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Yorodumi- EMDB-34428: Cryo-EM structure of the TnpB-omegaRNA-target DNA ternary complex -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-34428 | |||||||||
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Title | Cryo-EM structure of the TnpB-omegaRNA-target DNA ternary complex | |||||||||
Map data | ||||||||||
Sample |
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Keywords | CRISPR-Cas / Transposon-associated protein / RNA BINDING PROTEIN | |||||||||
Function / homology | Function and homology information transposition / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / DNA recombination / endonuclease activity / DNA binding / RNA binding / metal ion binding Similarity search - Function | |||||||||
Biological species | Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
Authors | Nakagawa R / Hirano H / Omura S / Nureki O | |||||||||
Funding support | Japan, 1 items
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Citation | Journal: Nature / Year: 2023 Title: Cryo-EM structure of the transposon-associated TnpB enzyme. Authors: Ryoya Nakagawa / Hisato Hirano / Satoshi N Omura / Suchita Nety / Soumya Kannan / Han Altae-Tran / Xiao Yao / Yuriko Sakaguchi / Takayuki Ohira / Wen Y Wu / Hiroshi Nakayama / Yutaro Shuto / ...Authors: Ryoya Nakagawa / Hisato Hirano / Satoshi N Omura / Suchita Nety / Soumya Kannan / Han Altae-Tran / Xiao Yao / Yuriko Sakaguchi / Takayuki Ohira / Wen Y Wu / Hiroshi Nakayama / Yutaro Shuto / Tatsuki Tanaka / Fumiya K Sano / Tsukasa Kusakizako / Yoshiaki Kise / Yuzuru Itoh / Naoshi Dohmae / John van der Oost / Tsutomu Suzuki / Feng Zhang / Osamu Nureki / Abstract: The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins. Recent studies have identified TnpB proteins as miniature ...The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_34428.map.gz | 2.4 MB | EMDB map data format | |
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Header (meta data) | emd-34428-v30.xml emd-34428.xml | 18.2 KB 18.2 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_34428_fsc.xml | 6.6 KB | Display | FSC data file |
Images | emd_34428.png | 48 KB | ||
Masks | emd_34428_msk_1.map | 2.6 MB | Mask map | |
Filedesc metadata | emd-34428.cif.gz | 6.4 KB | ||
Others | emd_34428_half_map_1.map.gz emd_34428_half_map_2.map.gz | 2.4 MB 2.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-34428 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-34428 | HTTPS FTP |
-Validation report
Summary document | emd_34428_validation.pdf.gz | 831.6 KB | Display | EMDB validaton report |
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Full document | emd_34428_full_validation.pdf.gz | 831.1 KB | Display | |
Data in XML | emd_34428_validation.xml.gz | 11 KB | Display | |
Data in CIF | emd_34428_validation.cif.gz | 14.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-34428 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-34428 | HTTPS FTP |
-Related structure data
Related structure data | 8h1jMC 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_34428.map.gz / Format: CCP4 / Size: 2.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 1.328 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_34428_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_34428_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_34428_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : TnpB-omegaRNA-target DNA ternary complex
Entire | Name: TnpB-omegaRNA-target DNA ternary complex |
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Components |
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-Supramolecule #1: TnpB-omegaRNA-target DNA ternary complex
Supramolecule | Name: TnpB-omegaRNA-target DNA ternary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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Source (natural) | Organism: Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant) |
-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 = ATCC 13939 = DSM 20539 (radioresistant) Strain: R1 |
Molecular weight | Theoretical: 46.628422 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: GSMIRNKAFV VRLYPNAAQT ELINRTLGSA RFVYNHFLAR RIAAYKESGK GLTYGQTSSE LTLLKQAEET SWLSEVDKFA LQNSLKNLE TAYKNFFRTV KQSGKKVGFP RFRKKRTGES YRTQFTNNNI QIGEGRLKLP KLGWVKTKGQ QDIQGKILNV T VRRIHEGH ...String: GSMIRNKAFV VRLYPNAAQT ELINRTLGSA RFVYNHFLAR RIAAYKESGK GLTYGQTSSE LTLLKQAEET SWLSEVDKFA LQNSLKNLE TAYKNFFRTV KQSGKKVGFP RFRKKRTGES YRTQFTNNNI QIGEGRLKLP KLGWVKTKGQ QDIQGKILNV T VRRIHEGH YEASVLCEVE IPYLPAAPKF AAGVDVGIKD FAIVTDGVRF KHEQNPKYYR STLKRLRKAQ QTLSRRKKGS AR YGKAKTK LARIHKRIVN KRQDFLHKLT TSLVREYEII GTEHLKPDNM RKNRRLALSI SDAGWGEFIR QLEYKAAWYG RLV SKVSPY FPSSQLCHDC GFKNPEVKNL AVRTWTCPNC GETHDRDENA ALNIRREALV AAGISDTLNA HGGYVRPASA GNGL RSENH ATLVV UniProtKB: RNA-guided DNA endonuclease TnpB |
-Macromolecule #2: Target strand
Macromolecule | Name: Target strand / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant) |
Molecular weight | Theoretical: 10.854021 KDa |
Sequence | String: (DA)(DT)(DT)(DT)(DG)(DA)(DA)(DT)(DG)(DG) (DG)(DC)(DG)(DC)(DC)(DA)(DA)(DG)(DG)(DG) (DA)(DC)(DT)(DC)(DA)(DT)(DC)(DA)(DA) (DT)(DA)(DG)(DA)(DA)(DA) |
-Macromolecule #4: Non-target strand
Macromolecule | Name: Non-target strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant) |
Molecular weight | Theoretical: 10.679866 KDa |
Sequence | String: (DT)(DT)(DT)(DC)(DT)(DA)(DT)(DT)(DG)(DA) (DT)(DG)(DA)(DG)(DT)(DC)(DC)(DC)(DT)(DT) (DG)(DG)(DC)(DG)(DC)(DC)(DC)(DA)(DT) (DT)(DC)(DA)(DA)(DA)(DT) |
-Macromolecule #3: omegaRNA (130-MER)
Macromolecule | Name: omegaRNA (130-MER) / type: rna / ID: 3 / Number of copies: 1 |
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Source (natural) | Organism: Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant) |
Molecular weight | Theoretical: 79.781219 KDa |
Sequence | String: GAUUCAAGAA UCCCGAAGUG AAGAAUCUUG CCGUCCGUAC AUGGACUUGC CCGAACUGUG GGGAAACCCA UGACCGAGAC GAGAACGCU GCGCUGAACA UUCGGCGUGA AGCGUUGGUG GCUGCGGGAA UCUCAGACAC CUUAAACGCU CAUGGAGGCU A UGUCAGAC ...String: GAUUCAAGAA UCCCGAAGUG AAGAAUCUUG CCGUCCGUAC AUGGACUUGC CCGAACUGUG GGGAAACCCA UGACCGAGAC GAGAACGCU GCGCUGAACA UUCGGCGUGA AGCGUUGGUG GCUGCGGGAA UCUCAGACAC CUUAAACGCU CAUGGAGGCU A UGUCAGAC CUGCUUCGGC GGGCAAUGGU CUGCGAAGUG AGAAUCACGC GACUUUAGUC GUGUGAGGUU CAAGAGUCCC UU GGCGCCC GENBANK: GENBANK: AE000513.1 |
-Macromolecule #5: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 5 / 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
Buffer | pH: 8 |
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Grid | Model: C-flat-1.2/1.3 / Material: GOLD / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 180 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 297 K |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 47.0 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: 1.6 µm / Nominal defocus min: 0.8 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |