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- PDB-8bf8: ISDra2 TnpB in complex with reRNA -

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Basic information

Entry
Database: PDB / ID: 8bf8
TitleISDra2 TnpB in complex with reRNA
Components
  • Deinococcus radiodurans R1 chromosome 1
  • RNA-guided DNA endonuclease TnpB
KeywordsRNA BINDING PROTEIN / Transposon / TnpB / 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
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 / RNA (> 10) / RNA (> 100) / RNA-guided DNA endonuclease TnpB
Similarity search - Component
Biological speciesDeinococcus radiodurans R1 (radioresistant)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsSasnauskas, G. / Tamulaitiene, G. / Carabias, A. / Siksnys, V. / Montoya, G. / Druteika, G. / Silanskas, A. / Venclovas, C. / Karvelis, T. / Kazlauskas, D.
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 24, 2022Deposition site: PDBE / Processing site: PDBE
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 ..._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

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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: Deinococcus radiodurans R1 chromosome 1


Theoretical massNumber of molelcules
Total (without water)94,8932
Polymers94,8932
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, SEC-MALS and mass photometry experiments
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4440 Å2
ΔGint-31 kcal/mol
Surface area31250 Å2
MethodPISA

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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 Deinococcus radiodurans R1 chromosome 1


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 / References: GenBank: 11612676

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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 binary complex / Type: COMPLEX / Details: ISDra2 TnpB in complex with reRNA / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.10 MDa / Experimental value: YES
Source (natural)Organism: Deinococcus radiodurans (radioresistant)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1125 mMsodium chlorideNaClSodium chloride1
210 mMTrisC4H11NO31
310 mMmagnesium chlorideMgCl21
SpecimenEmbedding 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 FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 38 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6688
Image scansWidth: 4000 / Height: 4000

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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
2EPUimage acquisition
4cryoSPARC3.4.0CTF correctionPatch CTF
13cryoSPARC3.4.03D reconstructionLocal refinement in cryoSPARC
CTF correctionType: NONE
Particle selectionNum. of particles selected: 4420084
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 985959 / Num. of class averages: 1 / Symmetry type: POINT
RefinementCross valid method: NONE

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