+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-16016 | |||||||||||||||
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Title | ISDra2 TnpB in complex with reRNA | |||||||||||||||
Map data | Sharpened map used for model building. Output of phenix.auto_refine v1.20.1-4487 | |||||||||||||||
Sample |
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Function / homology | Function and homology information transposition / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / endonuclease activity / DNA recombination / DNA binding / RNA binding / metal ion binding Similarity search - Function | |||||||||||||||
Biological species | Deinococcus radiodurans (radioresistant) / Deinococcus radiodurans R1 (radioresistant) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.8 Å | |||||||||||||||
Authors | Sasnauskas G / Tamulaitiene G / Carabias A / Siksnys V / Montoya G / Druteika G / Silanskas A / Venclovas C / Karvelis T / Kazlauskas D | |||||||||||||||
Funding support | Denmark, 4 items
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Citation | Journal: Nature / Year: 2023 Title: TnpB structure reveals minimal functional core of Cas12 nuclease family. Authors: Giedrius Sasnauskas / Giedre Tamulaitiene / Gytis Druteika / Arturo Carabias / Arunas Silanskas / Darius Kazlauskas / Česlovas Venclovas / Guillermo Montoya / Tautvydas Karvelis / Virginijus Siksnys / Abstract: The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic cells. Bioinformatic analysis ...The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic cells. Bioinformatic analysis identified TnpB proteins as the likely predecessors of Cas12 nucleases, which along with Cas9 are widely used for targeted genome manipulation. Whereas Cas12 family nucleases are well characterized both biochemically and structurally, the molecular mechanism of TnpB remains unknown. Here we present the cryogenic-electron microscopy structures of the Deinococcus radiodurans TnpB-reRNA (right-end transposon element-derived RNA) complex in DNA-bound and -free forms. The structures reveal the basic architecture of TnpB nuclease and the molecular mechanism for DNA target recognition and cleavage that is supported by biochemical experiments. Collectively, these results demonstrate that TnpB represents the minimal structural and functional core of the Cas12 protein family and provide a framework for developing TnpB-based genome editing tools. | |||||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_16016.map.gz | 67.6 MB | EMDB map data format | |
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Header (meta data) | emd-16016-v30.xml emd-16016.xml | 21.7 KB 21.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_16016_fsc.xml | 12.3 KB | Display | FSC data file |
Images | emd_16016.png | 161.1 KB | ||
Masks | emd_16016_msk_1.map | 75.1 MB | Mask map | |
Others | emd_16016_additional_1.map.gz emd_16016_half_map_1.map.gz emd_16016_half_map_2.map.gz | 37.4 MB 69.6 MB 69.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-16016 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-16016 | HTTPS FTP |
-Validation report
Summary document | emd_16016_validation.pdf.gz | 765.5 KB | Display | EMDB validaton report |
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Full document | emd_16016_full_validation.pdf.gz | 765.1 KB | Display | |
Data in XML | emd_16016_validation.xml.gz | 17.1 KB | Display | |
Data in CIF | emd_16016_validation.cif.gz | 22.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-16016 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-16016 | HTTPS FTP |
-Related structure data
Related structure data | 8bf8MC 8ex9C 8exaC 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_16016.map.gz / Format: CCP4 / Size: 75.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Sharpened map used for model building. Output of phenix.auto_refine v1.20.1-4487 | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.832 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_16016_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: Unsharpened map, output of CryoSPARC v3.4.0 local refinement job
File | emd_16016_additional_1.map | ||||||||||||
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Annotation | Unsharpened map, output of CryoSPARC v3.4.0 local refinement job | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map B, output of CryoSPARC v3.4.0 local refinement job
File | emd_16016_half_map_1.map | ||||||||||||
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Annotation | Half map B, output of CryoSPARC v3.4.0 local refinement job | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map A, output of CryoSPARC v3.4.0 local refinement job
File | emd_16016_half_map_2.map | ||||||||||||
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Annotation | Half map A, output of CryoSPARC v3.4.0 local refinement job | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : ISDra2 TnpB binary complex
Entire | Name: ISDra2 TnpB binary complex |
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Components |
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-Supramolecule #1: ISDra2 TnpB binary complex
Supramolecule | Name: ISDra2 TnpB binary complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all / Details: ISDra2 TnpB in complex with reRNA |
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Source (natural) | Organism: Deinococcus radiodurans (radioresistant) |
Molecular weight | Theoretical: 100 KDa |
-Macromolecule #1: RNA-guided DNA endonuclease TnpB
Macromolecule | Name: RNA-guided DNA endonuclease TnpB / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO EC number: Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters |
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Source (natural) | Organism: Deinococcus radiodurans R1 (radioresistant) Strain: ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422 |
Molecular weight | Theoretical: 46.484289 KDa |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) |
Sequence | String: MIRNKAFVVR LYPNAAQTEL INRTLGSARF VYNHFLARRI AAYKESGKGL TYGQTSSELT LLKQAEETSW LSEVDKFALQ NSLKNLETA YKNFFRTVKQ SGKKVGFPRF RKKRTGESYR TQFTNNNIQI GEGRLKLPKL GWVKTKGQQD IQGKILNVTV R RIHEGHYE ...String: MIRNKAFVVR LYPNAAQTEL INRTLGSARF VYNHFLARRI AAYKESGKGL TYGQTSSELT LLKQAEETSW LSEVDKFALQ NSLKNLETA YKNFFRTVKQ SGKKVGFPRF RKKRTGESYR TQFTNNNIQI GEGRLKLPKL GWVKTKGQQD IQGKILNVTV R RIHEGHYE ASVLCEVEIP YLPAAPKFAA GVDVGIKDFA IVTDGVRFKH EQNPKYYRST LKRLRKAQQT LSRRKKGSAR YG KAKTKLA RIHKRIVNKR QDFLHKLTTS LVREYEIIGT EHLKPDNMRK NRRLALSISD AGWGEFIRQL EYKAAWYGRL VSK VSPYFP SSQLCHDCGF KNPEVKNLAV RTWTCPNCGE THDRDENAAL NIRREALVAA GISDTLNAHG GYVRPASAGN GLRS ENHAT LVV |
-Macromolecule #2: Deinococcus radiodurans R1 chromosome 1
Macromolecule | Name: Deinococcus radiodurans R1 chromosome 1 / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: Deinococcus radiodurans R1 (radioresistant) |
Molecular weight | Theoretical: 48.408633 KDa |
Sequence | String: CAUUCGGCGU GAAGCGUUGG UGGCUGCGGG AAUCUCAGAC ACCUUAAACG CUCAUGGAGG CUAUGUCAGA CCUGCUUCGG CGGGCAAUG GUCUGCGAAG UGAGAAUCAC GCGACUUUAG UCGUGUGAGG UUCAAGAGUC CCUUGGCGCC C |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 8 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 1 / Number real images: 6688 / Average electron dose: 38.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm |
Sample stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |