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- PDB-8vcj: CryoEM structure of the TnsC(1-503)-TnsD(1-318)-DNA complex in a ... -

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Basic information

Entry
Database: PDB / ID: 8vcj
TitleCryoEM structure of the TnsC(1-503)-TnsD(1-318)-DNA complex in a 7:2:1 stoichiometry from E. coli Tn7 bound to ATPgS and ADP
Components
  • (DNA) x 2
  • (Transposon Tn7 transposition protein ...) x 2
KeywordsDNA BINDING PROTEIN/DNA / Transposon / AAA+ ATPase / Oligomer / Complex / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


transposition / DNA recombination / ATP hydrolysis activity / DNA binding / ATP binding
Similarity search - Function
Transposon Tn7 transposition protein TnsD, C-termianl / Tn7-like transposition protein D / Tn7 transposition regulator TnsC / Tn7 transposition regulator TnsC / TniQ / TniQ / : / AAA domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / Transposon Tn7 transposition protein TnsC / Transposon Tn7 transposition protein TnsD
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsShen, Y. / Guarne, A.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT-155941 Canada
CitationJournal: Mol Cell / Year: 2024
Title: Assembly of the Tn7 targeting complex by a regulated stepwise process.
Authors: Yao Shen / Shreya S Krishnan / Michael T Petassi / Mark A Hancock / Joseph E Peters / Alba Guarné /
Abstract: The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection ...The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection proteins from the TniQ family and the AAA+ adaptor TnsC to recruit and activate the transposase at specific target sites. Here, we report the cryoelectron microscopy (cryo-EM) structures of TnsC bound to the TniQ domain of TnsD from prototypical Tn7 and unveil key regulatory steps stemming from unique behaviors of ATP- versus ADP-bound TnsC. We show that TnsD recruits ADP-bound dimers of TnsC and acts as an exchange factor to release one protomer with exchange to ATP. This loading process explains how TnsC assembles a heptameric ring unidirectionally from the target site. This unique loading process results in functionally distinct TnsC protomers within the ring, providing a checkpoint for target immunity and explaining how insertions at programmed sites precisely occur in a specific orientation across Tn7 elements.
History
DepositionDec 14, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 31, 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: Transposon Tn7 transposition protein TnsC
B: Transposon Tn7 transposition protein TnsC
C: Transposon Tn7 transposition protein TnsC
Y: Transposon Tn7 transposition protein TnsD
D: Transposon Tn7 transposition protein TnsC
E: Transposon Tn7 transposition protein TnsC
F: Transposon Tn7 transposition protein TnsC
G: Transposon Tn7 transposition protein TnsC
X: Transposon Tn7 transposition protein TnsD
H: DNA
I: DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)522,79427
Polymers519,31011
Non-polymers3,48316
Water00
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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Transposon Tn7 transposition protein ... , 2 types, 9 molecules ABCDEFGYX

#1: Protein
Transposon Tn7 transposition protein TnsC / Protein E


Mass: 59318.926 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: tnsC / Production host: Escherichia coli (E. coli) / References: UniProt: P05846
#2: Protein Transposon Tn7 transposition protein TnsD


Mass: 36636.020 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: tnsD / Production host: Escherichia coli (E. coli) / References: UniProt: P13991

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DNA chain , 2 types, 2 molecules HI

#3: DNA chain DNA


Mass: 15438.931 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#4: DNA chain DNA


Mass: 15366.818 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 4 types, 16 molecules

#5: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, 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-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1Complex of TnsC(1-503) bound to TnsD(1-318) and DNA with cofactors ATPgS and ADPCOMPLEX#1, #3-#4, #20MULTIPLE SOURCES
2TnsC(1-503) and TnsD(1-318)COMPLEX#1-#21RECOMBINANT
3DNACOMPLEX#3-#41SYNTHETIC
Molecular weightValue: 0.55 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Details: 20 mM Tris pH 8.0, 150 mM NaCl, 1.4 mM beta-mercaptoethanol, 5 mM MgCl2
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: 2500 nm / Nominal defocus min: 1250 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.19.2_4158: / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 393901 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 8GLW

8glw


Accession code: 8GLW / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00333060
ELECTRON MICROSCOPYf_angle_d0.61545220
ELECTRON MICROSCOPYf_dihedral_angle_d15.9385217
ELECTRON MICROSCOPYf_chiral_restr0.0415091
ELECTRON MICROSCOPYf_plane_restr0.0045452

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