Activity and structure of EcoKMcrA.

Czapinska H, Kowalska M, Zagorskaite E, Manakova E, Slyvka A, Xu SY, Siksnys V, Sasnauskas G, Bochtler M, Nucleic Acids Res 46(18):9829-9841 (2018) Europe PMC

SASDJZ2 – N-terminal domain of modification dependent EcoKMcrA restriction endonuclease in complex with cognate hemimethylated 12bp oligoduplex

5-methylcytosine-specific restriction enzyme A (N-terminal domain)
cognate hemimethylated 12-bp oligoduplex
MWI(0) 21 kDa
MWexpected 28 kDa
VPorod 29 nm3
log I(s) 2.94×103 2.94×102 2.94×101 2.94×100
5-methylcytosine-specific restriction enzyme A (N-terminal domain) cognate hemimethylated 12-bp oligoduplex small angle scattering data  s, nm-1
ln I(s)
5-methylcytosine-specific restriction enzyme A (N-terminal domain) cognate hemimethylated 12-bp oligoduplex Guinier plot ln 2.94×103 Rg: 2.1 nm 0 (2.1 nm)-2 s2
(sRg)2I(s)/I(0)
5-methylcytosine-specific restriction enzyme A (N-terminal domain) cognate hemimethylated 12-bp oligoduplex Kratky plot 1.104 0 √3 sRg
p(r)
5-methylcytosine-specific restriction enzyme A (N-terminal domain) cognate hemimethylated 12-bp oligoduplex pair distance distribution function Rg: 2.1 nm 0 Dmax: 7.5 nm

Data validation

Fits and models

log I(s)
 s, nm-1
5-methylcytosine-specific restriction enzyme A (N-terminal domain) cognate hemimethylated 12-bp oligoduplex OTHER model
Synchrotron SAXS data from solutions of EcoKMcrA restriction endonuclease (N-terminal domain) bound to 12bp oligoduplex DNA in 20 mM Tris–HCl pH 7.5, 200 mM KCl, 0.1 mM EDTA, 0.01% (w/v) sodium azide and 1 mM DTT, were collected on the EMBL P12 beam line at PETRA III (DESY, Hamburg, Germany) using a Pilatus 2M detector at a sample-detector distance of 3.1 m and at a wavelength of λ = 0.124 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 1.14 mg/ml was measured at 20°C. 20 successive 0.050 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Slightly prone to aggregation. NOTE! The DNA used in the model is homologous, and does not correspond in sequence, to the deposited DNA sequence of the complex analysed using SAXS.

5-methylcytosine-specific restriction enzyme A (N-terminal domain) (EcoKMcrA-N)
Mol. type   Protein
Organism   Escherichia coli
Olig. state   Monomer
Mon. MW   20.6 kDa
 
UniProt   P24200 (1-175)
Sequence   FASTA
 
cognate hemimethylated 12-bp oligoduplex (12_5mC/12_C)
Mol. type   DNA
Olig. state   Monomer
Mon. MW   7.4 kDa
Sequence   FASTA