Conjuring up a ghost: structural and functional characterization of FhuF, a ferric siderophore reductase from E. coli

Trindade I, Hernandez G, Lebègue E, Barrière F, Cordeiro T, Piccioli M, Louro R, JBIC Journal of Biological Inorganic Chemistry (2021) DOI

SASDJ28 – Ferric Iron Reductase - FhuF∆1-17

Ferric iron reductase protein FhuF (∆1-17)
MWI(0) 26 kDa
MWexpected 28 kDa
VPorod 60 nm3
log I(s) 2.50×10-2 2.50×10-3 2.50×10-4 2.50×10-5
Ferric iron reductase protein FhuF (∆1-17) small angle scattering data  s, nm-1
ln I(s)
Ferric iron reductase protein FhuF (∆1-17) Guinier plot ln 2.50×10-2 Rg: 2.1 nm 0 (2.1 nm)-2 s2
(sRg)2I(s)/I(0)
Ferric iron reductase protein FhuF (∆1-17) Kratky plot 1.104 0 √3 sRg
p(r)
Ferric iron reductase protein FhuF (∆1-17) pair distance distribution function Rg: 2.2 nm 0 Dmax: 8.8 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Ferric iron reductase protein FhuF (∆1-17) DAMFILT model
Synchrotron SAXS data from FhuF in 20 mM Phosphate, 200 mM NaCl, pH 7.4 was collected on the B21 beam line at the Diamond Light Source (Didcot, UK) using a Pilatus 2M detector at a sample-detector distance of 2.7 m and at a wavelength of λ = 0.091 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed using an Agilent 1200 HPLC System by injecting a 50.00 μl sample at 8.9 mg/ml under a flow rate of 0.16 ml/min onto a Shodex KW402.5-4F column (Showa Denko) at 10°C collecting 915 successive 2-second frames. The data were normalized to the intensity of the transmitted beam and radially averaged; the solvent-blank scattering was subtracted from the sample elution’s peak frames. The displayed SAXS-generated ab initio molecular reconstruction was obtained using DAMMIF (DAMFILT occupancy and volume-corrected bead model) by clustering and averaging dummy residue models from 20 independent runs, with an averaged Normalized Spatial Discrepancy (NSD) of 0.727.

Ferric iron reductase protein FhuF (∆1-17) (FhuF(∆1-17))
Mol. type   Protein
Organism   Escherichia coli (strain K12)
Olig. state   Monomer
Mon. MW   28.0 kDa
 
UniProt   P39405 (18-262)
Sequence   FASTA