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

David Lambright.

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