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- EMDB-24783: CryoEM structure of Vibrio cholerae transposon Tn6677 AAA+ ATPase TnsC -

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Basic information

Entry
Database: EMDB / ID: EMD-24783
TitleCryoEM structure of Vibrio cholerae transposon Tn6677 AAA+ ATPase TnsC
Map datapost-processed map with deepEMhacer
Sample
  • Complex: VchTnsC
    • Protein or peptide: Tn6677 Vibrio cholerae transposon TnsC (VchTnsC)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsCRISPR / transposon / ATPase / AAA+ / TnsC / DNA BINDING PROTEIN
Biological speciesVibrio cholerae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsHoffmann FT / Kim M
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2HG011650-01 United States
CitationJournal: Nature / Year: 2022
Title: Selective TnsC recruitment enhances the fidelity of RNA-guided transposition.
Authors: Florian T Hoffmann / Minjoo Kim / Leslie Y Beh / Jing Wang / Phuc Leo H Vo / Diego R Gelsinger / Jerrin Thomas George / Christopher Acree / Jason T Mohabir / Israel S Fernández / Samuel H Sternberg /
Abstract: Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site ...Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.
History
DepositionAug 28, 2021-
Header (metadata) releaseJun 8, 2022-
Map releaseJun 8, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24783.png

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Map

FileDownload / File: emd_24783.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpost-processed map with deepEMhacer
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.36 Å/pix.
x 256 pix.
= 349.013 Å		1.36 Å/pix.
x 256 pix.
= 349.013 Å		1.36 Å/pix.
x 256 pix.
= 349.013 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.36333 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.062183864 - 1.8434111
Average (Standard dev.)0.0009426389 (±0.02060899)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 349.01334 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_24783_msk_1.map
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Additional map: unsharpened map

Fileemd_24783_additional_1.map
Annotationunsharpened map
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Additional map: Relion3 post-processed map

Fileemd_24783_additional_2.map
AnnotationRelion3 post-processed map
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Half map: half-map-1

Fileemd_24783_half_map_1.map
Annotationhalf-map-1
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Half map: half-map-2

Fileemd_24783_half_map_2.map
Annotationhalf-map-2
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Sample components

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Entire : VchTnsC

EntireName: VchTnsC
Components
  • Complex: VchTnsC
    • Protein or peptide: Tn6677 Vibrio cholerae transposon TnsC (VchTnsC)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: VchTnsC

SupramoleculeName: VchTnsC / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 238 KDa

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Macromolecule #1: Tn6677 Vibrio cholerae transposon TnsC (VchTnsC)

MacromoleculeName: Tn6677 Vibrio cholerae transposon TnsC (VchTnsC) / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 37.596043 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSETREARIS RAKRAFVSTP SVRKILSYMD RCRDLSDLES EPTCMMVYGA SGVGKTTVIK KYLNQNRRES EAGGDIIPVL HIELPDNAK PVDAARELLV EMGDPLALYE TDLARLTKRL TELIPAVGVK LIIIDEFQHL VEERSNRVLT QVGNWLKMIL N KTKCPIVI ...String:
MSETREARIS RAKRAFVSTP SVRKILSYMD RCRDLSDLES EPTCMMVYGA SGVGKTTVIK KYLNQNRRES EAGGDIIPVL HIELPDNAK PVDAARELLV EMGDPLALYE TDLARLTKRL TELIPAVGVK LIIIDEFQHL VEERSNRVLT QVGNWLKMIL N KTKCPIVI FGMPYSKVVL QANSQLHGRF SIQVELRPFS YNGGRGVFKT FLEYLDKALP FEKQAGLANE SLQKKLYAFS QG NMRSLRN LIYQASIEAI DNQHETITEE DFVFASKLTS GDKPNSWKNP FEEGVEVTED MLRPPPKDIG WEDYLRHSTP RVS KPGRNK NFFE

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

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

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

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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: 7.2
GridModel: Homemade / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 70.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 51.0 µm / Calibrated defocus max: 1.9000000000000001 µm / Calibrated defocus min: 0.55 µm / Calibrated magnification: 65000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 0.001 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 65000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 109000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: BACKBONE TRACE
Output model

PDB-7rzy:
CryoEM structure of Vibrio cholerae transposon Tn6677 AAA+ ATPase TnsC

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