How To Design Your Equilibrium SAXS Experiment

Small Angle X-ray Scattering (SAXS)

Equilibrium -

SAXS has emerged as a standard biophysical tool deployed routinely for characterizing macromolecules of biomedical interest. The relative logistical simplicity embodied in a technique that provides easy access to the size and low-resolution shape of macromolecules makes it an essential part of the biophysicists' tool kit. With the introduction of Size-Exclusion Chromatography (SEC)-SAXS at BioCAT, which ensures monodispersity of even the most biochemically challenging molecules, structural parameters such as the Radius of Gyration (Rg), Maximum Dimension (Dmax), Volume and Molecular weight estimates can be determined with a high degree of success for a large variety of samples.

There are three equilibrium data acquisition strategies available at BioCAT:

  1. SEC-SAXS -- The most common approach uses an AKTA-pure (GE Healthsciences) chromatography system equipped with a 5 loop-valve and a 5 column-valve that is also capable of automation. We also provide most generally used gel-filtration columns from GE healthsciences, namely superdex-200 increase, superdex-75 and superose-6 (both the 10/300 and 5/150 sizes), which can resolve proteins in sizes ranging from 3KDa to 5MDa from potential aggregates and other contaminants such as break down products.

  2. SEC-MALS-DLS-SAXS -- Also available is an Agilent Infinity II HPLC system which, in addition to the multi-wavelength UV detector, is combined with the HELEOS II (Wyatt Inc.) multi-angle light scattering (MALS), dynamic light scattering (DLS), and a tREX differential refractive index (dRI) detectors, which supplement the size and shape information from SAXS with reliable molecular weight estimates, hydrodynamic radius (Rh), and concentration. This method elegantly combines several biophysical tools along with the distinct advantage of minimizing sample consumption. We have a wide array of HPLC SEC columns from Wyatt manufactured specifically to suit the stringent requirements of the MALS and DLS detectors. The Agilent Infinity II also has an inbuilt auto-sampler which can load up to 166 samples without user intervention. Sample quality pre-requisites for this system are considerably more stringent than the simpler SEC-SAXS setup in approach 1 and the suitability of your sample must be determined through discussion with beam line personnel.

  3. Batch-mode -- For rare cases where sample concentration and volume are inadequate for SEC-SAXS, we are able to directly load the sample onto the SAXS flow cell (common for all three approaches) using a Hamilton syringe pump equipped with a multiway valve. We typically collect data while maintaining unidirectional flow of the sample through the capillary thus avoiding radiation damage. We also have an auto-sampler which can be deployed for large numbers of samples (in 96 well format) that need to be screened efficiently.

Selected Highlights -

The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts. O'Neil PK, Richardson LGL, Paila YD, Piszczek G, Chakravarthy S, Noinaj N, Schnell D. Proc Natl Acad Sci U S A. 2017 Jun 13;114(24):E4868-E4876. doi: 10.1073/pnas.1621179114. Epub 2017 May 30. PMID: 28559331

Stress-Triggered Phase Separation Is an Adaptive, Evolutionarily Tuned Response. Riback JA, Katanski CD, Kear-Scott JL, Pilipenko EV, Rojek AE, Sosnick TR, Drummond DA. Cell. 2017 Mar 9;168(6):1028-1040.e19. doi: 10.1016/j.cell.2017.02.027. PMID: 28283059

Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core. Gates ZP, Baxa MC, Yu W, Riback JA, Li H, Roux B, Kent SB, Sosnick TR. Proc Natl Acad Sci U S A. 2017 Feb 28;114(9):2241-2246. doi:10.1073/pnas.1609579114. Epub 2017 Feb 13.

Enzymatic hydrolysis by transition-metal-dependent nucleophilic aromatic substitution. Kalyoncu S, Heaner DP Jr, Kurt Z, Bethel CM, Ukachukwu CU, Chakravarthy S, Spain JC, Lieberman RL. Nat Chem Biol. 2016 Dec;12(12):1031-1036. doi: 10.1038/nchembio.2191. Epub 2016 Oct 3. PMID: 27694799

Crystal structures of the human elongation factor eEFSec suggest a non-canonical mechanism for selenocysteine incorporation. Dobosz-Bartoszek M, Pinkerton MH, Otwinowski Z, Chakravarthy S, Soll D, Copeland PR, Simonovi? M. Nat Commun. 2016 Oct 6;7:12941.

Identification of BECN1 and ATG14 Coiled-Coil Interface Residues That Are Important for Starvation-Induced Autophagy. Mei Y, Su M, Sanishvili R, Chakravarthy S, Colbert CL, Sinha SC. Biochemistry. 2016 Aug 2;55(30):4239-53. doi: 10.1021/acs.biochem.6b00246.

In Vitro Chromatin Assembly: Strategies and Quality Control. Muthurajan U, Mattiroli F, Bergeron S, Zhou K, Gu Y, Chakravarthy S, Dyer P, Irving T, Luger K. Methods Enzymol. 2016;573:3-41. doi: 10.1016/bs.mie.2016.01.002. Epub 2016 Feb 19. PMID: 27372747

Methods for analysis of size-exclusion chromatography-small-angle X-ray scattering and reconstruction of protein scattering. Malaby AW, Chakravarthy S, Irving TC, Kathuria SV, Bilsel O, Lambright DG. J Appl Crystallogr. 2015 Jul 8;48(Pt 4):1102-1113. eCollection 2015 Aug 1. PMID: 26306089

Structural mechanism of laforin function in glycogen dephosphorylation and lafora disease. Raththagala M, Brewer MK, Parker MW, Sherwood AR, Wong BK, Hsu S, Bridges TM, Paasch BC, Hellman LM, Husodo S, Meekins DA, Taylor AO, Turner BD, Auger KD, Dukhande VV, Chakravarthy S, Sanz P, Woods VL Jr, Li S, Vander Kooi CW, Gentry MS. Mol Cell. 2015 Jan 22;57(2):261-72. doi: 10.1016/j.molcel.2014.11.020. Epub 2014 Dec 24. PMID:25544560

Structural coupling of the EF hand and C-terminal GTPase domains in the mitochondrial protein Miro. Klosowiak JL, Focia PJ, Chakravarthy S, Landahl EC, Freymann DM, Rice SE. EMBO Rep. 2013 Nov;14(11):968-74. doi: 10.1038/embor.2013.151. Epub 2013 Sep 27. PMID: 24071720