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- EMDB-15294: Cryo-EM structure of the strand transfer complex of the TnsB tran... -

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Entry
Database: EMDB / ID: EMD-15294
TitleCryo-EM structure of the strand transfer complex of the TnsB transposase (type V-K CRISPR-associated transposon)
Map dataMap post-processed using DeepEMhancer and boxed out using Phenix.
Sample
  • Complex: Ternary complex of ShTnsB transposase with Strand Transfer Complex DNA.
    • Protein or peptide: TnsB
    • DNA: RE_Target
    • DNA: RE_PolyA
    • DNA: Target_1
    • DNA: LE_Target
    • DNA: LE_PolyA
    • DNA: Target_2
  • Ligand: water
Biological speciesScytonema hofmannii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.46 Å
AuthorsTenjo-Castano F / Sofos N / Lopez-Mendez B / Stutzke LS / Fuglsang A / Stella S / Montoya G
Funding support Denmark, 4 items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF0024386 Denmark
Novo Nordisk FoundationNNF14CC0001 Denmark
Novo Nordisk FoundationNNF17SA0030214 Denmark
Novo Nordisk FoundationNNF18OC0055061 Denmark
CitationJournal: Nat Commun / Year: 2022
Title: Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon.
Authors: Francisco Tenjo-Castaño / Nicholas Sofos / Blanca López-Méndez / Luisa S Stutzke / Anders Fuglsang / Stefano Stella / Guillermo Montoya /
Abstract: CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opted CRISPR-Cas systems for RNA-guided transposition. Here we present the 2.4 Å cryo-EM structure of the Scytonema ...CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opted CRISPR-Cas systems for RNA-guided transposition. Here we present the 2.4 Å cryo-EM structure of the Scytonema hofmannii (sh) TnsB transposase from Type V-K CAST, bound to the strand transfer DNA. The strand transfer complex displays an intertwined pseudo-symmetrical architecture. Two protomers involved in strand transfer display a catalytically competent active site composed by DDE residues, while other two, which play a key structural role, show active sites where the catalytic residues are not properly positioned for phosphodiester hydrolysis. Transposon end recognition is accomplished by the NTD1/2 helical domains. A singular in trans association of NTD1 domains of the catalytically competent subunits with the inactive DDE domains reinforces the assembly. Collectively, the structural features suggest that catalysis is coupled to protein-DNA assembly to secure proper DNA integration. DNA binding residue mutants reveal that lack of specificity decreases activity, but it could increase transposition in some cases. Our structure sheds light on the strand transfer reaction of DDE transposases and offers new insights into CAST transposition.
History
DepositionJun 30, 2022-
Header (metadata) releaseOct 19, 2022-
Map releaseOct 19, 2022-
UpdateOct 19, 2022-
Current statusOct 19, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15294.png

Downloads & links

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Map

FileDownload / File: emd_15294.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap post-processed using DeepEMhancer and boxed out using Phenix.
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.0017501548 - 1.8244389
Average (Standard dev.)0.01577485 (±0.080284886)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin141108147
Dimensions142178115
Spacing115142178
CellA: 95.68 Å / B: 118.144005 Å / C: 148.09601 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15294_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15294_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15294_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of ShTnsB transposase with Strand Transfer Comple...

EntireName: Ternary complex of ShTnsB transposase with Strand Transfer Complex DNA.
Components
  • Complex: Ternary complex of ShTnsB transposase with Strand Transfer Complex DNA.
    • Protein or peptide: TnsB
    • DNA: RE_Target
    • DNA: RE_PolyA
    • DNA: Target_1
    • DNA: LE_Target
    • DNA: LE_PolyA
    • DNA: Target_2
  • Ligand: water

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Supramolecule #1: Ternary complex of ShTnsB transposase with Strand Transfer Comple...

SupramoleculeName: Ternary complex of ShTnsB transposase with Strand Transfer Complex DNA.
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Scytonema hofmannii (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 359 KDa

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Macromolecule #1: TnsB

MacromoleculeName: TnsB / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 68.094609 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 FGSEFELENL YFQ

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Macromolecule #2: RE_Target

MacromoleculeName: RE_Target / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 24.402717 KDa
SequenceString: (DA)(DT)(DA)(DA)(DG)(DG)(DA)(DT)(DT)(DT) (DT)(DA)(DC)(DT)(DG)(DA)(DT)(DG)(DA)(DC) (DA)(DA)(DT)(DA)(DA)(DT)(DT)(DT)(DG) (DT)(DC)(DA)(DC)(DA)(DA)(DC)(DG)(DA)(DC) (DA) (DT)(DA)(DT)(DA)(DA)(DT) ...String:
(DA)(DT)(DA)(DA)(DG)(DG)(DA)(DT)(DT)(DT) (DT)(DA)(DC)(DT)(DG)(DA)(DT)(DG)(DA)(DC) (DA)(DA)(DT)(DA)(DA)(DT)(DT)(DT)(DG) (DT)(DC)(DA)(DC)(DA)(DA)(DC)(DG)(DA)(DC) (DA) (DT)(DA)(DT)(DA)(DA)(DT)(DT)(DA) (DG)(DT)(DC)(DA)(DC)(DT)(DG)(DT)(DA)(DC) (DA)(DC) (DG)(DT)(DA)(DG)(DA)(DG)(DA) (DC)(DG)(DT)(DA)(DG)(DC)(DA)(DA)(DT)(DG) (DC)(DT)

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Macromolecule #3: RE_PolyA

MacromoleculeName: RE_PolyA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 22.876809 KDa
SequenceString: (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DT)(DG)(DT)(DA)(DC) (DA)(DG)(DT)(DG)(DA)(DC)(DT)(DA)(DA) (DT)(DT)(DA)(DT)(DA)(DT)(DG)(DT)(DC)(DG) (DT) (DT)(DG)(DT)(DG)(DA)(DC) ...String:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DT)(DG)(DT)(DA)(DC) (DA)(DG)(DT)(DG)(DA)(DC)(DT)(DA)(DA) (DT)(DT)(DA)(DT)(DA)(DT)(DG)(DT)(DC)(DG) (DT) (DT)(DG)(DT)(DG)(DA)(DC)(DA)(DA) (DA)(DT)(DT)(DA)(DT)(DT)(DG)(DT)(DC)(DA) (DT)(DC) (DA)(DG)(DT)(DA)(DA)(DA)(DA) (DT)(DC)(DC)(DT)(DT)(DA)(DT)

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Macromolecule #4: Target_1

MacromoleculeName: Target_1 / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 4.559971 KDa
SequenceString:
(DA)(DG)(DC)(DA)(DT)(DT)(DG)(DC)(DT)(DA) (DC)(DG)(DT)(DC)(DT)

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Macromolecule #5: LE_Target

MacromoleculeName: LE_Target / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 24.63483 KDa
SequenceString: (DA)(DA)(DT)(DT)(DA)(DA)(DA)(DT)(DA)(DG) (DT)(DC)(DA)(DC)(DA)(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) ...String:
(DA)(DA)(DT)(DT)(DA)(DA)(DA)(DT)(DA)(DG) (DT)(DC)(DA)(DC)(DA)(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) (DC)(DT)(DA)(DC)(DG)(DC)(DC) (DT)(DT)(DT)(DT)(DG)(DT)(DG)(DG)(DA)(DG) (DA)(DT)(DG)

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Macromolecule #6: LE_PolyA

MacromoleculeName: LE_PolyA / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 23.238008 KDa
SequenceString: (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(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) ...String:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(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) (DT)(DG)(DT)(DG)(DA)(DC)(DT) (DA)(DT)(DT)(DT)(DA)(DA)(DT)(DT)

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Macromolecule #7: Target_2

MacromoleculeName: Target_2 / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 4.54699 KDa
SequenceString:
(DC)(DA)(DT)(DC)(DT)(DC)(DC)(DA)(DC)(DA) (DA)(DA)(DA)(DG)(DG)

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 2 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration3.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
300.0 mMNaClsodium chloride
50.0 mMHEPES2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
5.0 mMMgCl2magnesium chloride
1.0 mMTCEPTris(2-carboxyethyl)phosphine
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 4728 / Average exposure time: 40.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 96000
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

Particle selectionNumber selected: 9646000
CTF correctionSoftware - Name: cryoSPARC (ver. 3.3.2)
Startup modelType of model: NONE
Details: Ab initio reconstruction in cryoSPARC from the same data.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 208000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationNumber classes: 2 / Avg.num./class: 130000 / Software - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8aa5:
Cryo-EM structure of the strand transfer complex of the TnsB transposase (type V-K CRISPR-associated transposon)

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