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- PDB-8ex9: ISDra2 TnpB in complex with reRNA and cognate DNA, conformation 2... -

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Basic information

Entry
Database: PDB / ID: 8ex9
TitleISDra2 TnpB in complex with reRNA and cognate DNA, conformation 2 (RuvC domain unresolved)
Components
  • (DNA (43-MER)) x 2
  • RNA (150-MER)
  • RNA-guided DNA endonuclease TnpB
KeywordsRNA BINDING PROTEIN / Transposon / TnpB / RuvC domain / Cas12 / IS200/IS605 / reRNA
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
: / Transposase, putative, helix-turn-helix domain / Helix-turn-helix domain / Probable transposase, IS891/IS1136/IS1341 / Probable transposase / : / Transposase IS605, OrfB, C-terminal / Cas12f1-like, TNB 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 (radioresistant)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsSasnauskas, G. / Tamulaitiene, G. / Carabias, A. / Karvelis, T. / Druteika, G. / Silanskas, A. / Montoya, G. / Venclovas, C. / Kazlauskas, D. / Siksnys, V.
Funding support Denmark, 4items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
Novo Nordisk FoundationNNF0024386 Denmark
Novo Nordisk FoundationNNF17SA0030214 Denmark
Novo Nordisk FoundationNNF18OC0055061 Denmark
CitationJournal: 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
DepositionOct 25, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 19, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Apr 26, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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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
B: RNA (150-MER)
C: DNA (43-MER)
D: DNA (43-MER)


Theoretical massNumber of molelcules
Total (without water)121,4254
Polymers121,4254
Non-polymers00
Water00
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: 46484.289 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Deinococcus radiodurans R1 (radioresistant)
Strain: ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422
Gene: tnpB, orf2, tnpC, DR_0666, DR_0978, DR_1381, DR_1593, DR_1651, DR_1933, DR_2324
Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 / Variant (production host): AI
References: UniProt: Q7DF80, Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters
#2: RNA chain RNA (150-MER)


Mass: 48408.633 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Deinococcus radiodurans R1 (radioresistant)
Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 / Variant (production host): AI
#3: DNA chain DNA (43-MER)


Mass: 13310.532 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (43-MER)


Mass: 13221.472 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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: ISDra2 TnpB in complex with reRNA and cognate DNA / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Deinococcus radiodurans R1 (radioresistant)243230
31synthetic construct (others)32630
Source (recombinant)Organism: Escherichia coli BL21 (bacteria) / Strain: BL21-AI
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1125 mMsodium chlorideNaCl1
210 mMmagnesium chlorideMgCl21
310 mMTrisC4H11NO31
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 96000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 900 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 10580

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3.4.0particle selectionBlob picker
2EPU2.14.0image acquisition
4cryoSPARC3.4.0CTF correction
10cryoSPARC3.4.0initial Euler assignment
11cryoSPARC3.4.0final Euler assignment
13cryoSPARC3.4.03D reconstructionLocal refinement job
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 541638 / Symmetry type: POINT
RefinementCross valid method: NONE

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