Adding Size Exclusion Chromatography (SEC) and Light Scattering (LS) Devices to Obtain High-Quality Small Angle X-Ray Scattering (SAXS) Data

Graewert M, Da Vela S, Gräwert T, Molodenskiy D, Blanchet C, Svergun D, Jeffries C, Crystals 10(11):975 (2020) DOI

SASDFP8 – Carbonic anhydrase 2 from bovine erythrocytes - SEC-SAXS coupled to multiangle laser and quasi-elastic light scattering (MALLS and QELS)

Carbonic anhydrase 2
MWexperimental 28 kDa
MWexpected 29 kDa
VPorod 37 nm3
log I(s) 7.75×103 7.75×102 7.75×101 7.75×100
Carbonic anhydrase 2 small angle scattering data  s, nm-1
ln I(s)
Carbonic anhydrase 2 Guinier plot ln 7.75×103 Rg: 1.8 nm 0 (1.8 nm)-2 s2
Carbonic anhydrase 2 Kratky plot 1.104 0 3 sRg
Carbonic anhydrase 2 pair distance distribution function Rg: 1.8 nm 0 Dmax: 5.1 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Carbonic anhydrase 2 DAMMIN model
Carbonic anhydrase 2 DAMFILT model

log I(s)
 s, nm-1
Carbonic anhydrase 2 PDB (PROTEIN DATA BANK) model

Synchrotron SAXS data from carbonic anhydrase 2 in 50 mM HEPES, 150 mM NaCl, 2% v/v glycerol, pH 7 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 = 0.124 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 11.9 mg/ml was injected at a 0.50 ml/min flow rate onto a GE Superdex 75 Increase 10/300 column at 20°C. 41 successive 1 second frames were collected through the major SEC-elution peak and processed using CHROMIXS. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Carbonic anhydrase underwent pre-purification prior to SEC-SAXS using the following method. All procedures were performed at 4 oC. The protein (from Sigma; Gel Filtration Markers Kit MWGF1000) was made to approximately 25 mg/ml in 25 mM HEPES, 50 mM NaCl, 5 mM urea, 1% v/v glycerol, pH 7. Approximately 200 μl of sample were loaded onto a Superdex 75 Increase 10/300 column (GE Healthcare) equilibrated in the same buffer (flow rate = 0.4 ml/min). Fractionated aliquots corresponding to the highest absorbing peak (estimated using UV A280 and UV A245 nm) were pooled and concentrated (3 kDa centrifuge spin filter) to a final concentration of 11.9 mg/ml (the concentration was determined from triplicate UV A280 measurements using an E0.1% of 1.732 (= 1 g/l) calculated from the amino acid sequence (ProtParam)). Approximately 50 μl aliquots were snap-frozen in liquid nitrogen then stored at -80oC prior to the SEC-SAXS analysis that was performed at room temperature in 50 mM HEPES, 150 mM NaCl, 2% v/v glycerol, pH 7. The Rg-correlation through the SEC-SAXS peak, the individual unsubtracted SEC-SAXS frames as well as the results from coupled MALLS and QELS analysis are included in the full entry zip archive. The quoted experimental molecular weight was determined using MALLS in combination with refractive-index (RI) measurements that were recorded from the same sample eluting from the column using a split-flow SEC-SAXS-light scattering configuration (Graewert et al., (2015) Sci. Reports. 5, 10734: doi: 10.1038/srep10734). The average hydrodynamic radius of the protein is 2.4 nm.

Tags: benchmark
Carbonic anhydrase 2 (CA2)
Mol. type   Protein
Organism   Bos taurus
Olig. state   Monomer
Mon. MW   29.1 kDa
UniProt   P00921 (1-260)
Sequence   FASTA
PDB ID   5A25