AFM-based force spectroscopy unravels stepwise formation of the DNA transposition complex in the widespread Tn3 family mobile genetic elements.

Fernandez M, Shkumatov AV, Liu Y, Stulemeijer C, Derclaye S, Efremov RG, Hallet B, Alsteens D, Nucleic Acids Res (2023) Europe PMC

SASDMR5 – Tn3 family transposase (TnpA WT)

TnpA transposase
MWexperimental 270 kDa
MWexpected 234 kDa
VPorod 480 nm3
log I(s) 7.70×10-2 7.70×10-3 7.70×10-4 7.70×10-5
TnpA transposase small angle scattering data  s, nm-1
ln I(s)
TnpA transposase Guinier plot ln 7.70×10-2 Rg: 4.6 nm 0 (4.6 nm)-2 s2
(sRg)2I(s)/I(0)
TnpA transposase Kratky plot 1.104 0 √3 sRg
p(r)
TnpA transposase pair distance distribution function Rg: 4.6 nm 0 Dmax: 16 nm

Data validation

Fits and models

log I(s)
 s, nm-1
TnpA transposase GASBOR model
Small-angle X-ray scattering (SAXS) data collection and analysis. The SAXS data were collected on the SWING beamline36 at Soleil synchrotron (Gif-sur-Yvette, France). The TnpAWT containing fractions [50 mM HEPES (pH 7.9), 1 M NaCl, 10% glycerol, ~400mM imidazole] after elution from HisTrap column were concentrated to ~3.6 mg/ml and ~50ul of the sample was loaded onto a BioSEC3-300 (Agilent) column equilibrated in SEC buffer [50 mM HEPES (pH 7.9), 200 mM NaCl, 100 mM L-Arg HCL]. During the elution, 600 scattering measurements were taken with 1 s time frames. Data reduction, frame averaging, buffer subtraction and initial analysis were performed using FOXTROT and DATASW programs. Further interpretation of the SAXS data involved representation using the dimensionless Kratky plot and calculation of the pair-distance distribution function using the ATSAS program GNOM The excluded volume was estimated using DAMMIN in P1 and P(r) function. GASBOR program, which models the particle in solution as a protein-like assembly of dummy residues, was used to generate ab initio models with P2 symmetry constraint.

Notes - The TnpA WT protein used for SAXS includes a cMyc-His6 tag at the C-terminus.

TnpA transposase (TnpA WT)
Mol. type   Protein
Organism   Bacillus thuringiensis serovar thuringiensis str. IS5056
Olig. state   Dimer
Mon. MW   116.8 kDa
 
UniProt   M1QZ58 (1-987)
Sequence   FASTA