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Yorodumi- EMDB-29433: Cryo-EM structure of the Cas13bt3-crRNA-target RNA ternary comple... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-29433 | |||||||||
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Title | Cryo-EM structure of the Cas13bt3-crRNA-target RNA ternary complex in activated state | |||||||||
Map data | ||||||||||
Sample |
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Keywords | Cas13bt3-crRNA-target RNA ternary complex / activated state / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex | |||||||||
Function / homology | Uncharacterized protein Function and homology information | |||||||||
Biological species | Planctomycetota bacterium (bacteria) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.5 Å | |||||||||
Authors | Gao Y / Deng X | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Nat Commun / Year: 2023 Title: Structural basis for the activation of a compact CRISPR-Cas13 nuclease. Authors: Xiangyu Deng / Emmanuel Osikpa / Jie Yang / Seye J Oladeji / Jamie Smith / Xue Gao / Yang Gao / Abstract: The CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 ...The CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 effectors and their non-specific RNA cleavage upon target activation limit the adeno-associated virus based delivery of Cas13 systems for therapeutic applications. Herein, we report detailed biochemical and structural characterizations of a compact Cas13 (Cas13bt3) suitable for adeno-associated virus delivery. Distinct from many other Cas13 systems, Cas13bt3 cleaves the target and other nonspecific RNA at internal "UC" sites and is activated in a target length-dependent manner. The cryo-electron microscope structure of Cas13bt3 in a fully active state illustrates the structural basis of Cas13bt3 activation. Guided by the structure, we obtain engineered Cas13bt3 variants with minimal off-target cleavage yet maintained target cleavage activities. In conclusion, our biochemical and structural data illustrate a distinct mechanism for Cas13bt3 activation and guide the engineering of Cas13bt3 applications. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_29433.map.gz | 97.2 MB | EMDB map data format | |
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Header (meta data) | emd-29433-v30.xml emd-29433.xml | 15.5 KB 15.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_29433_fsc.xml | 9.9 KB | Display | FSC data file |
Images | emd_29433.png | 50.2 KB | ||
Filedesc metadata | emd-29433.cif.gz | 5.9 KB | ||
Others | emd_29433_half_map_1.map.gz emd_29433_half_map_2.map.gz | 95.7 MB 95.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-29433 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-29433 | HTTPS FTP |
-Validation report
Summary document | emd_29433_validation.pdf.gz | 1006.3 KB | Display | EMDB validaton report |
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Full document | emd_29433_full_validation.pdf.gz | 1005.9 KB | Display | |
Data in XML | emd_29433_validation.xml.gz | 18.2 KB | Display | |
Data in CIF | emd_29433_validation.cif.gz | 23.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29433 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29433 | HTTPS FTP |
-Related structure data
Related structure data | 8ftiMC 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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Related items in Molecule of the Month |
-Map
File | Download / File: emd_29433.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 0.87 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #1
File | emd_29433_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_29433_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Ternary complex of Cas13bt3 in complex with crRNA and 30 nt target RNA
Entire | Name: Ternary complex of Cas13bt3 in complex with crRNA and 30 nt target RNA |
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Components |
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-Supramolecule #1: Ternary complex of Cas13bt3 in complex with crRNA and 30 nt target RNA
Supramolecule | Name: Ternary complex of Cas13bt3 in complex with crRNA and 30 nt target RNA type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Planctomycetota bacterium (bacteria) |
Molecular weight | Theoretical: 123.7 KDa |
-Supramolecule #2: Integrase
Supramolecule | Name: Integrase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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-Supramolecule #3: crRNA-linker-target hairpin RNA
Supramolecule | Name: crRNA-linker-target hairpin RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
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-Macromolecule #1: Integrase
Macromolecule | Name: Integrase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Planctomycetota bacterium (bacteria) |
Molecular weight | Theoretical: 90.38468 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MAQVSKQTSK KRELSIDEYQ GARKWCFTIA FNKALVNRDK NDGLFVESLL RHEKYSKHDW YDEDTRALIK CSTQAANAKA EALANYFSA YRHSPGCLTF TAEDELRTIM ERAYERAIFE CRRRETEVII EFPSLFEGDR ITTAGVVFFV SFFVERRVLD R LYGAVSGL ...String: MAQVSKQTSK KRELSIDEYQ GARKWCFTIA FNKALVNRDK NDGLFVESLL RHEKYSKHDW YDEDTRALIK CSTQAANAKA EALANYFSA YRHSPGCLTF TAEDELRTIM ERAYERAIFE CRRRETEVII EFPSLFEGDR ITTAGVVFFV SFFVERRVLD R LYGAVSGL KKNEGQYKLT RKALSMYCLK DSRFTKAWDK RVLLFRDILA QLGRIPAEAY EYYHGEQGDK KRANDNEGTN PK RHKDKFI EFALHYLEAQ HSEICFGRRH IVREEAGAGD EHKKHRTKGK VVVDFSKKDE DQSYYISKNN VIVRIDKNAG PRS YRMGLN ELKYLVLLSL QGKGDDAIAK LYRYRQHVEN ILDVVKVTDK DNHVFLPRFV LEQHGIGRKA FKQRIDGRVK HVRG VWEKK KAATNEMTLH EKARDILQYV NENCTRSFNP GEYNRLLVCL VGKDVENFQA GLKRLQLAER IDGRVYSIFA QTSTI NEMH QVVCDQILNR LCRIGDQKLY DYVGLGKKDE IDYKQKVAWF KEHISIRRGF LRKKFWYDSK KGFAKLVEEH LESGGG QRD VGLDKKYYHI DAIGRFEGAN PALYETLARD RLCLMMAQYF LGSVRKELGN KIVWSNDSIE LPVEGSVGNE KSIVFSV SD YGKLYVLDDA EFLGRICEYF MPHEKGKIRY HTVYEKGFRA YNDLQKKCVE AVLAFEEKVV KAKKMSEKEG AHYIDFRE I LAQTMCKEAE KTAVNKVARA FFAHHLKFVI DEFGLFSDVM KKYGIEKEWK FPVK UniProtKB: Uncharacterized protein |
-Macromolecule #2: crRNA-linker-target hairpin RNA
Macromolecule | Name: crRNA-linker-target hairpin RNA / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 33.005457 KDa |
Sequence | String: GGAAGAAGAG UUUAUUCAGA UAGAUUUGUC CUUCUUCGGA CAAAUCUAUC UGAAUAAACU CUUCUUCGCU GGAGCAGCCC CCGAUUUGU GGGGUGAUUA CAGC |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.8 mg/mL |
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Buffer | pH: 7.5 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 49.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: OTHER |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.6 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |