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

SASDJW4 – X-ray repair cross-complementing proteins 5 and 6-DNA

X-ray repair cross-complementing protein 6
X-ray repair cross-complementing protein 5
Y-DNA
MWexperimental 170 kDa
MWexpected 171 kDa
VPorod 280 nm3
log I(s) 1.51×103 1.51×102 1.51×101 1.51×100
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 Y-DNA small angle scattering data  s, nm-1
ln I(s)
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 Y-DNA Guinier plot ln 1.52×103 Rg: 4.1 nm 0 (4.1 nm)-2 s2
(sRg)2I(s)/I(0)
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 Y-DNA Kratky plot 1.104 0 3 sRg
p(r)
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 Y-DNA pair distance distribution function Rg: 4.2 nm 0 Dmax: 14.8 nm

Data validation


Fits and models


log I(s)
 s, nm-1
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 Y-DNA BILBOMD model
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 Y-DNA BILBOMD model
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 Y-DNA BILBOMD model

Synchrotron SAXS data from solutions of X-ray repair cross-complementing proteins 5 and 6-DNA 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). Solute concentrations ranging between 1 and 10 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.

X-ray repair cross-complementing protein 6 (KU70)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   69.8 kDa
 
UniProt   P12956 (1-609)
Sequence   FASTA
 
X-ray repair cross-complementing protein 5 (KU80)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   82.7 kDa
 
UniProt   P13010 (1-732)
Sequence   FASTA
 
Y-DNA
Mol. type   DNA
Olig. state   Monomer
Mon. MW   18.2 kDa
Sequence   FASTA