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- EMDB-50933: CryoEM volume of a heptameric stretched ring conformation of the ... -

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Basic information

Entry
Database: EMDB / ID: EMD-50933
TitleCryoEM volume of a heptameric stretched ring conformation of the PmcTnsC-dsDNA-AMPPNP complex
Map data
Sample
  • Complex: TnsC heptameric stretched ring bound to a dsDNA and AMPPNP
KeywordsAAA+ ATPase / CRISPR / Tn7-like transposon / CAST / transposase / DNA BINDING PROTEIN
Biological speciesPeltigera membranacea (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.45 Å
AuthorsFinocchio G / Chanez C / Querques I / Speichert KJ / Jinek M
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Structural basis of TnsC oligomerization and transposase recruitment in type I-B CRISPR-associated transposons.
Authors: Giada Finocchio / Irma Querques / Christelle Chanez / Katarzyna J Speichert / Martin Jinek /
Abstract: CRISPR-associated transposon (CAST) systems employ CRISPR-Cas systems as RNA-directed targeting modules for site-specific transposon DNA insertion. Among them, type I CASTs rely on the coordinated ...CRISPR-associated transposon (CAST) systems employ CRISPR-Cas systems as RNA-directed targeting modules for site-specific transposon DNA insertion. Among them, type I CASTs rely on the coordinated action of the guide RNA-bound Cascade complex and the transposon proteins TniQ, TnsC, and TnsAB. The interaction between the transposase TnsAB and the ATPase TnsC is crucial for transposition activity, yet the underlying molecular details have remained elusive. Here, we investigate the type I-B CAST system from Peltigera membranacea cyanobiont. Cryo-electron microscopic structures of TnsC and its complex with the C-terminal region of TnsAB reveal that TnsC forms a heptameric ring that recruits TnsAB by interacting with its C-terminal tail. In vitro binding assays indicate that TnsAB exclusively interacts with the TnsC heptamer without inducing its disassembly, in contrast to type V-K CAST systems. Mutational analysis of key structural features corroborates the significance of TnsC multimerization and TnsB interaction for transposon activity in vivo. Altogether, these findings offer detailed structural and functional insights into the molecular mechanism of type I-B CAST, with the aim of facilitating their development as genome engineering tools.
History
DepositionJul 8, 2024-
Header (metadata) releaseApr 2, 2025-
Map releaseApr 2, 2025-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50933.png

Downloads & links

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Map

FileDownload / File: emd_50933.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
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AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 480 pix.
= 312. Å		0.65 Å/pix.
x 480 pix.
= 312. Å		0.65 Å/pix.
x 480 pix.
= 312. Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0355
Minimum - Maximum-0.0386824 - 0.1390101
Average (Standard dev.)0.00030660428 (±0.0062605925)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 312.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_50933_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_50933_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : TnsC heptameric stretched ring bound to a dsDNA and AMPPNP

EntireName: TnsC heptameric stretched ring bound to a dsDNA and AMPPNP
Components
  • Complex: TnsC heptameric stretched ring bound to a dsDNA and AMPPNP

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Supramolecule #1: TnsC heptameric stretched ring bound to a dsDNA and AMPPNP

SupramoleculeName: TnsC heptameric stretched ring bound to a dsDNA and AMPPNP
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Peltigera membranacea (fungus)

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 59.172 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.45 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 12092
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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