+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13489 | |||||||||
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Title | Scytonema hofmannii TnsC bound to AMPPNP and DNA | |||||||||
Map data | ||||||||||
Sample |
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Keywords | TnsC / Transposition protein / AAA+ ATPase / Tn7 / CRISPR / DNA BINDING PROTEIN | |||||||||
Biological species | Scytonema hofmannii (bacteria) / synthetic construct (others) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.57 Å | |||||||||
Authors | Querques I / 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_13489.map.gz | 152.2 MB | EMDB map data format | |
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Header (meta data) | emd-13489-v30.xml emd-13489.xml | 10.7 KB 10.7 KB | Display Display | EMDB header |
Images | emd_13489.png | 76.1 KB | ||
Filedesc metadata | emd-13489.cif.gz | 5.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13489 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13489 | HTTPS FTP |
-Validation report
Summary document | emd_13489_validation.pdf.gz | 611.1 KB | Display | EMDB validaton report |
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Full document | emd_13489_full_validation.pdf.gz | 610.7 KB | Display | |
Data in XML | emd_13489_validation.xml.gz | 6.9 KB | Display | |
Data in CIF | emd_13489_validation.cif.gz | 7.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13489 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13489 | HTTPS FTP |
-Related structure data
Related structure data | 7plhMC 7oxdC 7plaC 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_13489.map.gz / Format: CCP4 / Size: 166.4 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: 0.65 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 : Complex of ShTnsC with AMPPNP and dsDNA
Entire | Name: Complex of ShTnsC with AMPPNP and dsDNA |
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Components |
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-Supramolecule #1: Complex of ShTnsC with AMPPNP and dsDNA
Supramolecule | Name: Complex of ShTnsC with AMPPNP and dsDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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-Macromolecule #1: ShTnsC
Macromolecule | Name: ShTnsC / type: protein_or_peptide / ID: 1 / Number of copies: 7 / 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 |
-Macromolecule #2: DNA (5'-D(P*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP...
Macromolecule | Name: DNA (5'-D(P*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*T)-3') type: dna / ID: 2 / Number of copies: 2 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 6.746438 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) |
-Macromolecule #3: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 7 / Formula: ANP |
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Molecular weight | Theoretical: 506.196 Da |
Chemical component information | ChemComp-ANP: |
-Macromolecule #4: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 7 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | filament |
-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 BIOQUANTUM (6k x 4k) / Average electron dose: 66.39 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Applied symmetry - Helical parameters - Δz: 6.78 Å Applied symmetry - Helical parameters - Δ&Phi: 59.72 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Resolution.type: BY AUTHOR / Resolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 77544 |
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Startup model | Type of model: PDB ENTRY |
Final angle assignment | Type: NOT APPLICABLE |