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- EMDB-35323: Cryo-EM structure of the ISFba1 TnpB-reRNA-dsDNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-35323
TitleCryo-EM structure of the ISFba1 TnpB-reRNA-dsDNA complex
Map data
Sample
  • Complex: The complex of protein with gRNA and target DNA
    • Protein or peptide: Transposase
    • RNA: RNA (207-MER)
    • DNA: DNA (5'-D(P*AP*CP*AP*TP*GP*GP*AP*CP*CP*AP*TP*CP*AP*GP*CP*TP*CP*CP*TP*AP*AP*TP*GP*G)-3')
    • DNA: DNA (5'-D(P*CP*CP*AP*TP*TP*AP*GP*GP*AP*GP*CP*TP*GP*AP*TP*G)-3')
  • Ligand: ZINC ION
KeywordsRNA / Complex / RNA BINDING PROTEIN/RNA / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology:
Function and homology information
Biological speciesFirmicutes bacterium AM43-11BH (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsYin M / Zhou F / Zhu Y / Huang Z
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31825008 China
CitationJournal: Cell Res / Year: 2024
Title: Discovery and structural mechanism of DNA endonucleases guided by RAGATH-18-derived RNAs.
Authors: Kuan Ren / Fengxia Zhou / Fan Zhang / Mingyu Yin / Yuwei Zhu / Shouyu Wang / Yan Chen / Tengjin Huang / Zixuan Wu / Jiale He / Anqi Zhang / Changyou Guo / Zhiwei Huang /
Abstract: CRISPR-Cas systems and IS200/IS605 transposon-associated TnpBs have been utilized for the development of genome editing technologies. Using bioinformatics analysis and biochemical experiments, here ...CRISPR-Cas systems and IS200/IS605 transposon-associated TnpBs have been utilized for the development of genome editing technologies. Using bioinformatics analysis and biochemical experiments, here we present a new family of RNA-guided DNA endonucleases. Our bioinformatics analysis initially identifies the stable co-occurrence of conserved RAGATH-18-derived RNAs (reRNAs) and their upstream IS607 TnpBs with an average length of 390 amino acids. IS607 TnpBs form programmable DNases through interaction with reRNAs. We discover the robust dsDNA interference activity of IS607 TnpB systems in bacteria and human cells. Further characterization of the Firmicutes bacteria IS607 TnpB system (ISFba1 TnpB) reveals that its dsDNA cleavage activity is remarkably sensitive to single mismatches between the guide and target sequences in human cells. Our findings demonstrate that a length of 20 nt in the guide sequence of reRNA achieves the highest DNA cleavage activity for ISFba1 TnpB. A cryo-EM structure of the ISFba1 TnpB effector protein bound by its cognate RAGATH-18 motif-containing reRNA and a dsDNA target reveals the mechanisms underlying reRNA recognition by ISFba1 TnpB, reRNA-guided dsDNA targeting, and the sensitivity of the ISFba1 TnpB system to base mismatches between the guide and target DNA. Collectively, this study identifies the IS607 TnpB family of compact and specific RNA-guided DNases with great potential for application in gene editing.
History
DepositionFeb 9, 2023-
Header (metadata) releaseApr 17, 2024-
Map releaseApr 17, 2024-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_35323.png

Downloads & links

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Map

FileDownload / File: emd_35323.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 256 pix.
= 220.16 Å		0.86 Å/pix.
x 256 pix.
= 220.16 Å		0.86 Å/pix.
x 256 pix.
= 220.16 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.3040354 - 0.43897957
Average (Standard dev.)0.00007970471 (±0.011570509)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 220.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_35323_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35323_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : The complex of protein with gRNA and target DNA

EntireName: The complex of protein with gRNA and target DNA
Components
  • Complex: The complex of protein with gRNA and target DNA
    • Protein or peptide: Transposase
    • RNA: RNA (207-MER)
    • DNA: DNA (5'-D(P*AP*CP*AP*TP*GP*GP*AP*CP*CP*AP*TP*CP*AP*GP*CP*TP*CP*CP*TP*AP*AP*TP*GP*G)-3')
    • DNA: DNA (5'-D(P*CP*CP*AP*TP*TP*AP*GP*GP*AP*GP*CP*TP*GP*AP*TP*G)-3')
  • Ligand: ZINC ION

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Supramolecule #1: The complex of protein with gRNA and target DNA

SupramoleculeName: The complex of protein with gRNA and target DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Firmicutes bacterium AM43-11BH (bacteria)

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Macromolecule #1: Transposase

MacromoleculeName: Transposase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Firmicutes bacterium AM43-11BH (bacteria)
Molecular weightTheoretical: 44.549645 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: LLKSFKTEIN PSEEQKVKIH KTIGTCRFIY NFYLAHNKEL YDKGEKFMSG KSFSVWLNNE YLPQNPDKLW IKEVSSKSVK HSIENGCIA FTRFFKHQSA FPNLKKKGKS DVKMYFVKNN PKDCRCERHR INIPSLGWVR IKEKGYIPTT KDGYVIKSGT V SMKADRYY ...String:
LLKSFKTEIN PSEEQKVKIH KTIGTCRFIY NFYLAHNKEL YDKGEKFMSG KSFSVWLNNE YLPQNPDKLW IKEVSSKSVK HSIENGCIA FTRFFKHQSA FPNLKKKGKS DVKMYFVKNN PKDCRCERHR INIPSLGWVR IKEKGYIPTT KDGYVIKSGT V SMKADRYY VSVLVEISNN KIANNSNAGI GIDLGLKDFA IVSNGKTYKN INKSARLKKH EKQLIREQRS LSRKYENLKK GE STQKANI QKQRLKVQKL HHRMDNIRTD YINKTIAEIV KTKPSYITIE DLNVKGMMKN RHLSKAVASQ KFYEFRTKLQ AKC NENGIE LRVVDRWYPS SKTCHCCGAV KKDLKLSDRI FKCSCGYVED RAFNAALNLR DAITYEVA

UniProtKB: UNIPROTKB: A0A417B524

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Macromolecule #2: RNA (207-MER)

MacromoleculeName: RNA (207-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Firmicutes bacterium AM43-11BH (bacteria)
Molecular weightTheoretical: 66.616352 KDa
SequenceString: CUAAAACGCA AACGUAAGUA UGUACCGAAG GCUAUUUUCG GGAAUUUACG ACUAUGGAGU GUACAAGAAC UUGUGAGUAG AUAUGAUUU CGGUCAAUCC AAAGCAUACA CGAUGAAAUA GUAAGUAAUG UUCGUGAGAA CUUACAUUAU CUCGAUGUGA G CAUAUUUA ...String:
CUAAAACGCA AACGUAAGUA UGUACCGAAG GCUAUUUUCG GGAAUUUACG ACUAUGGAGU GUACAAGAAC UUGUGAGUAG AUAUGAUUU CGGUCAAUCC AAAGCAUACA CGAUGAAAUA GUAAGUAAUG UUCGUGAGAA CUUACAUUAU CUCGAUGUGA G CAUAUUUA AUCACAUUUU GAGUGGCAGC UGAUGGUCCA UGUCUGUUA

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Macromolecule #3: DNA (5'-D(P*AP*CP*AP*TP*GP*GP*AP*CP*CP*AP*TP*CP*AP*GP*CP*TP*CP*CP...

MacromoleculeName: DNA (5'-D(P*AP*CP*AP*TP*GP*GP*AP*CP*CP*AP*TP*CP*AP*GP*CP*TP*CP*CP*TP*AP*AP*TP*GP*G)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Firmicutes bacterium AM43-11BH (bacteria)
Molecular weightTheoretical: 7.338756 KDa
SequenceString:
(DA)(DC)(DA)(DT)(DG)(DG)(DA)(DC)(DC)(DA) (DT)(DC)(DA)(DG)(DC)(DT)(DC)(DC)(DT)(DA) (DA)(DT)(DG)(DG)

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Macromolecule #4: DNA (5'-D(P*CP*CP*AP*TP*TP*AP*GP*GP*AP*GP*CP*TP*GP*AP*TP*G)-3')

MacromoleculeName: DNA (5'-D(P*CP*CP*AP*TP*TP*AP*GP*GP*AP*GP*CP*TP*GP*AP*TP*G)-3')
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Firmicutes bacterium AM43-11BH (bacteria)
Molecular weightTheoretical: 4.938216 KDa
SequenceString:
(DC)(DC)(DA)(DT)(DT)(DA)(DG)(DG)(DA)(DG) (DC)(DT)(DG)(DA)(DT)(DG)

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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: 7.2
VitrificationCryogen name: NITROGEN

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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: 1.5 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 369379

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