Macromolecular Solution Small-Angle X-ray Scattering (SAXS)

A significant number of proteins can not be crystallized. For those proteins, it is possible to acquire valuable structural information (size and shape) by analyzing the manner in which they scatter X-rays known as Small Angle X-ray Scattering (SAXS). This technique is used to study how the macromolecules in our cells interact with each other and function as “molecular machines.”

Basic information obtained from the typical SAXS experiment is radius of gyration (Rg) and the electron pair distance distribution function (P(r)), which in turn can be used to generate ab initio low-resolution molecular envelopes of macromolecules in solution.

Equilibrium SAXS

The BioCAT standard experimental set-up includes a Pilatus 1M detector from Dectris (Switzerland) and a camera of 3.5 m sample-to-detector distance to access a range of momentum transfer, q, from ~0.004 to 0.38 Å-1. This range of q allows not only accurate determination of radius of gyration, but also detailed modeling using ab initio and rigid body approaches. A temperature controlled quartz capillary (1.5 mm ID, 1.52 mm OD) flow cell is used for sample measurement.

The BioCAT beamline provides the following three modes of equilibrium SAXS.


The standard mode of SAXS data collection uses in-line Size Exclusion Chromatography (SEC) coupled to SAXS (SEC-SAXS). The sample runs through a size exclusion column to separate potential aggregates or different oligomeric states immediately before flowing through the capillary for X-ray exposure.

BioCAT provides an AKTA Pure FPLC with two pumps, column and loop valves, and a multi-wavelength UV detector. The beamline also has the following GE columns available for users, though users are encouraged to bring their own columns to address potential cross contamination and reproducibility issues:

  • Superdex 200 Increase, both 10/300 and 5/150 (MW ~10-600 kDa)
  • Superdex 75, both 10/300 and 5/150 (MW ~3-70 kDa)
  • Superose 6, 10/300 (MW ~5-5,000 kDa)


BioCAT also provides a data collection mode where SEC is coupled to MALS (multi-angle light scattering), DLS (dynamic light scattering), and RI (refractive index) detectors in addition to the SAXS flow-cell. The additional light scattering detectors provide accurate measurement of molecular weight, which is often hard to determine via SAXS methods. The measurement of the hydrodynamic radius in combination with Rg from SAXS, informs on the particle shape. SAXS and MALS-DLS-RI are measured on the same SEC elution, which is split into 2 separate streams, one of which goes directly to the SAXS flow-cell and the other through the UV, MALS / DLS, and RI detectors in that order thus minimizing dilution due to a long fluid trajectory from the column to the SAXS flow-cell. This approach provides all of the sample prep benefits of SEC-SAXS and eliminates possible ambiguity about differences between non-identical SAXS and MALS-DLS measurements. Sample quality pre-requisites for this system are considerably more stringent than the simpler SEC-SAXS setup and the suitability of your sample must be determined through discussion with beamline personnel.

BioCAT provides an Agilent Infinity II HPLC, a Wyatt DAWN HELEOS II MALS+DLS (17 channels LS, plus 1 DLS) detector, and a Wyatt Optilab T-rEX dRI detector. The beamline also has the following Wyatt columns available for users:

  • 010S5 100Å (MW range 0.1-100 kDa)
  • 015S5 150Å (MW range 0.5-150 kDa)
  • 030S5 300Å (MW range 5-1,250 kDa)

Batch Mode

Batch mode samples are directly loaded into the sample cell, rather than first passing through a sizing column. This reduces the volume and concentration required, but aggregates and other large species are not separated from the sample, increasing requirements on sample prep. At BioCAT, these measurements take ~100 µl of sample. However, in some cases smaller volumes can also yield usable SAXS data. In order to reduce radiation damage, the sample flows through or oscillates in the X-ray beam using a Hamilton programmable dual-syringe pump.

Time-Resolved SAXS

The high flux and efficient detectors at BioCAT enable time-resolved SAXS experiments that investigate the dynamic behavior of the macromolecules during processes such as protein and RNA folding, and enzyme-substrate/co-factor binding. BioCAT provides two different modes of time-resolved SAXS.

Continuous Flow

BioCAT has been developing advanced microfluidic mixers, including a chaotic/turbulent mixer and a laminar flow mixer, to collect SAXS data on reactions as fast as ~100 µs. Rapid mixing devices for SAXS have fallen into two broad categories — chaotic/turbulent and laminar. These devices facilitate rapid and efficient mixing events between multiple fluid streams containing the biological macromolecule of interest and small solutes that engender structural changes in the macromolecule.

Laminar mixing utilizes hydrodynamic focusing to reduce the central flow channel to a narrow (typically ~0.1-10 µm) sheath. A version of this mixer is currently under development at BioCAT. For chaotic/turbulent mixing, chaotic/turbulent flow breaks the solution into eddies small enough for reactants to diffuse rapidly. Chaotic/turbulent flow mixers have the advantage of being able to use all of the delivered photon flux in the X-ray beam for the best signal to noise ratio. The plug flow in this kind of mixer also makes the reaction time uniform orthogonally to the flow-direction thus making its incorporation into a SAXS camera quite straight forward. In its current iteration, the BioCAT mixer can access time regimes as low as 50µs and a complete experiment can be performed with as little as 10 mg of sample.

Currently experiments are collaborations with beamline staff, and users are encouraged to discuss possible experiments with the SAXS scientific contact.

Stopped Flow

The BioCAT stopped flow setup uses a Biologic SFM-400 stopped flow mixer with an MEC 22998 micro-volume mixer, allowing 0.5 ms dead time, and an x-ray observation cell. Because of the limitations in time resolution and possibility of radiation damage, unless you specifically know your experiment requires stopped flow mixing, BioCAT recommends using the continuous flow systems.

Instrumentation for SAXS

In addition to the instrumentation described above, BioCAT has a fully equipped wet lab for sample preparation. In addition to the beamline instrumentation described elsewhere, a set of in-vacuum JJ x-ray slits are used as the collimating beam slits, and a set of in-vacuum Xenocs scatterless x-ray slits are used as the guard slits. An in-line sample camera is located just after the guard slits, using a mirror with a 6 mm through hole for the x-ray beam. BioCAT also has two ISCO model 500D and four Harvard Instrument model PHD 4400 programmable, high-pressure pumps for the continuous flow mixer project. Normalization of data is done using an ion chamber just downstream of the collimator slits.