Structural Organization and Dynamics of Homodimeric Cytohesin Family Arf GTPase Exchange Factors in Solution and on Membranes.

Das S, Malaby AW, Nawrotek A, Zhang W, Zeghouf M, Maslen S, Skehel M, Chakravarthy S, Irving TC, Bilsel O, Cherfils J, Lambright DG, Structure (2019) Europe PMC

SASDG84 – Autoinhibited dimer of truncated 6xHis Cytohesin-2 (ARNO, amino acids 2-400) with Inositol 1,3,4,5-tetrakis phosphate (DAMMIF, GASBOR and antiparallel CORAL models)

Cytohesin-2
MWexperimental 95 kDa
MWexpected 95 kDa
VPorod 180 nm3
log I(s) 1.03×100 1.03×10-1 1.03×10-2 1.03×10-3
Cytohesin-2 small angle scattering data  s, nm-1
ln I(s)
Cytohesin-2 Guinier plot ln 1.03×100 Rg: 5.3 nm 0 (5.3 nm)-2 s2
(sRg)2I(s)/I(0)
Cytohesin-2 Kratky plot 1.104 0 √3 sRg
p(r)
Cytohesin-2 pair distance distribution function Rg: 5.6 nm 0 Dmax: 27 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Cytohesin-2 DAMMIF model
Cytohesin-2 DAMFILT model
log I(s)
 s, nm-1
Cytohesin-2 GASBOR model
Cytohesin-2 DAMFILT model
log I(s)
 s, nm-1
Cytohesin-2 CORAL model
Synchrotron SAXS data from solutions of the autoinhibited dimer Cytohesin-2 (ARNO, amino acids 2-400) in 20 mM Tris, 150 mM NaCl, 2 mM MgCl2, 0.1% 2-mercaptoethanol, 5% glycerol, 0.001 mM insitol 1,3,4,5-tetrakis phosphate, pH 8 were collected on the BioCAT 18ID beam line at the Advanced Photon Source (APS), Argonne National Laboratory (Lemont, IL, USA) using a MAR 165 CCD detector at a sample-detector distance of 2.5 m and at a wavelength of λ = 0.10332 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 100.00 μl sample at 10 mg/ml was injected at a 0.25 ml/min flow rate onto a GE Superdex 200 Increase 3.2/300 column at 20°C. SAXS data sets were acquired with 1 s exposures at 5 s intervals during the elution. Raw SAXS images were radially averaged on a log scale over the s range 0.0621-3.33 nm-1 and normalized by the incident beam intensity. The protein scattering profile was reconstructed by singular value decomposition and linear combination (SVD-LC) as described in Malaby et al. (2015) Methods for analysis of size-exclusion chromatography-small angle X-ray scattering and reconstruction of protein scattering. J Appl Crystallogr 48: 1102-1113.

The protein was equilibrated with a 1.2 molar excess of inositol 1,3,4,5-tetrakis phosphate (IP4) and concentrated to 10 mg/ml prior to injection.

Cytohesin-2 (Cyth2, ARNO)
Mol. type   Protein
Organism   Mus musculus
Olig. state   Dimer
Mon. MW   47.6 kDa
 
UniProt   P63034 (2-400)
Sequence   FASTA
 
PDB ID   2R09