+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-15976 | |||||||||||||||
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Title | Cas12k-sgRNA-dsDNA-TnsC non-productive complex | |||||||||||||||
Map data | Cas12k-TnsC non productive complex | |||||||||||||||
Sample |
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Keywords | Cas12k / sgRNA / TnsC / CRISPR-Cas / Tn7-like transposons / transposition / RNA BINDING PROTEIN | |||||||||||||||
Function / homology | Bacterial TniB / Bacterial TniB protein / : / : / P-loop containing nucleoside triphosphate hydrolase / TnsC / Cas12k Function and homology information | |||||||||||||||
Biological species | Scytonema hofmannii (bacteria) / synthetic construct (others) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.1 Å | |||||||||||||||
Authors | Schmitz M / Querques I / Oberli S / Chanez C / Jinek M | |||||||||||||||
Funding support | Switzerland, European Union, United States, 4 items
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Citation | Journal: Cell / Year: 2022 Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex. Authors: Michael Schmitz / Irma Querques / Seraina Oberli / Christelle Chanez / Martin Jinek / Abstract: CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the ...CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads. | |||||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_15976.map.gz | 334.8 MB | EMDB map data format | |
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Header (meta data) | emd-15976-v30.xml emd-15976.xml | 20 KB 20 KB | Display Display | EMDB header |
Images | emd_15976.png | 105 KB | ||
Filedesc metadata | emd-15976.cif.gz | 6.7 KB | ||
Others | emd_15976_half_map_1.map.gz emd_15976_half_map_2.map.gz | 337.8 MB 337.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-15976 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-15976 | HTTPS FTP |
-Validation report
Summary document | emd_15976_validation.pdf.gz | 967.3 KB | Display | EMDB validaton report |
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Full document | emd_15976_full_validation.pdf.gz | 966.8 KB | Display | |
Data in XML | emd_15976_validation.xml.gz | 17.9 KB | Display | |
Data in CIF | emd_15976_validation.cif.gz | 21.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-15976 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-15976 | HTTPS FTP |
-Related structure data
Related structure data | 8bd6MC 8bd4C 8bd5C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_15976.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Cas12k-TnsC non productive complex | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.65 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: Half1
File | emd_15976_half_map_1.map | ||||||||||||
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Annotation | Half1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half2
File | emd_15976_half_map_2.map | ||||||||||||
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Annotation | Half2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Target DNA bound Cas12k-sgRNA-TnsC complex in non-productive state
Entire | Name: Target DNA bound Cas12k-sgRNA-TnsC complex in non-productive state |
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Components |
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-Supramolecule #1: Target DNA bound Cas12k-sgRNA-TnsC complex in non-productive state
Supramolecule | Name: Target DNA bound Cas12k-sgRNA-TnsC complex in non-productive state type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3, #6, #4-#5 / Details: Cas12k, sgRNA, target DNA, TnsC |
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Source (natural) | Organism: Scytonema hofmannii (bacteria) |
-Macromolecule #1: Cas12k
Macromolecule | Name: Cas12k / 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: 79.156773 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MGSSHHHHHH SGGGSGGSAW SHPQFEKGGG SGGGSGGSAW SHPQFEKSGG GENLYFQSNA SQITIQARLI SFESNRQQLW KLMADLNTP LINELLCQLG QHPDFEKWQQ KGKLPSTVVS QLCQPLKTDP RFAGQPSRLY MSAIHIVDYI YKSWLAIQKR L QQQLDGKT ...String: MGSSHHHHHH SGGGSGGSAW SHPQFEKGGG SGGGSGGSAW SHPQFEKSGG GENLYFQSNA SQITIQARLI SFESNRQQLW KLMADLNTP LINELLCQLG QHPDFEKWQQ KGKLPSTVVS QLCQPLKTDP RFAGQPSRLY MSAIHIVDYI YKSWLAIQKR L QQQLDGKT RWLEMLNSDA ELVELSGDTL EAIRVKAAEI LAIAMPASES DSASPKGKKG KKEKKPSSSS PKRSLSKTLF DA YQETEDI KSRSAISYLL KNGCKLTDKE EDSEKFAKRR RQVEIQIQRL TEKLISRMPK GRDLTNAKWL ETLLTATTTV AED NAQAKR WQDILLTRSS SLPFPLVFET NEDMVWSKNQ KGRLCVHFNG LSDLIFEVYC GNRQLHWFQR FLEDQQTKRK SKNQ HSSGL FTLRNGHLVW LEGEGKGEPW NLHHLTLYCC VDNRLWTEEG TEIVRQEKAD EITKFITNMK KKSDLSDTQQ ALIQR KQST LTRINNSFER PSQPLYQGQS HILVGVSLGL EKPATVAVVD AIANKVLAYR SIKQLLGDNY ELLNRQRRQQ QYLSHE RHK AQKNFSPNQF GASELGQHID RLLAKAIVAL ARTYKAGSIV LPKLGDMREV VQSEIQAIAE QKFPGYIEGQ QKYAKQY RV NVHRWSYGRL IQSIQSKAAQ TGIVIEEGKQ PIRGSPHDKA KELALSAYNL RLTRRS UniProtKB: Cas12k |
-Macromolecule #5: TnsC
Macromolecule | Name: TnsC / type: protein_or_peptide / ID: 5 / Number of copies: 9 / Enantiomer: LEVO |
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Source (natural) | Organism: Scytonema hofmannii (bacteria) |
Molecular weight | Theoretical: 31.444617 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTV PVVYIRPHQK CGPKDLFKKI TEYLKYRVTK GTVSDFRDRT IEVLKGCGVE MLIIDEADRL KPETFADVRD I AEDLGIAV ...String: MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTV PVVYIRPHQK CGPKDLFKKI TEYLKYRVTK GTVSDFRDRT IEVLKGCGVE MLIIDEADRL KPETFADVRD I AEDLGIAV VLVGTDRLDA VIKRDEQVLE RFRAHLRFGK LSGEDFKNTV EMWEQMVLKL PVSSNLKSKE MLRILTSATE GY IGRLDEI LREAAIRSLS RGLKKIDKAV LQEVAKEYK UniProtKB: TnsC |
-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: 14.916607 KDa |
Sequence | String: (DA)(DT)(DA)(DT)(DC)(DT)(DA)(DC)(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) |
-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: 15.125739 KDa |
Sequence | String: (DG)(DT)(DA)(DG)(DG)(DA)(DG)(DG)(DT)(DT) (DC)(DT)(DC)(DT)(DT)(DC)(DA)(DG)(DT)(DA) (DT)(DT)(DA)(DA)(DT)(DA)(DA)(DG)(DG) (DC)(DC)(DA)(DC)(DT)(DG)(DT)(DT)(DA)(DA) (DA) (DC)(DG)(DT)(DA)(DC)(DT)(DA)(DT) (DA) |
-Macromolecule #6: DNA
Macromolecule | Name: DNA / type: dna / ID: 6 / Number of copies: 2 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 12.303033 KDa |
Sequence | String: (DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT) (DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT) (DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA) (DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA) (DT) |
-Macromolecule #7: ADENOSINE-5'-TRIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 9 / Formula: ATP |
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Molecular weight | Theoretical: 507.181 Da |
Chemical component information | ChemComp-ATP: |
-Macromolecule #8: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 9 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-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 |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 67.68 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: EMDB MAP EMDB ID: |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 133000 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |