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- EMDB-40221: CryoEM structure of the TnsC(1-503)-TnsD(1-318)-DNA complex in a ... -

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Basic information

Entry
Database: EMDB / ID: EMD-40221
TitleCryoEM structure of the TnsC(1-503)-TnsD(1-318)-DNA complex in a 7:2:1 stoichiometry from E. coli Tn7
Map data
Sample
  • Complex: Complex of TnsC(1-503) bound to TnsD(1-318) and DNA with cofactors ATP and ADP
    • Protein or peptide: Transposon Tn7 transposition protein TnsC
    • Protein or peptide: Transposon Tn7 transposition protein TnsD
    • DNA: DNA (50-MER)
    • DNA: DNA (50-MER)
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ZINC ION
KeywordsTransposon / 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
Transposon Tn7 transposition protein TnsC / Transposon Tn7 transposition protein TnsD
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.51 Å
AuthorsShen Y / Guarne A
Funding support Canada, 1 items
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
DepositionMar 23, 2023-
Header (metadata) releaseJul 31, 2024-
Map releaseJul 31, 2024-
UpdateJul 31, 2024-
Current statusJul 31, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40221.png

Downloads & links

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Map

FileDownload / File: emd_40221.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.86 Å/pix.
x 400 pix.
= 342. Å		0.86 Å/pix.
x 400 pix.
= 342. Å		0.86 Å/pix.
x 400 pix.
= 342. Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.855 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.3
Minimum - Maximum-37.079352999999998 - 50.798824000000003
Average (Standard dev.)0.000000000000125 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 342.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #2

Fileemd_40221_additional_1.map
Projections & Slices
AxesZYX

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Additional map: #1

Fileemd_40221_additional_2.map
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Half map: #2

Fileemd_40221_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_40221_half_map_2.map
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Sample components

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Entire : Complex of TnsC(1-503) bound to TnsD(1-318) and DNA with cofactor...

EntireName: Complex of TnsC(1-503) bound to TnsD(1-318) and DNA with cofactors ATP and ADP
Components
  • Complex: Complex of TnsC(1-503) bound to TnsD(1-318) and DNA with cofactors ATP and ADP
    • Protein or peptide: Transposon Tn7 transposition protein TnsC
    • Protein or peptide: Transposon Tn7 transposition protein TnsD
    • DNA: DNA (50-MER)
    • DNA: DNA (50-MER)
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ZINC ION

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Supramolecule #1: Complex of TnsC(1-503) bound to TnsD(1-318) and DNA with cofactor...

SupramoleculeName: Complex of TnsC(1-503) bound to TnsD(1-318) and DNA with cofactors ATP and ADP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 550 KDa

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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: 59.318926 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 QIANAHALGL 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

UniProtKB: Transposon Tn7 transposition protein TnsC

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Macromolecule #2: Transposon Tn7 transposition protein TnsD

MacromoleculeName: Transposon Tn7 transposition protein TnsD / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 36.63602 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRNFPVPYSN ELIYSTIARA GVYQGIVSPK QLLDEVYGNR KVVATLGLPS HLGVIARHLH QTGRYAVQQL IYEHTLFPLY APFVGKERR DEAIRLMEYQ AQGAVHLMLG VAASRVKSDN RFRYCPDCVA LQLNRYGEAF WQRDWYLPAL PYCPKHGALV F FDRAVDDH ...String:
MRNFPVPYSN ELIYSTIARA GVYQGIVSPK QLLDEVYGNR KVVATLGLPS HLGVIARHLH QTGRYAVQQL IYEHTLFPLY APFVGKERR DEAIRLMEYQ AQGAVHLMLG VAASRVKSDN RFRYCPDCVA LQLNRYGEAF WQRDWYLPAL PYCPKHGALV F FDRAVDDH RHQFWALGHT ELLSDYPKDS LSQLTALAAY IAPLLDAPRA QELSPSLEQW TLFYQRLAQD LGLTKSKHIR HD LVAERVR QTFSDEALEK LDLKLAENKD TCWLKSIFRK HRKAFSYLQH SIVWQALLPK LTVIEALQQA SALTEHSITT R

UniProtKB: Transposon Tn7 transposition protein TnsD

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Macromolecule #3: DNA (50-MER)

MacromoleculeName: DNA (50-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 15.438931 KDa
SequenceString:
(DA)(DT)(DA)(DC)(DT)(DG)(DT)(DG)(DG)(DA) (DC)(DC)(DA)(DG)(DA)(DA)(DC)(DC)(DC)(DT) (DG)(DA)(DT)(DA)(DA)(DA)(DT)(DG)(DC) (DA)(DA)(DC)(DG)(DC)(DT)(DC)(DA)(DT)(DA) (DG) (DC)(DG)(DG)(DG)(DC)(DA)(DG)(DA) (DC)(DG)

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Macromolecule #4: DNA (50-MER)

MacromoleculeName: DNA (50-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 15.366818 KDa
SequenceString:
(DC)(DG)(DT)(DC)(DT)(DG)(DC)(DC)(DC)(DG) (DC)(DT)(DA)(DT)(DG)(DA)(DG)(DC)(DG)(DT) (DT)(DG)(DC)(DA)(DT)(DT)(DT)(DA)(DT) (DC)(DA)(DG)(DG)(DG)(DT)(DT)(DC)(DT)(DG) (DG) (DT)(DC)(DC)(DA)(DC)(DA)(DG)(DT) (DA)(DT)

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Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 7 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 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

Concentration0.5 mg/mL
BufferpH: 8
Details: 20 mM Tris pH 8.0, 150 mM NaCl, 1.4 mM beta-mercaptoethanol, 5 mM MgCl2
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 79.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.75 µm / Nominal defocus min: 1.25 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.51 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 364564
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8glu


Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: TnsC and TnsD were modelled using PDB 8GLU.
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8glw:
CryoEM structure of the TnsC(1-503)-TnsD(1-318)-DNA complex in a 7:2:1 stoichiometry from E. coli Tn7

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