Methods for analysis of size-exclusion chromatography–small-angle X-ray scattering and reconstruction of protein scattering

Abstract

Size-exclusion chromatography in line with small-angle X-ray scattering (SEC-SAXS) has emerged as an important method for investigation of heterogeneous and self-associating systems, but presents specific challenges for data processing including buffer subtraction and analysis of overlapping peaks. This paper presents novel methods based on singular value decomposition (SVD) and Guinier-optimized linear combination (LC) to facilitate analysis of SEC-SAXS data sets and high-quality reconstruction of protein scattering directly from peak regions. It is shown that Guinier-optimized buffer subtraction can reduce common subtraction artifacts and that Guinier-optimized linear combination of significant SVD basis components improves signal-to-noise and allows reconstruction of protein scattering, even in the absence of matching buffer regions. In test cases with conventional SAXS data sets for cytochrome c and SEC-SAXS data sets for the small GTPase Arf6 and the Arf GTPase exchange factors Grp1 and cytohesin-1, SVD-LC consistently provided higher quality reconstruction of protein scattering than either direct or Guinier-optimized buffer subtraction. These methods have been implemented in the context of a Python-extensible Mac OS X application known as (), which provides convenient tools for data-set selection, beam intensity normalization, SVD, and other relevant processing and analytical procedures, as well as automated Python scripts for common SAXS analyses and Guinier-optimized reconstruction of protein scattering.

Coincidence detection and bi-directional transmembrane signaling control a bacterial second messenger receptor

“…To avoid complications associated with such polydisperse samples and to capitalize on the observation that the predominant species could be separated by gel filtration, these samples were injected into an in-line size-exclusion chromatography system mirroring our SEC-MALS experiments. Guinier analysis of these samples collected on the SEC-SAXS setup (Malaby et al, 2015) revealed that they were monodisperse, and were therefore suitable for further analysis. Interpretation of the SAXS data was facilitated by having experimentally determined crystallographic structures of isolated domains of LapD and by the recently developed program MEMPROT, which models a detergent corona around transmembrane domains (Pérez and Koutsioubas, 2015).…”

Structural Organization and Dynamics of Homodimeric Cytohesin Family Arf GTPase Exchange Factors in Solution and on Membranes

“…Basic SAXS Analyses and Ab Initio Modeling For the diglycine variants of Grp1 and ARNO 2-400 , Guinier analyses and dimensionless Kratky plots (Durand et al, 2010) were calculated in DELA (Malaby et al, 2015). P(r) distributions were calculated using GNOM (Svergun, 1992) in PRIMUS (Konarev et al, 2003) and MEM with a sine prior in DELA (Malaby et al, 2015(Malaby et al, , 2018. Ab initio bead envelopes were calculated using DAMMIF (Franke and Svergun, 2009) and GASBOR (Svergun et al, 2001).…”

“…Minor peaks before and after the main peak may represent a higher-order oligomer and monomer, respectively. Singular value decomposition (SVD) of the SAXS profiles from the main peak and a post-peak buffer region revealed two significant components from which a high-quality protein scattering profile was reconstructed by Guinier-optimized linear combination (SVD-LC) as described previously (Malaby et al, 2015). Guinier analysis of the low q region yielded an R G of 54.5 Å (Figure 2A; Table S2), which is approximately twice the value of 28 Å for monomeric Grp1 63-399 , which lacks the CC domain (Malaby et al, 2018).…”

Structural assembly of the human Miro1/2 GTPases based on the crystal structure of the N-terminal GTPase domain

“…Samples of HsMiro1 (residues 1-592 with a C-terminal 6xHis tag) and HsMiro2 (residues 1-588 with a C-terminal 6xHis tag), prepared in 25mM Tris pH 8.5, 500mM NaCl, 1mM MgCl2, 1mM CaCl2, 0.5 mM TCEP-HCl, and 2.5% sucrose, were injected onto the column (HsMiro1: 300 microliters at 2.2 mg/ml and 350 microliters at 5.8 mg/ml; HsMiro2: 400 microliters at 2.7 mg/ml). SAXS was performed in-line with this size exclusion column setup (Malaby et al, 2015). Photons scattered from the 12 keV X-ray beam were detected with a Dectris Pilatus 1M detector at a distance of 3.5 m from the sample.…”

“…However, in those studies of the Drosophila Miro we noted some heterogeneity in the SAXS data from the full-length protein likely arising from aggregation and/or formation of multimers. To obviate this previously noted heterogeneity, we took advantage of a SEC-SAXS facility available at the BioCAT beamline that allowed us to readily resolve scattering from dimers and aggregates from that of the monomeric full length protein (Malaby et al, 2015) (Suppl. Fig.…”