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Yorodumi- EMDB-27971: V-K CAST Transpososome from Scytonema hofmanni, major configuration -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27971 | |||||||||
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Title | V-K CAST Transpososome from Scytonema hofmanni, major configuration | |||||||||
Map data | ||||||||||
Sample |
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Keywords | DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex | |||||||||
Function / homology | Function and homology information rRNA binding / ribosome / structural constituent of ribosome / ribonucleoprotein complex / translation / cytoplasm Similarity search - Function | |||||||||
Biological species | Scytonema hofmannii (bacteria) / synthetic construct (others) / Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.7 Å | |||||||||
Authors | Rizo AN / Park J-U / Tsai AW / Kellogg EH | |||||||||
Funding support | United States, 2 items
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Citation | Journal: Nature / Year: 2023 Title: Structures of the holo CRISPR RNA-guided transposon integration complex. Authors: Jung-Un Park / Amy Wei-Lun Tsai / Alexandrea N Rizo / Vinh H Truong / Tristan X Wellner / Richard D Schargel / Elizabeth H Kellogg / Abstract: CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR ...CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_27971.map.gz | 567.4 MB | EMDB map data format | |
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Header (meta data) | emd-27971-v30.xml emd-27971.xml | 33.1 KB 33.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_27971_fsc.xml | 17.9 KB | Display | FSC data file |
Images | emd_27971.png | 96.9 KB | ||
Masks | emd_27971_msk_1.map | 600.7 MB | Mask map | |
Filedesc metadata | emd-27971.cif.gz | 7.7 KB | ||
Others | emd_27971_additional_1.map.gz emd_27971_additional_2.map.gz emd_27971_additional_3.map.gz emd_27971_half_map_1.map.gz emd_27971_half_map_2.map.gz | 301.6 MB 563.4 MB 565.2 MB 556.8 MB 556.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27971 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27971 | HTTPS FTP |
-Validation report
Summary document | emd_27971_validation.pdf.gz | 1.2 MB | Display | EMDB validaton report |
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Full document | emd_27971_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | emd_27971_validation.xml.gz | 27.7 KB | Display | |
Data in CIF | emd_27971_validation.cif.gz | 36.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27971 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27971 | HTTPS FTP |
-Related structure data
Related structure data | 8ea3MC 7svuC 8ea4C C: citing same article (ref.) M: atomic model generated by this map |
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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_27971.map.gz / Format: CCP4 / Size: 600.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 1.067 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_27971_msk_1.map | ||||||||||||
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Density Histograms |
-Additional map: #3
File | emd_27971_additional_1.map | ||||||||||||
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Density Histograms |
-Additional map: #2
File | emd_27971_additional_2.map | ||||||||||||
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-Additional map: #1
File | emd_27971_additional_3.map | ||||||||||||
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Density Histograms |
-Half map: #2
File | emd_27971_half_map_1.map | ||||||||||||
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Density Histograms |
-Half map: #1
File | emd_27971_half_map_2.map | ||||||||||||
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Density Histograms |
-Sample components
+Entire : 12_TnsC ShCAST Transpososome
+Supramolecule #1: 12_TnsC ShCAST Transpososome
+Macromolecule #1: Target_LE
+Macromolecule #2: Non-target_R
+Macromolecule #9: LE_R
+Macromolecule #10: RE_F
+Macromolecule #11: RE_R1
+Macromolecule #12: RE_R2
+Macromolecule #3: sg_RNA
+Macromolecule #4: TnsC
+Macromolecule #5: Cas12k
+Macromolecule #6: TniQ
+Macromolecule #7: 30S ribosomal protein S15
+Macromolecule #8: TnsB
+Macromolecule #13: MAGNESIUM ION
+Macromolecule #14: ADENOSINE-5'-TRIPHOSPHATE
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.4 |
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Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | TFS KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 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: 2.5 µm / Nominal defocus min: 1.0 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |