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- EMDB-27140: TnsBhook-TnsC complex -

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Basic information

Entry
Database: EMDB / ID: EMD-27140
TitleTnsBhook-TnsC complex
Map dataSharpened map
Sample
  • Complex: AMPPNP-bound TnsBhook-TnsC filament from ShCAST element
    • DNA: DNA
    • DNA: DNA
    • Protein or peptide: TnsC filament
    • Protein or peptide: TnsB hook peptide
KeywordsCAST / transposase / AAA+ ATPase / AAA+ / CRISPR / Cas / DNA BINDING PROTEIN-DNA complex / DNA BINDING PROTEIN
Biological species[Scytonema hofmanni] UTEX 2349 (bacteria) / synthetic construct (others)
Methodhelical reconstruction / cryo EM / Resolution: 3.77 Å
AuthorsPark J / Tsai AWT / Kellogg EH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00-GM124463 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM.
Authors: Jung-Un Park / Amy Wei-Lun Tsai / Tiffany H Chen / Joseph E Peters / Elizabeth H Kellogg /
Abstract: CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the ...CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from (ShCAST). Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. However, key structural differences in the C-terminal domains indicate that TnsB's tetrameric architecture is stabilized by a different set of protein-protein interactions compared with MuA. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. We observe that melting of the 5' nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor-DNA integration. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. The C-terminal end of TnsB adopts a short, structured 15-residue "hook" that decorates TnsC filaments. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools.
History
DepositionMay 27, 2022-
Header (metadata) releaseAug 10, 2022-
Map releaseAug 10, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27140.png

Downloads & links

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Map

FileDownload / File: emd_27140.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.33 Å/pix.
x 200 pix.
= 266. Å		1.33 Å/pix.
x 200 pix.
= 266. Å		1.33 Å/pix.
x 200 pix.
= 266. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.33 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.28
Minimum - Maximum-1.0881915 - 1.9084463
Average (Standard dev.)0.021296637 (±0.10348976)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 266.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unprocessed full map

Fileemd_27140_additional_1.map
Annotationunprocessed full map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap A

Fileemd_27140_half_map_1.map
Annotationhalfmap A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap B

Fileemd_27140_half_map_2.map
Annotationhalfmap B
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : AMPPNP-bound TnsBhook-TnsC filament from ShCAST element

EntireName: AMPPNP-bound TnsBhook-TnsC filament from ShCAST element
Components
  • Complex: AMPPNP-bound TnsBhook-TnsC filament from ShCAST element
    • DNA: DNA
    • DNA: DNA
    • Protein or peptide: TnsC filament
    • Protein or peptide: TnsB hook peptide

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Supramolecule #1: AMPPNP-bound TnsBhook-TnsC filament from ShCAST element

SupramoleculeName: AMPPNP-bound TnsBhook-TnsC filament from ShCAST element
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: DNA

MacromoleculeName: DNA / type: dna / ID: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #2: DNA

MacromoleculeName: DNA / type: dna / ID: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)

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Macromolecule #3: TnsC filament

MacromoleculeName: TnsC filament / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTV PVVYIRPHQK CGPKDLFKKI TEYLKYRVTK GTVSDFRDRT IEVLKGCGVE MLIIDEADRL KPETFADVRD I AEDLGIAV ...String:
MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTV PVVYIRPHQK CGPKDLFKKI TEYLKYRVTK GTVSDFRDRT IEVLKGCGVE MLIIDEADRL KPETFADVRD I AEDLGIAV VLVGTDRLDA VIKRDEQVLE RFRAHLRFGK LSGEDFKNTV EMWEQMVLKL PVSSNLKSKE MLRILTSATE GY IGRLDEI LREAAIRSLS RGLKKIDKAV LQEVAKEYK

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Macromolecule #4: TnsB hook peptide

MacromoleculeName: TnsB hook peptide / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
SequenceString:
IEVWDYEQLR EEYGF

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
2.0 mMC10H16N5O13P3ATP
200.0 mMNaClSodium Chloride
2.0 mMMgCl2Magnesium Chloride
2.0 %C3H8O3Glycerol
1.0 mMC4H10O2S2DTT
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 7.001 Å
Applied symmetry - Helical parameters - Δ&Phi: 59.391 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 562516
Startup modelType of model: EMDB MAP
EMDB ID:

23720

Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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