DC)(DG)(DT) (DA)(DG) |
-Macromolecule #4: DNA non-target strand
Macromolecule | Name: DNA non-target strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 15.497987 KDa |
Sequence | String: (DC)(DT)(DA)(DC)(DG)(DA)(DT)(DA)(DC)(DG) (DT)(DA)(DG)(DG)(DA)(DG)(DG)(DT)(DT)(DG) (DA)(DG)(DA)(DA)(DG)(DT)(DC)(DA)(DT) (DT)(DT)(DA)(DA)(DT)(DA)(DA)(DG)(DG)(DC) (DC) (DA)(DC)(DT)(DG)(DT)(DT)(DA)(DA) (DA)(DC) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV |
-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: 51.81 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: NONE |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 138000 |
Initial angle assignment | Type: OTHER |
Final angle assignment | Type: OTHER |