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- EMDB-28655: ISDra2 TnpB in complex with reRNA and cognate DNA, conformation 2... -

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Basic information

Entry
Database: EMDB / ID: EMD-28655
TitleISDra2 TnpB in complex with reRNA and cognate DNA, conformation 2 (RuvC domain unresolved)
Map dataSharpened map, output of phenix.auto_sharpen
Sample
  • Complex: ISDra2 TnpB in complex with reRNA and cognate DNA
    • Protein or peptide: RNA-guided DNA endonuclease TnpB
    • RNA: RNA (150-MER)
    • DNA: DNA (43-MER)
    • DNA: DNA (43-MER)
KeywordsTransposon / TnpB / RuvC domain / Cas12 / IS200/IS605 / reRNA / RNA BINDING 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
: / 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
RNA-guided DNA endonuclease TnpB
Similarity search - Component
Biological speciesDeinococcus radiodurans R1 (radioresistant) / synthetic construct (others)
Methodsingle 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, 4 items
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, 2022-
Header (metadata) releaseApr 5, 2023-
Map releaseApr 5, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28655.png

Downloads & links

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Map

FileDownload / File: emd_28655.map.gz / Format: CCP4 / Size: 75.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map, output of phenix.auto_sharpen
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.83 Å/pix.
x 270 pix.
= 224.64 Å		0.83 Å/pix.
x 270 pix.
= 224.64 Å		0.83 Å/pix.
x 270 pix.
= 224.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.5
Minimum - Maximum-47.852609999999999 - 69.926100000000005
Average (Standard dev.)-0.000000000004966 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions270270270
Spacing270270270
CellA=B=C: 224.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28655_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map. Output of CryoSPARC local refinement job.

Fileemd_28655_additional_1.map
AnnotationUnsharpened map. Output of CryoSPARC local refinement job.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Output of CryoSPARC local refinement job.

Fileemd_28655_half_map_1.map
AnnotationOutput of CryoSPARC local refinement job.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Output of CryoSPARC local refinement job.

Fileemd_28655_half_map_2.map
AnnotationOutput of CryoSPARC local refinement job.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ISDra2 TnpB in complex with reRNA and cognate DNA

EntireName: ISDra2 TnpB in complex with reRNA and cognate DNA
Components
  • Complex: ISDra2 TnpB in complex with reRNA and cognate DNA
    • Protein or peptide: RNA-guided DNA endonuclease TnpB
    • RNA: RNA (150-MER)
    • DNA: DNA (43-MER)
    • DNA: DNA (43-MER)

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Supramolecule #1: ISDra2 TnpB in complex with reRNA and cognate DNA

SupramoleculeName: ISDra2 TnpB in complex with reRNA and cognate DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Deinococcus radiodurans R1 (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 (radioresistant)
Strain: ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422
Molecular weightTheoretical: 46.484289 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MIRNKAFVVR LYPNAAQTEL INRTLGSARF VYNHFLARRI AAYKESGKGL TYGQTSSELT LLKQAEETSW LSEVDKFALQ NSLKNLETA YKNFFRTVKQ SGKKVGFPRF RKKRTGESYR TQFTNNNIQI GEGRLKLPKL GWVKTKGQQD IQGKILNVTV R RIHEGHYE ...String:
MIRNKAFVVR LYPNAAQTEL INRTLGSARF VYNHFLARRI AAYKESGKGL TYGQTSSELT LLKQAEETSW LSEVDKFALQ NSLKNLETA YKNFFRTVKQ SGKKVGFPRF RKKRTGESYR TQFTNNNIQI GEGRLKLPKL GWVKTKGQQD IQGKILNVTV R RIHEGHYE ASVLCEVEIP YLPAAPKFAA GVDVGIKDFA IVTDGVRFKH EQNPKYYRST LKRLRKAQQT LSRRKKGSAR YG KAKTKLA RIHKRIVNKR QDFLHKLTTS LVREYEIIGT EHLKPDNMRK NRRLALSISD AGWGEFIRQL EYKAAWYGRL VSK VSPYFP SSQLCHDCGF KNPEVKNLAV RTWTCPNCGE THDRDENAAL NIRREALVAA GISDTLNAHG GYVRPASAGN GLRS ENHAT LVV

UniProtKB: RNA-guided DNA endonuclease TnpB

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Macromolecule #2: RNA (150-MER)

MacromoleculeName: RNA (150-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Deinococcus radiodurans R1 (radioresistant)
Molecular weightTheoretical: 48.408633 KDa
SequenceString:
CAUUCGGCGU GAAGCGUUGG UGGCUGCGGG AAUCUCAGAC ACCUUAAACG CUCAUGGAGG CUAUGUCAGA CCUGCUUCGG CGGGCAAUG GUCUGCGAAG UGAGAAUCAC GCGACUUUAG UCGUGUGAGG UUCAAGAGUC CCUUGGCGCC C

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Macromolecule #3: DNA (43-MER)

MacromoleculeName: DNA (43-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.310532 KDa
SequenceString:
(DG)(DT)(DC)(DA)(DT)(DG)(DG)(DG)(DC)(DG) (DC)(DC)(DA)(DA)(DG)(DG)(DG)(DA)(DC)(DT) (DC)(DA)(DT)(DC)(DA)(DA)(DG)(DC)(DG) (DA)(DC)(DG)(DG)(DT)(DT)(DG)(DA)(DT)(DC) (DT) (DC)(DA)(DG)

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Macromolecule #4: DNA (43-MER)

MacromoleculeName: DNA (43-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.221472 KDa
SequenceString:
(DG)(DT)(DC)(DA)(DT)(DG)(DG)(DG)(DC)(DG) (DC)(DC)(DA)(DA)(DG)(DG)(DG)(DA)(DC)(DT) (DC)(DA)(DT)(DC)(DA)(DA)(DC)(DC)(DG) (DT)(DC)(DG)(DC)(DT)(DT)(DG)(DA)(DT)(DC) (DT) (DC)(DA)(DG)

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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

Concentration0.7 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
125.0 mMNaClsodium chloride
10.0 mMMgCl2magnesium chloride
10.0 mMC4H11NO3Tris
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Number real images: 10580 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 96000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: Ab-initio model generated in cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.4.0) / Software - details: Local refinement job / Number images used: 541638
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.4.0)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.4.0)
FSC plot (resolution estimation)

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