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- PDB-9bw1: TnsABCD-DNA transpososome -

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Basic information

Entry
Database: PDB / ID: 9bw1
TitleTnsABCD-DNA transpososome
Components
  • Integrase
  • LE_polyA
  • RE_polyA
  • RE_target
  • Target_downstream
  • Target_val
  • TnsC
  • TnsD
  • tRNA_LE_LUEGO
KeywordsDNA BINDING PROTEIN/DNA / Tn7-like transposon / type I-B2 CAST / DNA binding protein / transpososome / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


DNA integration / nucleic acid binding / ATP hydrolysis activity
Similarity search - Function
TnsA endonuclease C-terminal / TnsA endonuclease, N-terminal / TnsA endonuclease C terminal / TnsA-like endonuclease N terminal / Transposase-like, Mu, C-terminal / Mu transposase, C-terminal / TniQ / TniQ / : / AAA domain ...TnsA endonuclease C-terminal / TnsA endonuclease, N-terminal / TnsA endonuclease C terminal / TnsA-like endonuclease N terminal / Transposase-like, Mu, C-terminal / Mu transposase, C-terminal / TniQ / TniQ / : / AAA domain / Cro/C1-type HTH domain profile. / Cro/C1-type helix-turn-helix domain / Integrase, catalytic core / Integrase catalytic domain profile. / Ribonuclease H superfamily / Ribonuclease H-like superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA (> 100) / ORC1/DEAH AAA+ ATPase domain-containing protein / Integrase / HTH cro/C1-type domain-containing protein
Similarity search - Component
Biological speciesPeltigera membranacea (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.65 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
DepositionMay 20, 2024Deposition 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
2: LE_polyA
4: RE_target
5: RE_polyA
6: Target_downstream
H: Target_val
I: tRNA_LE_LUEGO
J: TnsD
M: Integrase
N: Integrase
O: Integrase
P: Integrase
Q: TnsC
R: TnsC
S: TnsC
T: TnsC
U: TnsC
V: TnsC
W: TnsC
a: Integrase
b: Integrase
c: Integrase
d: Integrase
x: TnsC
y: TnsC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,495,47044
Polymers1,491,52224
Non-polymers3,94820
Water1267
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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DNA chain , 6 types, 6 molecules 2456HI

#1: DNA chain LE_polyA


Mass: 26308.939 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Peltigera membranacea (fungus)
#2: DNA chain RE_target


Mass: 61828.516 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Peltigera membranacea (fungus)
#3: DNA chain RE_polyA


Mass: 53267.184 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Peltigera membranacea (fungus)
#4: DNA chain Target_downstream


Mass: 11471.370 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Peltigera membranacea (fungus)
#5: DNA chain Target_val


Mass: 21278.615 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Peltigera membranacea (fungus)
#6: DNA chain tRNA_LE_LUEGO


Mass: 48725.188 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Peltigera membranacea (fungus)

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Protein , 3 types, 18 molecules JMNOPabcdQRSTUVWxy

#7: Protein TnsD


Mass: 52821.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Peltigera membranacea (fungus) / Gene: CDG76_08985 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IGG7
#8: Protein
Integrase / TnsAB


Mass: 103015.461 Da / Num. of mol.: 8 / Mutation: E62A, D519A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Peltigera membranacea (fungus) / Gene: CDG76_08995 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IFR8
#9: Protein
TnsC


Mass: 43521.934 Da / Num. of mol.: 9
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

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Non-polymers , 4 types, 27 molecules

#10: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#12: 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
#13: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 7 / 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

Component
IDNameTypeEntity IDParent-IDSource
1TnsABCD-DNA complexCOMPLEX#1-#90MULTIPLE SOURCES
2TnsABCDCOMPLEX#7-#91RECOMBINANT
3DNACOMPLEX#1-#61SYNTHETIC
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Peltigera membranacea (fungus)161997
33Peltigera membranacea (fungus)161997
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

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 38326 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00548869
ELECTRON MICROSCOPYf_angle_d0.70267389
ELECTRON MICROSCOPYf_dihedral_angle_d19.6978434
ELECTRON MICROSCOPYf_chiral_restr0.047436
ELECTRON MICROSCOPYf_plane_restr0.0057681

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