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- PDB-7mcs: Cryo-electron microscopy structure of TnsC(1-503)A225V bound to DNA -

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Basic information

Entry
Database: PDB / ID: 7mcs
TitleCryo-electron microscopy structure of TnsC(1-503)A225V bound to DNA
Components
  • DNA (5'-D(P*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*C)-3')
  • DNA (5'-D(P*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*G)-3')
  • Transposon Tn7 transposition protein TnsC
KeywordsDNA BINDING PROTEIN/DNA / AAA+ ATPase / DNA binding / Transposition / 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
Tn7 transposition regulator TnsC / Tn7 transposition regulator TnsC / AAA domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / Transposon Tn7 transposition protein TnsC
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.56 Å
AuthorsShen, Y. / Ortega, J. / Guarne, A.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT-155941 Canada
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Structural basis for DNA targeting by the Tn7 transposon.
Authors: Yao Shen / Josue Gomez-Blanco / Michael T Petassi / Joseph E Peters / Joaquin Ortega / Alba Guarné /
Abstract: Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ ...Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ adaptor (TnsC) to coordinate target-site selection with transpososome assembly and to prevent insertions at sites already containing a Tn7 element. Owing to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. TnsC forms an asymmetric ring on target DNA that segregates target-site selection and interaction with the paired-end complex to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.
History
DepositionApr 2, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 23, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 2, 2022Group: Database references / Category: citation
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

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Structure viewerMolecule:
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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
D: Transposon Tn7 transposition protein TnsC
E: Transposon Tn7 transposition protein TnsC
F: Transposon Tn7 transposition protein TnsC
G: Transposon Tn7 transposition protein TnsC
H: DNA (5'-D(P*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*C)-3')
I: DNA (5'-D(P*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)427,79821
Polymers424,6159
Non-polymers3,18312
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 59346.980 Da / Num. of mol.: 7 / Mutation: S2G, A225V
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: DNA chain DNA (5'-D(P*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*C)-3')


Mass: 4572.937 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(P*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*G)-3')


Mass: 4612.961 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / 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

Component
IDNameTypeEntity IDParent-IDSource
1Complex of TnsC bound to DNA in the presence of AMPPnP.COMPLEX#1-#30MULTIPLE SOURCES
2TnsC(1-503)A225V bound to DNACOMPLEX#1-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.43 MDaNO
210.43 MDaNO
310.03 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Escherichia coli (E. coli)562
21synthetic construct (others)32630
32Escherichia coli (E. coli)562
42synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Escherichia coli (E. coli)562
22Escherichia coli (E. coli)562
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2750 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 78 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
3SerialEMimage acquisition
5GctfCTF correction
8UCSF Chimeramodel fitting
9Cootmodel fitting
12RELION3.1initial Euler assignment
14RELION3.1final Euler assignment
16RELION3.1classification
18RELION3.13D reconstruction
20PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 905856
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 155988 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00422051
ELECTRON MICROSCOPYf_angle_d0.70729998
ELECTRON MICROSCOPYf_dihedral_angle_d12.363265
ELECTRON MICROSCOPYf_chiral_restr0.0453401
ELECTRON MICROSCOPYf_plane_restr0.0053747

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