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- EMDB-23757: Cryo-electron microscopy structure of TnsC(1-503)A225V bound to DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-23757
TitleCryo-electron microscopy structure of TnsC(1-503)A225V bound to DNA
Map data
Sample
  • Complex: Complex of TnsC bound to DNA in the presence of AMPPnP.
    • Complex: TnsC(1-503)A225V bound to DNA
      • Protein or peptide: Transposon Tn7 transposition protein TnsC
      • DNA: DNA (5'-D(P*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*C)-3')
      • DNA: DNA (5'-D(P*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*G)-3')
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
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
Transposon Tn7 transposition protein TnsC
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.56 Å
AuthorsShen Y / Ortega J / Guarne A
Funding support Canada, 1 items
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, 2021-
Header (metadata) releaseFeb 23, 2022-
Map releaseFeb 23, 2022-
UpdateMar 2, 2022-
Current statusMar 2, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0145
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0145
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mcs
  • Surface level: 0.0145
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23757.png

Downloads & links

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Map

FileDownload / File: emd_23757.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.855 Å
Density
Contour LevelBy AUTHOR: 0.0145 / Movie #1: 0.0145
Minimum - Maximum-0.05757957 - 0.10468085
Average (Standard dev.)4.4002394e-05 (±0.003446926)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 256.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8550.8550.855
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z256.500256.500256.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0580.1050.000

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Supplemental data

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Sample components

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Entire : Complex of TnsC bound to DNA in the presence of AMPPnP.

EntireName: Complex of TnsC bound to DNA in the presence of AMPPnP.
Components
  • Complex: Complex of TnsC bound to DNA in the presence of AMPPnP.
    • Complex: TnsC(1-503)A225V bound to DNA
      • Protein or peptide: Transposon Tn7 transposition protein TnsC
      • DNA: DNA (5'-D(P*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*C)-3')
      • DNA: DNA (5'-D(P*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*G)-3')
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Complex of TnsC bound to DNA in the presence of AMPPnP.

SupramoleculeName: Complex of TnsC bound to DNA in the presence of AMPPnP.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 30 KDa

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Supramolecule #2: TnsC(1-503)A225V bound to DNA

SupramoleculeName: TnsC(1-503)A225V bound to DNA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Transposon Tn7 transposition protein TnsC

MacromoleculeName: Transposon Tn7 transposition protein TnsC / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 59.34698 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGATRIQAVY RDTGVEAYRD NPFIEALPPL QESVNSAASL KSSLQLTSSD LQKSRVIRAH TICRIPDDYF QPLGTHLLLS ERISVMIRG GYVGRNPKTG DLQKHLQNGY ERVQTGELET FRFEEARSTA QSLLLIGCSG SGKTTSLHRI LATYPQVIYH R ELNVEQVV ...String:
MGATRIQAVY RDTGVEAYRD NPFIEALPPL QESVNSAASL KSSLQLTSSD LQKSRVIRAH TICRIPDDYF QPLGTHLLLS ERISVMIRG GYVGRNPKTG DLQKHLQNGY ERVQTGELET FRFEEARSTA QSLLLIGCSG SGKTTSLHRI LATYPQVIYH R ELNVEQVV YLKIDCSHNG SLKEICLNFF RALDRALGSN YERRYGLKRH GIETMLALMS QIANAHVLGL LVIDEIQHLS RS RSGGSQE MLNFFVTMVN IIGVPVMLIG TPKAREIFEA DLRSARRGAG FGAIFWDPIQ QTQRGKPNQE WIAFTDNLWQ LQL LQRKDA LLSDEVRDVW YELSQGVMDI VVKLFVLAQL RALALGNERI TAGLLRQVYQ DELKPVHPML EALRSGIPER IARY SDLVV PEIDKRLIQL QLDIAAIQEQ TPEEKALQEL DTEDQRHLYL MLKEDYDSSL LIPTIKKAFS QNPTMTRQKL LPLVL QWLM EGETVVSELE KPSKSKKVSP NSSSVDKLAA ALEHHHHHH

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Macromolecule #2: DNA (5'-D(P*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*C)-3')

MacromoleculeName: DNA (5'-D(P*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*C)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.572937 KDa
SequenceString:
(DC)(DG)(DC)(DG)(DC)(DG)(DC)(DG)(DC)(DG) (DC)(DG)(DC)(DG)(DC)

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Macromolecule #3: DNA (5'-D(P*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*G)-3')

MacromoleculeName: DNA (5'-D(P*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*G)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.612961 KDa
SequenceString:
(DG)(DC)(DG)(DC)(DG)(DC)(DG)(DC)(DG)(DC) (DG)(DC)(DG)(DC)(DG)

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Macromolecule #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 6 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.75 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 78.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 905856
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: Stochastic Gradient Descent (SGD)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 155988
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

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

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