+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-15540 | |||||||||
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Title | Structure of the type I-G CRISPR effector | |||||||||
Map data | Main map | |||||||||
Sample |
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Keywords | CRISPR effector / IMMUNE SYSTEM | |||||||||
Function / homology | CRISPR-associated protein GSU0053 / CRISPR-associated protein GSU0053 (Cas_GSU0053) / Type I-U CRISPR-associated protein Cas7 Function and homology information | |||||||||
Biological species | Thioalkalivibrio sulfidiphilus HL-EbGr7 (bacteria) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
Authors | Shangguan Q / Graham S / Sundaramoorthy R / White MF | |||||||||
Funding support | United Kingdom, 1 items
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Citation | Journal: Nucleic Acids Res / Year: 2022 Title: Structure and mechanism of the type I-G CRISPR effector. Authors: Qilin Shangguan / Shirley Graham / Ramasubramanian Sundaramoorthy / Malcolm F White / Abstract: Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 ...Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3' end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_15540.map.gz | 11.2 MB | EMDB map data format | |
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Header (meta data) | emd-15540-v30.xml emd-15540.xml | 31.5 KB 31.5 KB | Display Display | EMDB header |
Images | emd_15540.png | 30.4 KB | ||
Filedesc metadata | emd-15540.cif.gz | 6.5 KB | ||
Others | emd_15540_additional_1.map.gz emd_15540_additional_2.map.gz emd_15540_additional_3.map.gz emd_15540_additional_4.map.gz emd_15540_additional_5.map.gz emd_15540_additional_6.map.gz emd_15540_half_map_1.map.gz emd_15540_half_map_2.map.gz | 128.2 MB 118.4 MB 120.4 MB 128.4 MB 120.4 MB 128.6 MB 129.8 MB 129.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-15540 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-15540 | HTTPS FTP |
-Validation report
Summary document | emd_15540_validation.pdf.gz | 728 KB | Display | EMDB validaton report |
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Full document | emd_15540_full_validation.pdf.gz | 727.6 KB | Display | |
Data in XML | emd_15540_validation.xml.gz | 14.5 KB | Display | |
Data in CIF | emd_15540_validation.cif.gz | 17.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-15540 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-15540 | HTTPS FTP |
-Related structure data
Related structure data | 8aneMC 8b2xC 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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-Map
File | Download / File: emd_15540.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Main map | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.008 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: First body of Multibody refinement
File | emd_15540_additional_1.map | ||||||||||||
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Annotation | First body of Multibody refinement | ||||||||||||
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Density Histograms |
-Additional map: Second body of Multibody refinement
File | emd_15540_additional_2.map | ||||||||||||
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Annotation | Second body of Multibody refinement | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: Half map of body2 of Multibody
File | emd_15540_additional_3.map | ||||||||||||
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Annotation | Half map of body2 of Multibody | ||||||||||||
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-Additional map: #2
File | emd_15540_additional_4.map | ||||||||||||
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-Additional map: #1
File | emd_15540_additional_5.map | ||||||||||||
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Density Histograms |
-Additional map: Half map of body1 of Multibody
File | emd_15540_additional_6.map | ||||||||||||
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Annotation | Half map of body1 of Multibody | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half Map for the main map
File | emd_15540_half_map_1.map | ||||||||||||
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Annotation | Half Map for the main map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half Map for the main map
File | emd_15540_half_map_2.map | ||||||||||||
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Annotation | Half Map for the main map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Type IG cascade core cas7 with CrRNA
Entire | Name: Type IG cascade core cas7 with CrRNA |
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Components |
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-Supramolecule #1: Type IG cascade core cas7 with CrRNA
Supramolecule | Name: Type IG cascade core cas7 with CrRNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Thioalkalivibrio sulfidiphilus HL-EbGr7 (bacteria) |
Molecular weight | Theoretical: 35 KDa |
-Macromolecule #1: Cas7
Macromolecule | Name: Cas7 / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO |
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Source (natural) | Organism: Thioalkalivibrio sulfidiphilus HL-EbGr7 (bacteria) Strain: HL-EbGR7 |
Molecular weight | Theoretical: 34.833082 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MKLEPLLSDV PRLLMEADLV PVQGTRFQPT GFPDLGAAHY EGPDGRPMLL VESAQSMANR LETVCWDKDA DDWVVPLRGL PVVKVLDKA GKPLTNSVLE AHRLNSPYIL EGKDKTLFDL LKQELAHMEE GPVDIRKLAE TLLKVDANAV LHGVFLAKKE L AGGRLRLP ...String: MKLEPLLSDV PRLLMEADLV PVQGTRFQPT GFPDLGAAHY EGPDGRPMLL VESAQSMANR LETVCWDKDA DDWVVPLRGL PVVKVLDKA GKPLTNSVLE AHRLNSPYIL EGKDKTLFDL LKQELAHMEE GPVDIRKLAE TLLKVDANAV LHGVFLAKKE L AGGRLRLP RALSAFIEAE DVRVASSGGV KNDHVNPSGD TSRGFGNVPF ARDEYVSPRI KAYFNLDLAQ IRAFGLGEQV DR LLIALAL YKVRRFLVHG LRLRTACDLD CQALRVTRPE GWEVPELSEL EAALPGLIEA VAGEGRFAQP AVTIVTYEK UniProtKB: Type I-U CRISPR-associated protein Cas7 |
-Macromolecule #2: RNA (66-MER)
Macromolecule | Name: RNA (66-MER) / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 21.082465 KDa |
Sequence | String: AUUGAAGCAA GCUGUCCCUG AUGGUCGUCA UCUACCUGCC UGGAGUCAUC CGCGGCAUUU AGCCGC |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 7.5 / Details: 20 mM Tris-HCl, 250 mM NaCl, pH 7.5 |
Grid | Model: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.0001 kPa |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV / Details: blot time 3.5 blot force 4. |
Details | Size exclusion purified monodisperse sample in solution |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 77.0 K / Max: 183.0 K |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 1 / Number real images: 15710 / Average exposure time: 3.0 sec. / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.8000000000000003 µm / Calibrated defocus min: 1.6 µm / Calibrated magnification: 105000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.6 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
Refinement | Space: REAL / Protocol: AB INITIO MODEL / Overall B value: 188 / Target criteria: correlation coefficient |
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Output model | PDB-8ane: |