Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders

Kevin Haubrich.

SASDU52 – Cereblon-midi (CRBNmidi), an engineered Cereblon construct for crystallographic and biophysical studies, apo form

Cereblon-midi
MWexperimental 40 kDa
MWexpected 37 kDa
VPorod 61 nm3
log I(s) 2.82×10-2 2.82×10-3 2.82×10-4 2.82×10-5
Cereblon-midi small angle scattering data  s, nm-1
ln I(s)
Cereblon-midi Guinier plot ln 2.82×10-2 Rg: 2.7 nm 0 (2.7 nm)-2 s2
(sRg)2I(s)/I(0)
Cereblon-midi Kratky plot 1.104 0 3 sRg
p(r)
Cereblon-midi pair distance distribution function Rg: 2.7 nm 0 Dmax: 8.6 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Cereblon-midi SWISSMODEL model

log I(s)
 s, nm-1
Cereblon-midi PYMOL model

Synchrotron SAXS data from solutions of Cereblon-midi (CRBNmidi), an engineered Cereblon construct for crystallographic and biophysical studies, apo form in 20 mM Hepes, 500 mM NaCl, 0.5 mM TCEP, pH 7.5 were collected on the B21 beam line at the Diamond Light Source storage ring (Didcot, UK) using a Eiger 4M detector at a sample-detector distance of 3.7 m and at a wavelength of λ = 0.09464 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 6.5 mg/ml was injected at a 0.08 ml/min flow rate onto a Cytiva Superdex 200 Increase 3.2/300 column at 15°C. 600 successive 3 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

SEC-SAXS of CRBN-midi apo

Cereblon-midi (CRBNmidi)
Mol. type   Protein
Olig. state   Monomer
Mon. MW   37.4 kDa
Sequence   FASTA