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

SASDJZ4 – DNA-dependent protein kinase/ X-ray repair cross-complementing protein 5 and 6 complex bound to DNA (DNA-PK monomer )

X-ray repair cross-complementing protein 6
X-ray repair cross-complementing protein 5
DNA-dependent protein kinase catalytic subunit
dsDNA
MWexperimental 590 kDa
MWexpected 642 kDa
VPorod 1090 nm3
log I(s) 2.51×102 2.51×101 2.51×100 2.51×10-1
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 DNA-dependent protein kinase catalytic subunit dsDNA small angle scattering data  s, nm-1
ln I(s)
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 DNA-dependent protein kinase catalytic subunit dsDNA Guinier plot ln 2.52×102 Rg: 6.5 nm 0 (6.5 nm)-2 s2
(sRg)2I(s)/I(0)
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 DNA-dependent protein kinase catalytic subunit dsDNA Kratky plot 1.104 0 3 sRg
p(r)
X-ray repair cross-complementing protein 6 X-ray repair cross-complementing protein 5 DNA-dependent protein kinase catalytic subunit dsDNA pair distance distribution function Rg: 6.6 nm 0 Dmax: 23.1 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 DNA-dependent protein kinase catalytic subunit dsDNA BILBOMD model

Synchrotron SAXS data from solutions of DNA-dependent protein kinase/ X-ray repair cross-complementing protein 5 and 6 complex bound to DNA (DNA-PK monomer ) 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 5 mg/ml was injected at a 0.25 ml/min flow rate onto a Shodex KW400 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 (KU80)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   82.7 kDa
 
UniProt   P13010 (1-732)
Sequence   FASTA
 
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
 
dsDNA
Mol. type   DNA
Olig. state   Dimer
Mon. MW   10.6 kDa
Sequence   FASTA