+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-40296 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | CRISPR-Cas type III-D effector complex local refinement map | |||||||||
Map data | Type III-D Cas7-insertion local refinement map | |||||||||
Sample |
| |||||||||
Keywords | CRISPR / CRISPR-Cas / type III / complex / RNA / crRNA / RNA BINDING PROTEIN | |||||||||
Biological species | Synechocystis sp. PCC 6803 (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.5 Å | |||||||||
Authors | Schwartz EA / Taylor DW | |||||||||
Funding support | United States, 1 items
| |||||||||
Citation | Journal: Nat Commun / Year: 2024 Title: RNA targeting and cleavage by the type III-Dv CRISPR effector complex. Authors: Evan A Schwartz / Jack P K Bravo / Mohd Ahsan / Luis A Macias / Caitlyn L McCafferty / Tyler L Dangerfield / Jada N Walker / Jennifer S Brodbelt / Giulia Palermo / Peter C Fineran / Robert D ...Authors: Evan A Schwartz / Jack P K Bravo / Mohd Ahsan / Luis A Macias / Caitlyn L McCafferty / Tyler L Dangerfield / Jada N Walker / Jennifer S Brodbelt / Giulia Palermo / Peter C Fineran / Robert D Fagerlund / David W Taylor / Abstract: CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular ...CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular intervals is characteristic of type III effector complexes. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes. | |||||||||
History |
|
-Structure visualization
Supplemental images |
---|
-Downloads & links
-EMDB archive
Map data | emd_40296.map.gz | 306.8 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-40296-v30.xml emd-40296.xml | 15.3 KB 15.3 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_40296_fsc.xml | 14.5 KB | Display | FSC data file |
Images | emd_40296.png | 47.5 KB | ||
Filedesc metadata | emd-40296.cif.gz | 4.5 KB | ||
Others | emd_40296_half_map_1.map.gz emd_40296_half_map_2.map.gz | 301.2 MB 301.2 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-40296 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-40296 | HTTPS FTP |
-Validation report
Summary document | emd_40296_validation.pdf.gz | 1004.6 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_40296_full_validation.pdf.gz | 1004.2 KB | Display | |
Data in XML | emd_40296_validation.xml.gz | 23.5 KB | Display | |
Data in CIF | emd_40296_validation.cif.gz | 30.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40296 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40296 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|
-Map
File | Download / File: emd_40296.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Type III-D Cas7-insertion local refinement map | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.81 Å | ||||||||||||||||||||
Density |
| ||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
|
-Supplemental data
-Half map: Half map B
File | emd_40296_half_map_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half map B | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: Half map A
File | emd_40296_half_map_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half map A | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Sample components
-Entire : CRISPR-Cas type III-D effector complex
Entire | Name: CRISPR-Cas type III-D effector complex |
---|---|
Components |
|
-Supramolecule #1: CRISPR-Cas type III-D effector complex
Supramolecule | Name: CRISPR-Cas type III-D effector complex / type: complex / ID: 1 / Parent: 0 |
---|---|
Source (natural) | Organism: Synechocystis sp. PCC 6803 (bacteria) |
Molecular weight | Theoretical: 351 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.3 mg/mL | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Buffer | pH: 7.5 Component:
| |||||||||||||||
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV | |||||||||||||||
Details | Particles were monodisperse and homogeneous. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
---|---|
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 80.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 105000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model | Chain - Source name: AlphaFold / Chain - Initial model type: in silico model |
---|