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

SASDJX4 – DNA-dependent protein kinase catalytic subunit (DNA-PKcs)

DNA-dependent protein kinase catalytic subunit
MWexperimental 480 kDa
MWexpected 468 kDa
VPorod 1040 nm3
log I(s) 4.77×103 4.77×102 4.77×101 4.77×100
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 4.78×103 Rg: 5.6 nm 0 (5.6 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: 5.4 nm 0 Dmax: 15.6 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 DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in 50 mM Tris-HCl, 100 mM NaCl, 5% glycerol, 0.01% sodium azide, pH 7.5 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). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 50.00 μl sample at 10 mg/ml was injected at a 0.25 ml/min flow rate onto a Shodex KW403 column 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.

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