Biophysical characterization of the complex between the iron-responsive transcription factor Fep1 and DNA.

Miele AE, Cervoni L, Le Roy A, Cutone A, Musci G, Ebel C, Bonaccorsi di Patti MC, Eur Biophys J (2021) Europe PMC

SASDJ86 – Fep1 wild type

GATA-type iron responsive transcription factor Fep1
MWI(0) 28 kDa
MWexpected 22 kDa
VPorod 41 nm3
log I(s) 4.23×100 4.23×10-1 4.23×10-2 4.23×10-3
GATA-type iron responsive transcription factor Fep1 small angle scattering data  s, nm-1
ln I(s)
GATA-type iron responsive transcription factor Fep1 Guinier plot ln 4.23×100 Rg: 3.5 nm 0 (3.5 nm)-2 s2
(sRg)2I(s)/I(0)
GATA-type iron responsive transcription factor Fep1 Kratky plot 1.104 0 3 sRg
p(r)
GATA-type iron responsive transcription factor Fep1 pair distance distribution function Rg: 3.7 nm 0 Dmax: 11.8 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Fep1 wild type Rg histogram Rg, nm
GATA-type iron responsive transcription factor Fep1 EOM/RANCH model

log I(s)
 s, nm-1
Fep1 wild type Rg histogram Rg, nm
GATA-type iron responsive transcription factor Fep1 EOM/RANCH model

log I(s)
 s, nm-1
Fep1 wild type Rg histogram Rg, nm
GATA-type iron responsive transcription factor Fep1 EOM/RANCH model

Synchrotron SAXS data from solutions of Fep1 wild type in 50 mM MOPS, 50 mM NaCl, pH 7 were collected on the BM29 beam line at the ESRF storage ring (Grenoble, France) using a Pilatus 1M detector at a sample-detector distance of 2.9 m and at a wavelength of λ = 0.0999 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 40.00 μl sample at 9.5 mg/ml was injected at a 0.40 ml/min flow rate onto a GE Superdex 75 Increase 5/150 column at 20°C. 900 successive 1 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.

SAS data derived from merging the elution peaks of 2 successive HPLC run at ESRF BM29 (Grenoble, France). Column Superdex75 5/150 (GE Healthcare) run at 20 °C, flow of 0.4 ml/min. 900 frames (1/sec) were collected per column run. Chromixs and US-SOMO HPLC-SAXS were used to convert the I(q) vs t in I(q) vs q. For the buffer subtraction the first 20 frames of the column run were merged, after inspection of absence of capillary fouling.

GATA-type iron responsive transcription factor Fep1 (Fep1)
Mol. type   Protein
Organism   Komagataella pastoris
Olig. state   Monomer
Mon. MW   22.1 kDa
 
UniProt   A4VAR6 (1-208)
Sequence   FASTA