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Yorodumi- EMDB-27972: V-K CAST Transpososome from Scytonema hofmanni, minor configuration -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27972 | |||||||||
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Title | V-K CAST Transpososome from Scytonema hofmanni, minor configuration | |||||||||
Map data | ||||||||||
Sample |
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Keywords | DNA BINDING PROTEIN / DNA BINDING PROTEIN-RNA-DNA complex | |||||||||
Function / homology | Function and homology information rRNA binding / ribosome / structural constituent of ribosome / ribonucleoprotein complex / translation / cytoplasm Similarity search - Function | |||||||||
Biological species | [Scytonema hofmanni] UTEX 2349 (bacteria) / Scytonema hofmannii (bacteria) / Escherichia coli (E. coli) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.0 Å | |||||||||
Authors | Rizo AR / Park J-U / Tsai AW / Kellogg EK | |||||||||
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_27972.map.gz | 301 MB | EMDB map data format | |
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Header (meta data) | emd-27972-v30.xml emd-27972.xml | 28.8 KB 28.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_27972_fsc.xml | 17.9 KB | Display | FSC data file |
Images | emd_27972.png | 89.9 KB | ||
Masks | emd_27972_msk_1.map | 600.7 MB | Mask map | |
Filedesc metadata | emd-27972.cif.gz | 7.8 KB | ||
Others | emd_27972_additional_1.map.gz emd_27972_half_map_1.map.gz emd_27972_half_map_2.map.gz | 568 MB 557.4 MB 557.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27972 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27972 | HTTPS FTP |
-Validation report
Summary document | emd_27972_validation.pdf.gz | 1 MB | Display | EMDB validaton report |
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Full document | emd_27972_full_validation.pdf.gz | 1 MB | Display | |
Data in XML | emd_27972_validation.xml.gz | 27.7 KB | Display | |
Data in CIF | emd_27972_validation.cif.gz | 36.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27972 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27972 | HTTPS FTP |
-Related structure data
Related structure data | 8ea4MC 7svuC 8ea3C 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_27972.map.gz / Format: CCP4 / Size: 600.7 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: 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_27972_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: #1
File | emd_27972_additional_1.map | ||||||||||||
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Density Histograms |
-Half map: #2
File | emd_27972_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_27972_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Sh_CAST Transpososome
+Supramolecule #1: Sh_CAST Transpososome
+Macromolecule #1: TnsC
+Macromolecule #2: Cas12k
+Macromolecule #3: TniQ
+Macromolecule #4: 30S ribosomal protein S15
+Macromolecule #5: TnsB
+Macromolecule #6: Target-LE
+Macromolecule #7: LE_R
+Macromolecule #8: Non-target_R
+Macromolecule #9: RE_F
+Macromolecule #10: RE_R1
+Macromolecule #11: RE_R2
+Macromolecule #12: sg_RNA
+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 |
-Electron microscopy
Microscope | FEI TITAN 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 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |