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- EMDB-15344: Type V-K CAST TnsB bound to LTR-SR -

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Basic information

Entry
Database: EMDB / ID: EMD-15344
TitleType V-K CAST TnsB bound to LTR-SR
Map data
Sample
  • Complex: Type V-K CAST TnsB bound to LTR-SR
    • Protein or peptide: TnsB
    • DNA: RightElement
    • DNA: LeftElement
Biological speciesScytonema hofmannii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.2 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
DepositionJul 5, 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_15344.png

Downloads & links

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Map

FileDownload / File: emd_15344.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.98 Å/pix.
x 400 pix.
= 392. Å		0.98 Å/pix.
x 400 pix.
= 392. Å		0.98 Å/pix.
x 400 pix.
= 392. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.98 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0504
Minimum - Maximum-0.13324593 - 0.46563247
Average (Standard dev.)-1.9994433e-05 (±0.013075983)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 392.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_15344_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15344_half_map_2.map
Projections & Slices
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Sample components

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Entire : Type V-K CAST TnsB bound to LTR-SR

EntireName: Type V-K CAST TnsB bound to LTR-SR
Components
  • Complex: Type V-K CAST TnsB bound to LTR-SR
    • Protein or peptide: TnsB
    • DNA: RightElement
    • DNA: LeftElement

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Supramolecule #1: Type V-K CAST TnsB bound to LTR-SR

SupramoleculeName: Type V-K CAST TnsB bound to LTR-SR / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
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 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNSQQNPDLA VHPLAIPMEG LLGESATTLE KNVIATQLSE EAQVKLEVIQ SLLEPCDRTT YGQKLREAAE KLNVSLRTVQ RLVKNWEQDG LVGLTQTSRA DKGKHRIGEF WEN FITKTY KEGNKGSKRM TPKQVALRVE AKARELKDSK PPNYKTVLRV LAPILEKQQK ...String:
MNSQQNPDLA VHPLAIPMEG LLGESATTLE KNVIATQLSE EAQVKLEVIQ SLLEPCDRTT YGQKLREAAE KLNVSLRTVQ RLVKNWEQDG LVGLTQTSRA DKGKHRIGEF WEN FITKTY KEGNKGSKRM TPKQVALRVE AKARELKDSK PPNYKTVLRV LAPILEKQQK AKSIRSPGWR GTTLSVKTRE GKDLSVDYSN HVWQCDHTRV DVLLVDQHGE ILSRPWLT T VIDTYSRCIM GINLGFDAPS SGVVALALRH AILPKRYGSE YKLHCEWGTY GKPEHFYTDG GKDFRSNHLS QIGAQLGFVC HLRDRPSEGG VVERPFKTLN DQLFSTLPGY TGSN VQERP EDAEKDARLT LRELEQLLVR YIVDRYNQSI DARMGDQTRF ERWEAGLPTV PVPIPERDLD ICLMKQSRRT VQRGGCLQFQ NLMYRGEYLA GYAGETVNLR FDPRDITTI LVYRQENNQE VFLTRAHAQG LETEQLALDE AEAASRRLRT AGKTISNQSL LQEVVDRDAL VATKKSRKER QKLEQTVLRS AAVDESNRES LPSQIVEPDE VESTETVHSQ YEDIEVWDYE QLREEYGFGS EFELENLYFQ

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Macromolecule #2: RightElement

MacromoleculeName: RightElement / type: dna / ID: 2 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
SequenceString: TGCTACGTCT CTACGTGTAC AGTGACTAAT TATATGTCGT TGTGACAAAT TATTGTCATC AGTAAAATCC TTATACAGTA TAGATTATAG CGCTTTGGCA GTTTTAGCAT AACCTCTTTG CAGTGACAAA ATAGATGTCG TTGTCCGTGA TTGTGACAAA TTAGCTGTCG ...String:
TGCTACGTCT CTACGTGTAC AGTGACTAAT TATATGTCGT TGTGACAAAT TATTGTCATC AGTAAAATCC TTATACAGTA TAGATTATAG CGCTTTGGCA GTTTTAGCAT AACCTCTTTG CAGTGACAAA ATAGATGTCG TTGTCCGTGA TTGTGACAAA TTAGCTGTCG CTTTGCAAGA TAGGAAAAAG CTTTTGTGTA TTTTCATAAT GACAAATTGA CTGTCGCAGG AGGTAA

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Macromolecule #3: LeftElement

MacromoleculeName: LeftElement / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
SequenceString:
ATTATATTGA TGACATTTAA TTTGTCATCA ATTAATTAAG CAACGCTGAT GGGTCACGAC GACAATTAAA TAGTCACAAT GACATTAATC TGTCACCGAC GACAGATAAT TTGTCACTGT ACACTACGCC TTTTGTGG

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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
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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 738 / Average exposure time: 40.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 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: 2.5 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 150000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 38871
CTF correctionSoftware - Name: cryoSPARC (ver. 3.3.2)
Startup modelType of model: NONE
Details: Ab initio reconstruction in cryoSPARC from the same data.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 11.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 5554
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL

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