+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-35154 | |||||||||
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Title | Cryo-EM structure of Cas12g-sgRNA binary complex | |||||||||
Map data | ||||||||||
Sample |
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Keywords | CRISPR-Cas / RNA BINDING PROTEIN | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
Authors | Ouyang SY / Liu MX / Li ZK | |||||||||
Funding support | China, 1 items
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Citation | Journal: PLoS Genet / Year: 2023 Title: Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage. Authors: Mengxi Liu / Zekai Li / Jing Chen / Jinying Lin / Qiuhua Lu / Yangmiao Ye / Hongmin Zhang / Bo Zhang / Songying Ouyang / Abstract: Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC ...Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. The conserved zinc finger motifs in Cas12g undergo an ordered-to-disordered transition from the apo to the sgRNA-bound state and their mutations negatively impact on target RNA cleavage. Moreover, we identified a lid motif in the RuvC domain that undergoes transformation from a helix to loop to regulate the access to the RuvC active site and subsequent cleavage of the RNA substrate. Overall, our study provides valuable insights into the mechanisms by which Cas12g recognizes sgRNA and the conformational changes it undergoes from sgRNA binding to the activation of the RNase active site, thereby laying a foundation for the potential repurposing of Cas12g as a tool for RNA-editing. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_35154.map.gz | 4.2 MB | EMDB map data format | |
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Header (meta data) | emd-35154-v30.xml emd-35154.xml | 15.7 KB 15.7 KB | Display Display | EMDB header |
Images | emd_35154.png | 37.8 KB | ||
Masks | emd_35154_msk_1.map | 52.7 MB | Mask map | |
Filedesc metadata | emd-35154.cif.gz | 5.9 KB | ||
Others | emd_35154_half_map_1.map.gz emd_35154_half_map_2.map.gz | 40.8 MB 40.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-35154 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-35154 | HTTPS FTP |
-Validation report
Summary document | emd_35154_validation.pdf.gz | 695 KB | Display | EMDB validaton report |
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Full document | emd_35154_full_validation.pdf.gz | 694.5 KB | Display | |
Data in XML | emd_35154_validation.xml.gz | 11.7 KB | Display | |
Data in CIF | emd_35154_validation.cif.gz | 13.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-35154 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-35154 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_35154.map.gz / Format: CCP4 / Size: 52.7 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: 0.84 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_35154_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_35154_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_35154_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Cas12g-sgRNA binary complex
Entire | Name: Cas12g-sgRNA binary complex |
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Components |
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-Supramolecule #1: Cas12g-sgRNA binary complex
Supramolecule | Name: Cas12g-sgRNA binary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
-Supramolecule #2: Cas12g
Supramolecule | Name: Cas12g / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2 |
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-Supramolecule #3: RNA
Supramolecule | Name: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1 |
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-Macromolecule #1: RNA (139-MER)
Macromolecule | Name: RNA (139-MER) / type: rna / ID: 1 / Number of copies: 1 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 44.771703 KDa |
Sequence | String: GGGAUGCUUA CUUAGUCAUC UGGUUGGCAA ACCUCCGCGG ACCUUCGGGA CCAAUGGAGA GGAACCCAGC CGAGAAGCAU CGAGCCGGU AAAUGAAUUU ACCGGCUCUG ACACCAAUUC GAAAUUAACA CAAACAAGCU |
-Macromolecule #2: Cas12g
Macromolecule | Name: Cas12g / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 89.847672 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MAQASSTPAV SPRPRPRYRE ERTLVRKLLP RPGQSKQEFR ENVKKLRKAF LQFNADVSGV CQWAIQFRPR YGKPAEPTET FWKFFLEPE TSLPPNDSRS PEFRRLQAFE AAAGINGAAA LDDPAFTNEL RDSILAVASR PKTKEAQRLF SRLKDYQPAH R MILAKVAA ...String: MAQASSTPAV SPRPRPRYRE ERTLVRKLLP RPGQSKQEFR ENVKKLRKAF LQFNADVSGV CQWAIQFRPR YGKPAEPTET FWKFFLEPE TSLPPNDSRS PEFRRLQAFE AAAGINGAAA LDDPAFTNEL RDSILAVASR PKTKEAQRLF SRLKDYQPAH R MILAKVAA EWIESRYRRA HQNWERNYEE WKKEKQEWEQ NHPELTPEIR EAFNQIFQQL EVKEKRVRIC PAARLLQNKD NC QYAGKNK HSVLCNQFNE FKKNHLQGKA IKFFYKDAEK YLRCGLQSLK PNVQGPFRED WNKYLRYMNL KEETLRGKNG GRL PHCKNL GQECEFNPHT ALCKQYQQQL SSRPDLVQHD ELYRKWRREY WREPRKPVFR YPSVKRHSIA KIFGENYFQA DFKN SVVGL RLDSMPAGQY LEFAFAPWPR NYRPQPGETE ISSVHLHFVG TRPRIGFRFR VPHKRSRFDC TQEELDELRS RTFPR KAQD QKFLEAARKR LLETFPGNAE QELRLLAVAL GTDSARAAFF IGKTFQQAFP LKIVKIEKLY EQWPNQKQAG DRRDAS SKQ PRPGLSRDHV GRHLQKMRAQ ASEIAQKRQE LTGTPAPETT TDQAAKKATL QPFDLRGLTV HTARMIRDWA RLNARQI IQ LAEENQVDLI VLESLRGFRP PGYENLDQEK KRRVAFFAHG RIRRKVTEKA VERGMRVVTV PYLASSKVCA ECRKKQKD N KQWEKNKKRG LFKCEGCGSQ AQVDENAARV LGRVFWGEIE LPTAIP |
-Macromolecule #3: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 3 mg/mL |
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Buffer | pH: 8.5 |
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: 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: 1.8 µm / Nominal defocus min: 1.2 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: INSILICO MODEL |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 88696 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |