EFAMIX , a tool to decompose inline chromatography SAXS data from partially overlapping components

Konarev P, Graewert M, Jeffries C, Fukuda M, Cheremnykh T, Volkov V, Svergun D, Protein Science (2021) DOI

SASDEX9 – Class II pyruvate aldolase (HpcH/HpaI aldolase), wild-type hexamer in HEPES isolated using SEC-SAXS

HpcH/HpaI aldolase
MWexperimental 157 kDa
MWexpected 165 kDa
VPorod 233 nm3
log I(s) 1.32×103 1.32×102 1.32×101 1.32×100
HpcH/HpaI aldolase small angle scattering data  s, nm-1
ln I(s)
HpcH/HpaI aldolase Guinier plot ln 1.32×103 Rg: 3.3 nm 0 (3.3 nm)-2 s2
(sRg)2I(s)/I(0)
HpcH/HpaI aldolase Kratky plot 1.104 0 √3 sRg
p(r)
HpcH/HpaI aldolase pair distance distribution function Rg: 3.3 nm 0 Dmax: 9.4 nm

Data validation

Fits and models

log I(s)
 s, nm-1
HpcH/HpaI aldolase GASBOR model
log I(s)
 s, nm-1
HpcH/HpaI aldolase PDB (PROTEIN DATA BANK) model
Synchrotron SAXS data from solutions of hydroxypyruvate aldolase in 20 mM HEPES, pH 7.5 were collected using size exclusion chromatography SAXS (SEC-SAXS) on the EMBL P12 beam line at PETRA III (Hamburg, Germany) equipped with a Pilatus 6M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.124 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). The SEC parameters were as follows: An 80 μl sample at 8 mg/ml was injected at room temperature onto a GE Healthcare Superdex 200 Increase 10/300 column at a flow rate of 0.4 mL/min. 51 successive 1 second frames were collected through the lagging region of the SEC-elution peak that corresponded to the molecular weight (MW) of the hexamer (as assessed using multi-angle laser light scattering/refractive index measurements; MALLS/RI). The data were normalized to the intensity of the transmitted beam and radially averaged and the scattering of an appropriate solvent-blank was subtracted and the data scaled and averaged to produce the final SAXS profile displayed in this entry. CHROMIXS was used to process the SEC-SAXS data and assess the MW and Rg through the SEC peak (Rg and MW estimates and unsubtracted individual SEC-SAXS data frames are included in the full entry zip archive).

The quoted experimental MW is that determined from MALLS.

HpcH/HpaI aldolase
Mol. type   Protein
Organism   Rhizorhabdus wittichii RW1
Olig. state   Hexamer
Mon. MW   27.4 kDa
Sequence   FASTA
 
PDB ID   6R62