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- PDB-8v32: TnsD-TnsC-DNA complex -

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Basic information

Entry
Database: PDB / ID: 8v32
TitleTnsD-TnsC-DNA complex
Components
  • (DNA (41-MER)) x 2
  • TnsC
  • TnsD
KeywordsDNA BINDING PROTEIN/DNA / TnsC / TnsD / Tn7-like transposon / type I-B2 CAST / DNA BINDING PROTEIN-DNA complex
Function / homology: / AAA domain / ATP hydrolysis activity / P-loop containing nucleoside triphosphate hydrolase / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / ORC1/DEAH AAA+ ATPase domain-containing protein
Function and homology information
Biological speciesPeltigera membranacea (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsChang, L. / Wang, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Cell / Year: 2024
Title: Structure of TnsABCD transpososome reveals mechanisms of targeted DNA transposition.
Authors: Shukun Wang / Romana Siddique / Mark C Hall / Phoebe A Rice / Leifu Chang /
Abstract: Tn7-like transposons are characterized by their ability to insert specifically into host chromosomes. Recognition of the attachment (att) site by TnsD recruits the TnsABC proteins to form the ...Tn7-like transposons are characterized by their ability to insert specifically into host chromosomes. Recognition of the attachment (att) site by TnsD recruits the TnsABC proteins to form the transpososome and facilitate transposition. Although this pathway is well established, atomic-level structural insights of this process remain largely elusive. Here, we present the cryo-electron microscopy (cryo-EM) structures of the TnsC-TnsD-att DNA complex and the TnsABCD transpososome from the Tn7-like transposon in Peltigera membranacea cyanobiont 210A, a type I-B CRISPR-associated transposon. Our structures reveal a striking bending of the att DNA, featured by the intercalation of an arginine side chain of TnsD into a CC/GG dinucleotide step. The TnsABCD transpososome structure reveals TnsA-TnsB interactions and demonstrates that TnsC not only recruits TnsAB but also directly participates in the transpososome assembly. These findings provide mechanistic insights into targeted DNA insertion by Tn7-like transposons, with implications for improving the precision and efficiency of their genome-editing applications.
History
DepositionNov 26, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 16, 2024Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
Q: TnsC
R: TnsC
S: TnsC
T: TnsC
U: TnsC
V: TnsC
W: TnsC
H: DNA (41-MER)
I: DNA (41-MER)
J: TnsD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)410,66226
Polymers406,81110
Non-polymers3,85116
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 8 molecules QRSTUVWJ

#1: Protein
TnsC


Mass: 43521.934 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Peltigera membranacea (fungus) / Gene: CDG76_08990 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IFM2
#4: Protein TnsD


Mass: 52821.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Peltigera membranacea (fungus) / Production host: Escherichia coli (E. coli)

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DNA chain , 2 types, 2 molecules HI

#2: DNA chain DNA (41-MER)


Mass: 24727.807 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Peltigera membranacea (fungus)
#3: DNA chain DNA (41-MER)


Mass: 24608.814 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Peltigera membranacea (fungus)

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Non-polymers , 4 types, 18 molecules

#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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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: Ternary complex of TnsC, TnsD, and DNA in type I-B2 CRISPR-associated transposon system
Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Source (natural)Organism: Peltigera membranacea (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 154947 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00325272
ELECTRON MICROSCOPYf_angle_d0.55634616
ELECTRON MICROSCOPYf_dihedral_angle_d16.0233957
ELECTRON MICROSCOPYf_chiral_restr0.0393879
ELECTRON MICROSCOPYf_plane_restr0.0054117

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