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- EMDB-25455: Strand-transfer complex of TnsB from ShCAST -

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Basic information

Entry
Database: EMDB / ID: EMD-25455
TitleStrand-transfer complex of TnsB from ShCAST
Map datasharpened map
Sample
  • Complex: strand-transfer complex of TnsB from ShCAST element
    • DNA: STC_LE_For
    • DNA: STC_LE_Rev1
    • DNA: STC_LE_Rev2
    • Protein or peptide: TnsB
  • Ligand: MAGNESIUM ION
KeywordsCAST / transposase / strand-transfer complex / CRISPR / Cas / DNA BINDING PROTEIN-DNA complex
Biological species[Scytonema hofmanni] UTEX 2349 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.69 Å
AuthorsPark J / Tsai AWT
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
DepositionNov 19, 2021-
Header (metadata) releaseAug 10, 2022-
Map releaseAug 10, 2022-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25455.png

Downloads & links

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Map

FileDownload / File: emd_25455.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Voxel sizeX=Y=Z: 1.33 Å
Density
Contour LevelBy AUTHOR: 0.017
Minimum - Maximum-0.0629231 - 0.10650018
Average (Standard dev.)0.00011830115 (±0.0017765776)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 399.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25455_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: full map

Fileemd_25455_additional_1.map
Annotationfull map
Projections & Slices
AxesZYX

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Half map: halfmap2

Fileemd_25455_half_map_1.map
Annotationhalfmap2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: halfmap1

Fileemd_25455_half_map_2.map
Annotationhalfmap1
Projections & Slices
AxesZYX

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Sample components

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Entire : strand-transfer complex of TnsB from ShCAST element

EntireName: strand-transfer complex of TnsB from ShCAST element
Components
  • Complex: strand-transfer complex of TnsB from ShCAST element
    • DNA: STC_LE_For
    • DNA: STC_LE_Rev1
    • DNA: STC_LE_Rev2
    • Protein or peptide: TnsB
  • Ligand: MAGNESIUM ION

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Supramolecule #1: strand-transfer complex of TnsB from ShCAST element

SupramoleculeName: strand-transfer complex of TnsB from ShCAST element / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Molecular weightTheoretical: 350 KDa

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Macromolecule #1: STC_LE_For

MacromoleculeName: STC_LE_For / type: dna / ID: 1 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 21.57983 KDa
SequenceString: (DA)(DT)(DG)(DA)(DC)(DA)(DT)(DT)(DA)(DA) (DT)(DC)(DT)(DG)(DT)(DC)(DA)(DC)(DC)(DG) (DA)(DC)(DG)(DA)(DC)(DA)(DG)(DA)(DT) (DA)(DA)(DT)(DT)(DT)(DG)(DT)(DC)(DA)(DC) (DT) (DG)(DT)(DA)(DC)(DA)(DG) ...String:
(DA)(DT)(DG)(DA)(DC)(DA)(DT)(DT)(DA)(DA) (DT)(DC)(DT)(DG)(DT)(DC)(DA)(DC)(DC)(DG) (DA)(DC)(DG)(DA)(DC)(DA)(DG)(DA)(DT) (DA)(DA)(DT)(DT)(DT)(DG)(DT)(DC)(DA)(DC) (DT) (DG)(DT)(DA)(DC)(DA)(DG)(DG)(DC) (DC)(DC)(DT)(DA)(DG)(DG)(DT)(DC)(DT)(DA) (DC)(DG) (DG)(DT)(DT)(DA)(DG)(DA)(DG) (DG)(DC)(DT)

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Macromolecule #2: STC_LE_Rev1

MacromoleculeName: STC_LE_Rev1 / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.905951 KDa
SequenceString:
(DT)(DG)(DT)(DA)(DC)(DA)(DG)(DT)(DG)(DA) (DC)(DA)(DA)(DA)(DT)(DT)(DA)(DT)(DC)(DT) (DG)(DT)(DC)(DG)(DT)(DC)(DG)(DG)(DT) (DG)(DA)(DC)(DA)(DG)(DA)(DT)(DT)(DA)(DA) (DT) (DG)(DT)(DC)(DA)(DT)

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Macromolecule #3: STC_LE_Rev2

MacromoleculeName: STC_LE_Rev2 / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.062945 KDa
SequenceString:
(DA)(DG)(DC)(DC)(DT)(DC)(DT)(DA)(DA)(DC) (DC)(DG)(DT)(DA)(DG)(DA)(DC)(DC)(DT)(DA)

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Macromolecule #4: TnsB

MacromoleculeName: TnsB / type: protein_or_peptide / ID: 4 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Molecular weightTheoretical: 66.63707 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNSQQNPDLA VHPLAIPMEG LLGESATTLE KNVIATQLSE EAQVKLEVIQ SLLEPCDRTT YGQKLREAAE KLNVSLRTVQ RLVKNWEQD GLVGLTQTSR ADKGKHRIGE FWENFITKTY KEGNKGSKRM TPKQVALRVE AKARELKDSK PPNYKTVLRV L APILEKQQ ...String:
MNSQQNPDLA VHPLAIPMEG LLGESATTLE KNVIATQLSE EAQVKLEVIQ SLLEPCDRTT YGQKLREAAE KLNVSLRTVQ RLVKNWEQD GLVGLTQTSR ADKGKHRIGE FWENFITKTY KEGNKGSKRM TPKQVALRVE AKARELKDSK PPNYKTVLRV L APILEKQQ KAKSIRSPGW RGTTLSVKTR EGKDLSVDYS NHVWQCDHTR VDVLLVDQHG EILSRPWLTT VIDTYSRCIM GI NLGFDAP SSGVVALALR HAILPKRYGS EYKLHCEWGT YGKPEHFYTD GGKDFRSNHL SQIGAQLGFV CHLRDRPSEG GVV ERPFKT LNDQLFSTLP GYTGSNVQER PEDAEKDARL TLRELEQLLV RYIVDRYNQS IDARMGDQTR FERWEAGLPT VPVP IPERD LDICLMKQSR RTVQRGGCLQ FQNLMYRGEY LAGYAGETVN LRFDPRDITT ILVYRQENNQ EVFLTRAHAQ GLETE QLAL DEAEAASRRL RTAGKTISNQ SLLQEVVDRD ALVATKKSRK ERQKLEQTVL RSAAVDESNR ESLPSQIVEP DEVEST ETV HSQYEDIEVW DYEQLREEYG F

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / 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

Concentration0.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
26.0 mMC8H18N2O4SHEPES
20.0 mMKClPotassium Chloride
100.0 mMNaClSodium chlorideSodium Chloride
0.2 mMMgCl2Magnesium Chloride
15.0 mMMg(CH3COO)2Magnesium Acetate
3.0 %C3H8O3Glycerol
1.5 mMC4H10O2S2DTT
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 49.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 186121
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio generated from cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.69 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 46568
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7svw:
Strand-transfer complex of TnsB from ShCAST

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