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

SASDJV4 – X-ray repair cross-complementing proteins 5ΔCTR and 6

X-ray repair cross-complementing protein 6
X-ray repair cross-complementing protein 5 ΔCTR
MWexperimental 140 kDa
MWexpected 134 kDa
VPorod 240 nm3
log I(s) 4.28×101 4.28×100 4.28×10-1 4.28×10-2
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 ΔCTR small angle scattering data  s, nm-1
ln I(s)
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 ΔCTR Guinier plot ln 4.29×101 Rg: 3.8 nm 0 (3.8 nm)-2 s2
(sRg)2I(s)/I(0)
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 ΔCTR Kratky plot 1.104 0 √3 sRg
p(r)
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 ΔCTR pair distance distribution function Rg: 3.8 nm 0 Dmax: 12.5 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 ΔCTR BILBOMD model
Synchrotron SAXS data from solutions of X-ray repair cross-complementing proteins 5ΔCTR and 6 in 50 mM Hepes, 50 mM KCl, 5 mM MgCl2, 5% glycerol and 0.2 mM DTT, 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 Pilatus3 X 2M detector at a sample-detector distance of 2.1 m and at a wavelength of λ = 0.1127 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 55.00 μl sample at 5 mg/ml was injected at a 0.50 ml/min flow rate onto a Shodex KW-800 series 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.

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 ΔCTR (KU80ΔCTR)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   64.1 kDa
 
UniProt   P13010 (1-565)
Sequence   FASTA