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- PDB-8aa5: Cryo-EM structure of the strand transfer complex of the TnsB tran... -

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Basic information

Entry
Database: PDB / ID: 8aa5
TitleCryo-EM structure of the strand transfer complex of the TnsB transposase (type V-K CRISPR-associated transposon)
Components
  • LE_PolyA
  • LE_Target
  • RE_PolyA
  • RE_Target
  • Target_1
  • Target_2
  • TnsB
KeywordsDNA BINDING PROTEIN / Transposase / complex / CRISPR / transposition / DNA cleavage / ligation
Function / homologyDNA / DNA (> 10)
Function and homology information
Biological speciesScytonema hofmannii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.46 Å
AuthorsTenjo-Castano, F. / Sofos, N. / Lopez-Mendez, B. / Stutzke, L.S. / Fuglsang, A. / Stella, S. / Montoya, G.
Funding support Denmark, 4items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 19, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
AP1: TnsB
BP1: TnsB
CP1: TnsB
DP1: TnsB
I: RE_Target
J: RE_PolyA
K: Target_1
L: LE_Target
M: LE_PolyA
N: Target_2


Theoretical massNumber of molelcules
Total (without water)376,63810
Polymers376,63810
Non-polymers00
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering, SEC-MALS, electron microscopy, isothermal titration calorimetry, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 6 types, 6 molecules IJKLMN

#2: DNA chain RE_Target


Mass: 24402.717 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria)
#3: DNA chain RE_PolyA


Mass: 22876.809 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria)
#4: DNA chain Target_1


Mass: 4559.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria)
#5: DNA chain LE_Target


Mass: 24634.830 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria)
#6: DNA chain LE_PolyA


Mass: 23238.008 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria)
#7: DNA chain Target_2


Mass: 4546.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria)

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Protein / Non-polymers , 2 types, 6 molecules AP1BP1CP1DP1

#1: Protein
TnsB


Mass: 68094.609 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli)
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ternary complex of ShTnsB transposase with Strand Transfer Complex DNA.
Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT
Molecular weightValue: 0.359 MDa / Experimental value: YES
Source (natural)Organism: Scytonema hofmannii (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1300 mMsodium chlorideNaClSodium chloride1
250 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHEPES1
35 mMmagnesium chlorideMgCl21
41 mMTris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 3.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 96000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 40 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4728

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
2EPUimage acquisition
4cryoSPARC3.3.2CTF correction
7UCSF ChimeraXmodel fitting
8Coot0.9.6model fitting
10PHENIXmodel refinement
11cryoSPARC3.3.2initial Euler assignment
12cryoSPARC3.3.2final Euler assignment
13cryoSPARC3.3.2classification
14cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 9646000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 208000 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 50.8 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003616164
ELECTRON MICROSCOPYf_angle_d0.573722599
ELECTRON MICROSCOPYf_chiral_restr0.03762500
ELECTRON MICROSCOPYf_plane_restr0.00432328
ELECTRON MICROSCOPYf_dihedral_angle_d24.19443370

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