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- EMDB-34428: Cryo-EM structure of the TnpB-omegaRNA-target DNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-34428
TitleCryo-EM structure of the TnpB-omegaRNA-target DNA ternary complex
Map data
Sample
  • Complex: TnpB-omegaRNA-target DNA ternary complex
    • Protein or peptide: RNA-guided DNA endonuclease TnpB
    • DNA: Target strand
    • RNA: omegaRNA (130-MER)
    • DNA: Non-target strand
  • Ligand: ZINC ION
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
Probable transposase, IS891/IS1136/IS1341 / Transposase, putative, helix-turn-helix domain / Probable transposase / Helix-turn-helix domain / Transposase IS605, OrfB, C-terminal / Putative transposase DNA-binding domain / ARFGAP/RecO-like zinc finger
Similarity search - Domain/homology
RNA-guided DNA endonuclease TnpB
Similarity search - Component
Biological speciesDeinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsNakagawa R / Hirano H / Omura S / Nureki O
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP22ama121012 Japan
CitationJournal: 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
DepositionOct 3, 2022-
Header (metadata) releaseApr 12, 2023-
Map releaseApr 12, 2023-
UpdateApr 26, 2023-
Current statusApr 26, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34428.png

Downloads & links

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Map

FileDownload / File: emd_34428.map.gz / Format: CCP4 / Size: 2.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.328 Å
Density
Contour LevelBy AUTHOR: 0.323
Minimum - Maximum-1.461808 - 2.2795746
Average (Standard dev.)0.004098875 (±0.13364525)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin565656
Dimensions888888
Spacing888888
CellA=B=C: 116.864 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_34428_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_34428_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34428_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : TnpB-omegaRNA-target DNA ternary complex

EntireName: TnpB-omegaRNA-target DNA ternary complex
Components
  • Complex: TnpB-omegaRNA-target DNA ternary complex
    • Protein or peptide: RNA-guided DNA endonuclease TnpB
    • DNA: Target strand
    • RNA: omegaRNA (130-MER)
    • DNA: Non-target strand
  • Ligand: ZINC ION

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Supramolecule #1: TnpB-omegaRNA-target DNA ternary complex

SupramoleculeName: TnpB-omegaRNA-target DNA ternary complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant)

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Macromolecule #1: RNA-guided DNA endonuclease TnpB

MacromoleculeName: 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
Source (natural)Organism: Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant)
Strain: R1
Molecular weightTheoretical: 46.628422 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #2: Target strand

MacromoleculeName: Target strand / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant)
Molecular weightTheoretical: 10.854021 KDa
SequenceString:
(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)

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Macromolecule #4: Non-target strand

MacromoleculeName: Non-target strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant)
Molecular weightTheoretical: 10.679866 KDa
SequenceString:
(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)

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Macromolecule #3: omegaRNA (130-MER)

MacromoleculeName: omegaRNA (130-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant)
Molecular weightTheoretical: 79.781219 KDa
SequenceString: 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

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
GridModel: C-flat-1.2/1.3 / Material: GOLD / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 180 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 297 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 47.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 131006
FSC plot (resolution estimation)

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