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- PDB-8iaz: Cryo-EM structure of the ISFba1 TnpB-reRNA-dsDNA complex -

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Basic information

Entry
Database: PDB / ID: 8iaz
TitleCryo-EM structure of the ISFba1 TnpB-reRNA-dsDNA complex
Components
  • 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 (5'-D(P*CP*CP*AP*TP*TP*AP*GP*GP*AP*GP*CP*TP*GP*AP*TP*G)-3')
  • RNA (207-MER)
  • Transposase
KeywordsRNA BINDING PROTEIN/RNA/DNA / RNA / Complex / RNA BINDING PROTEIN/RNA / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / :
Function and homology information
Biological speciesFirmicutes bacterium AM43-11BH (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsYin, M. / Zhou, F. / Zhu, Y. / Huang, Z.
Funding support China, 1items
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, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Apr 17, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transposase
E: RNA (207-MER)
B: 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')
C: DNA (5'-D(P*CP*CP*AP*TP*TP*AP*GP*GP*AP*GP*CP*TP*GP*AP*TP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)123,5085
Polymers123,4434
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 Transposase /


Mass: 44549.645 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Firmicutes bacterium AM43-11BH (bacteria)
Gene: DW928_16025 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A417B524
#2: RNA chain RNA (207-MER)


Mass: 66616.352 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Firmicutes bacterium AM43-11BH (bacteria)
#3: DNA chain 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')


Mass: 7338.756 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Firmicutes bacterium AM43-11BH (bacteria)
#4: DNA chain DNA (5'-D(P*CP*CP*AP*TP*TP*AP*GP*GP*AP*GP*CP*TP*GP*AP*TP*G)-3')


Mass: 4938.216 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Firmicutes bacterium AM43-11BH (bacteria)
#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: The complex of protein with gRNA and target DNA / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Source (natural)Organism: Firmicutes bacterium AM43-11BH (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: NITROGEN

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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: 1500 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 369379 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047960
ELECTRON MICROSCOPYf_angle_d0.65811666
ELECTRON MICROSCOPYf_dihedral_angle_d22.672753
ELECTRON MICROSCOPYf_chiral_restr0.041428
ELECTRON MICROSCOPYf_plane_restr0.005736

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