Large-scale conformational rearrangement of the α5-helix of Gα subunits in complex with the guanine nucleotide exchange factor Ric8A.

Srivastava D, Artemyev NO, J Biol Chem (2019) Europe PMC

SASDGB6 – Resistance to inhibitors of cholinesterase 8 homolog A (Ric8A) miniGi complex

Resistance to inhibitors of cholinesterase 8 homolog A
miniGi
MWexperimental 84 kDa
MWexpected 80 kDa
log I(s) 2.60×10-3 2.60×10-4 2.60×10-5 2.60×10-6
Resistance to inhibitors of cholinesterase 8 homolog A miniGi small angle scattering data  s, nm-1
ln I(s)
Resistance to inhibitors of cholinesterase 8 homolog A miniGi Guinier plot ln 2.60×10-3 Rg: 3.2 nm 0 (3.2 nm)-2 s2
(sRg)2I(s)/I(0)
Resistance to inhibitors of cholinesterase 8 homolog A miniGi Kratky plot 1.104 0 3 sRg
p(r)
Resistance to inhibitors of cholinesterase 8 homolog A miniGi pair distance distribution function Rg: 3.3 nm 0 Dmax: 10.7 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Resistance to inhibitors of cholinesterase 8 homolog A miniGi OTHER model

log I(s)
 s, nm-1
Resistance to inhibitors of cholinesterase 8 homolog A miniGi OTHER model

Synchrotron SAXS data from solutions of the Ric8A-miniGi complex in 20 mM Tris, 150 mM KCl, 5 % glycerol, 1 mM TCEP, pH 8 were collected on the BioCAT 18ID beam line at the Advanced Photon Source (APS), Argonne National Laboratory storage ring (Lemont, IL, USA) using a Pilatus3 X 1M detector (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 250.00 μl sample at 10 mg/ml was injected at a 0.90 ml/min flow rate onto a GE Superdex 200 Increase 10/300 column . The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Purified Ric8a1-492 was mixed with excess of purified miniGi and the complex was purified by size-exclusion chromatography (SEC). The complex fractions were pooled together, concentrated and additional SEC-SAXS data was collected as described above. X-ray wavelength = UNKNOWN.

Resistance to inhibitors of cholinesterase 8 homolog A (Ric8a)
Mol. type   Protein
Organism   Bos taurus
Olig. state   Monomer
Mon. MW   55.6 kDa
 
UniProt   Q5E9J8 (1-492)
Sequence   FASTA
 
miniGi
Mol. type   Protein
Organism   synthetic construct
Olig. state   Monomer
Mon. MW   24.5 kDa
Sequence   FASTA
 
PDB code   6N85