Automated pipeline for purification, biophysical and x-ray analysis of biomacromolecular solutions

Abstract

Small angle X-ray scattering (SAXS), an increasingly popular method for structural analysis of biological macromolecules in solution, is often hampered by inherent sample polydispersity. We developed an all-in-one system combining in-line sample component separation with parallel biophysical and SAXS characterization of the separated components. The system coupled to an automated data analysis pipeline provides a novel tool to study difficult samples at the P12 synchrotron beamline (PETRA-3, EMBL/DESY, Hamburg).

Figure 1. SEC-SAXS/TDA set-up at the EMBL-P12 beamline.

(a) Using a micro-splitter valve, the eluting stream is divided in equal parts enabling in-parallel SAXS and light-scattering measurements.(b) Automated SEC-SAXS pipeline trace of I(0) vs. frame number. Bovine serum albumin was analyzed without TDA detectors (SEC-SAXS; ◯), with a parallel SEC-SAXS/TDA split stream set-up (●) and in-line SEC-TDA-SAXS set-up (△). *, ** indicate the peaks corresponding to the monomeric and dimeric fraction, respectively. (c) Schematic of the P12 data processing pipeline with integrated SEC-SAXS/TDA modules.

Figure 2. Biophysical and structural characterization of Lpn-PlaB.

(a) The SEC-SAXS/TDA elution profile of Lpn-PlaB. RALS (black), RI (light gray) and the corresponding MW_RALS correlations (gray) across the elution peak. The oligomerization states are indicated and the 3D reconstructions presented. SAXS I(0) values determined from the SEC-SAXS pipeline are shown as open circles. (b+c) Determination of R_g and MW across the SAXS I(0) peak. The MW_RALS (230 ± 15 kDa) corresponds to the MW determined from the zero angle scattering by SAXS (225 ± 15 kDa). Due to weak scattering intensities only the tetrameric peak I(0) was detected automatically by the pipeline. (d) SAXS profiles of Lpn-PlaB collected in batch mode (black) and after component separation (tetramer (gray)) (e) the corresponding 3D reconstructions (inlay, as mesh and bead model, respectively).

Figure 3. Analysis of dimeric PlaB with SAXS and TDA data.

(a) SAXS scattering profiles of Lpn-PlaB collected from the tetrameric fraction (black, frames 2000-2160) and dimeric fraction (gray, frames 2350-2360). (b) 3D reconstructions of the tetrameric fraction (dark mesh) and bead model of the dimer fraction (gray). For these, the SAXS profiles shown in (a) were merged with data collected at higher concentration in batch mode (4.5 mg/ml, Fig. 2d) and 10 runs of DAMMIF were performed to generate a starting model for a final run of DAMMIN. (c) Estimation of the volume fraction of PlaB dimeric species. The volume fraction of the dimeric component in the respective SAXS frames was determined with Oligomer (crosses). For this 10 frames were averaged and fitted by distinct ratios of the form factors derived from the scattering profiles corresponding to the tetrameric and dimeric species as shown in (a). This coincides very well with the volume fractions derived from the TDA data (black curve); the % volume fraction of dimeric species was determined by the ratio (MW_tetramer – MW_RALS) to MW_dimer.