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- PDB-8h1j: Cryo-EM structure of the TnpB-omegaRNA-target DNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 8h1j
TitleCryo-EM structure of the TnpB-omegaRNA-target DNA ternary complex
Components
  • Non-target strand
  • RNA-guided DNA endonuclease TnpB
  • Target strand
  • omegaRNA (130-MER)
KeywordsRNA BINDING PROTEIN / CRISPR-Cas / Transposon-associated protein
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
: / DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / RNA-guided DNA endonuclease TnpB
Similarity search - Component
Biological speciesDeinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsNakagawa, R. / Hirano, H. / Omura, S. / Nureki, O.
Funding support Japan, 1items
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, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 12, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 19, 2023Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Apr 26, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-guided DNA endonuclease TnpB
C: Target strand
B: omegaRNA (130-MER)
D: Non-target strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)148,0095
Polymers147,9444
Non-polymers651
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein RNA-guided DNA endonuclease TnpB


Mass: 46628.422 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant)
Strain: R1 / Gene: tnpB / Production host: Escherichia coli (E. coli)
References: UniProt: Q7DF80, Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters
#2: DNA chain Target strand


Mass: 10854.021 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant)
#3: RNA chain omegaRNA (130-MER)


Mass: 79781.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant)
Production host: Escherichia coli (E. coli) / References: GenBank: 11612676
#4: DNA chain Non-target strand


Mass: 10679.866 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant)
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: TnpB-omegaRNA-target DNA ternary complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 (radioresistant)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: C-flat-1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 47 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 131006 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0056167
ELECTRON MICROSCOPYf_angle_d0.4958952
ELECTRON MICROSCOPYf_dihedral_angle_d16.5421935
ELECTRON MICROSCOPYf_chiral_restr0.0341071
ELECTRON MICROSCOPYf_plane_restr0.004648

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