Visualizing functional dynamicity in the DNA-dependent protein kinase holoenzyme DNA-PK complex by integrating SAXS with cryo-EM.

Hammel M, Rosenberg DJ, Bierma J, Hura GL, Lees-Miller SP, Tainer JA, Prog Biophys Mol Biol (2020) Europe PMC

SASDJY4 – Autophosphorylated DNA-dependent protein kinase (DNA-PKcs)

DNA-dependent protein kinase catalytic subunit
MWexperimental 480 kDa
MWexpected 468 kDa
VPorod 918 nm3
log I(s) 5.55×102 5.55×101 5.55×100 5.55×10-1
DNA-dependent protein kinase catalytic subunit small angle scattering data  s, nm-1
ln I(s)
DNA-dependent protein kinase catalytic subunit Guinier plot ln 5.55×102 Rg: 5.5 nm 0 (5.5 nm)-2 s2
(sRg)2I(s)/I(0)
DNA-dependent protein kinase catalytic subunit Kratky plot 1.104 0 3 sRg
p(r)
DNA-dependent protein kinase catalytic subunit pair distance distribution function Rg: 5.6 nm 0 Dmax: 16.1 nm

Data validation


Fits and models


log I(s)
 s, nm-1
DNA-dependent protein kinase catalytic subunit BILBOMD model
DNA-dependent protein kinase catalytic subunit BILBOMD model

Synchrotron SAXS data from solutions of Autophosphorylated DNA-dependent protein kinase (DNA-PKcs) in 50 mM Tris-HCl, 100 mM KCl, 5% (v/v) glycerol, 0.2 mM EDTA containing 0.1 mM benzamidine, 0.2 mM PMSF and 0.2 µg/ml pepstatin, pH 8 were collected on the 12.3.1 (SIBYLS) beam line at the Advanced Light Source (ALS) storage ring (Berkeley, CA, USA) using a MAR 165 CCD detector at a sample-detector distance of 1.5 m and at a wavelength of λ = 0.103 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Solute concentrations ranging between 1.5 and 15 mg/ml were measured at 20°C. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted. The low angle data collected at lower concentration were merged with the highest concentration high angle data to yield the final composite scattering curve.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   468.3 kDa
 
UniProt   P78527 (10-4128)
Sequence   FASTA