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Yorodumi- EMDB-15975: Cas12k-sgRNA-dsDNA-S15-TniQ-TnsC transposon recruitment complex -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-15975 | |||||||||||||||
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Title | Cas12k-sgRNA-dsDNA-S15-TniQ-TnsC transposon recruitment complex | |||||||||||||||
Map data | Cas12k transposon recruitment complex | |||||||||||||||
Sample |
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Keywords | Cas12k / sgRNA / S15 / TniQ / TnsC / CRISPR-Cas / Tn7-like transposons / transposition / RNA BINDING PROTEIN | |||||||||||||||
Function / homology | Function and homology information small ribosomal subunit rRNA binding / regulation of translation / ribosomal small subunit assembly / cytosolic small ribosomal subunit / cytoplasmic translation / rRNA binding / structural constituent of ribosome / translation / cytosol / cytoplasm Similarity search - Function | |||||||||||||||
Biological species | Scytonema hofmannii (bacteria) / Escherichia coli K-12 (bacteria) / synthetic construct (others) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 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_15975.map.gz | 338.1 MB | EMDB map data format | |
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Header (meta data) | emd-15975-v30.xml emd-15975.xml | 22.3 KB 22.3 KB | Display Display | EMDB header |
Images | emd_15975.png | 114.7 KB | ||
Filedesc metadata | emd-15975.cif.gz | 7.1 KB | ||
Others | emd_15975_half_map_1.map.gz emd_15975_half_map_2.map.gz | 339.3 MB 337.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-15975 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-15975 | HTTPS FTP |
-Validation report
Summary document | emd_15975_validation.pdf.gz | 1009.4 KB | Display | EMDB validaton report |
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Full document | emd_15975_full_validation.pdf.gz | 1009 KB | Display | |
Data in XML | emd_15975_validation.xml.gz | 17.9 KB | Display | |
Data in CIF | emd_15975_validation.cif.gz | 21.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-15975 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-15975 | HTTPS FTP |
-Related structure data
Related structure data | 8bd5MC 8bd4C 8bd6C 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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Related items in Molecule of the Month |
-Map
File | Download / File: emd_15975.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Cas12k transposon recruitment 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: Half2
File | emd_15975_half_map_1.map | ||||||||||||
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Annotation | Half2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half1
File | emd_15975_half_map_2.map | ||||||||||||
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Annotation | Half1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Target DNA bound Cas12k-sgRNA-S15-TniQ-TnsC transposon recruitmen...
+Supramolecule #1: Target DNA bound Cas12k-sgRNA-S15-TniQ-TnsC transposon recruitmen...
+Macromolecule #1: ShCas12k
+Macromolecule #5: TnsC
+Macromolecule #6: TniQ (Homology model)
+Macromolecule #7: 30S ribosomal protein S15
+Macromolecule #2: sgRNA
+Macromolecule #3: DNA target strand
+Macromolecule #4: DNA non-target strand
+Macromolecule #8: ADENOSINE-5'-TRIPHOSPHATE
+Macromolecule #9: MAGNESIUM ION
+Macromolecule #10: ZINC ION
-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 / Chamber humidity: 75 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-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: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.2) / Number images used: 75000 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |