AF4-to-SAXS: Expanded Characterization of Nanoparticles and Proteins at the P12 Biosaxs Beamline

Melissa Graewert.

SASDWA4 – Yeast alcohol dehydrogenase 1 from AF4-SAXS measurements

Alcohol dehydrogenase 1
MWexperimental 144 kDa
MWexpected 147 kDa
VPorod 197 nm3
log I(s) 1.67×103 1.67×102 1.67×101 1.67×100
Alcohol dehydrogenase 1 small angle scattering data  s, nm-1
ln I(s)
Alcohol dehydrogenase 1 Guinier plot ln 1.68×103 Rg: 3.4 nm 0 (3.4 nm)-2 s2
(sRg)2I(s)/I(0)
Alcohol dehydrogenase 1 Kratky plot 1.104 0 3 sRg
p(r)
Alcohol dehydrogenase 1 pair distance distribution function Rg: 3.3 nm 0 Dmax: 9.5 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Alcohol dehydrogenase 1 PDB (PROTEIN DATA BANK) model

Synchrotron SAXS data from solutions of alcohol dehydrogenase 1 separated using asymmetric flow field flow fractionation (AF4) in phosphate buffered saline, 1% glycerol, pH 7.2 were collected on the EMBL P12 beam line at PETRA III (DESY; Hamburg, Germany) using a Pilatus 6M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.12 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). AF4 separation was performed at 20°C using a flow rate of 0.5 mL/min with an applied constant cross flow rate of 4 mL/min. 50 µL of alcohol dehydrogenase 1 at a concentration of 7.7 mg/mL were injected. 1500 successive 1 second SAXS data frames were collected through the entire AF4 elution (45 min), where 10 sample data frames were selected through the tetrameric separated elution peak. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Tags: AFFFF
Alcohol dehydrogenase 1
Mol. type   Protein
Organism   Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Olig. state   Tetramer
Mon. MW   36.8 kDa
 
UniProt   P00330 (1-348)
Sequence   FASTA
 
PDB ID   4W6Z