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. To learn more about designing and carrying out SAXS experiments at BioCAT see our user guides.
The BioCAT standard experimental set-up includes a Pilatus3 X 1M detector from Dectris and a camera of ~3.7 m sample-to-detector distance at 12 keV to access a range of momentum transfer, q, from ~0.0045 to 0.36 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 (1.0 mm ID) flow cell with coflow sample geometry is used for batch mode SAXS, SEC-SAXS and SEC-MALS-SAXS sample measurement. The coflow setup minimizes radiation damage on the sample, ensuring optimum signal to noise and baseline stability for consistently high quality data. Temperature control for the entire system (both the SEC and SAXS instruments) is available from 10 to 55 C upon request.
The BioCAT beamline provides the following four 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 Increase, both 10/300 and 5/150 (MW ~3-70 kDa)
- Superose 6 Increase, both 10/300 and 5/150 (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 goes through the UV, MALS/DLS, and RI detectors and then 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 separate SAXS and MALS-DLS measurements. Sample quality prerequisites 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 two Agilent Infinity II HPLCs each with 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 columns available for users:
- Superdex 200 Increase 10/300 (MW ~10-600 kDa)
- Superdex 75 Increase 10/300 (MW ~3-70 kDa)
- Superose 6 Increase 10/300 (MW ~5-5,000 kDa)
- Wyatt 010S5 100Å (MW range 0.1-100 kDa)
- Wyatt 015S5 150Å (MW range 0.5-150 kDa)
- Wyatt 030S5 300Å (MW range 5-1,250 kDa)
Ion exchange chromatography (IEC) allows separation of particles by charge rather than size, making it useful in cases where SEC cannot resolve between different components in solution. BioCAT offers IEC in-line with SAXS (IEC-SAXS) for samples that are not separable by SEC-SAXS. Because IEC requires a changing buffer during elution (typically a slope or step gradient in salt or pH), data analysis is more involved than for SEC-SAXS, but analysis algorithms are now widely available, making this a routine technique at the beamline. These experiments are more involved, and often require some work to optimize an appropriate gradient, so it is important to discuss your potential IEC-SAXS experiments with beamline personnel before requesting beamtime.
The AKTA Pure used for SEC-SAXS is also capable of IEC-SAXS using the two pumps to create the necessary gradient for elution. BioCAT also has the following columns available for users:
- HiTrap Q HP anion exchange, 1 mL and 5 mL
- HiTrap SP HP cation exchange, 1 mL and 5 mL
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 ~50 µl of sample. However, in some cases smaller volumes can also yield usable SAXS data. These experiments are also done in the coflow sample cell, to minimize radiation damage.
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.
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 available at BioCAT and can provide access to time ranges from ~1 ms to 1.5 s. These experiments use modest amounts of sample, ~1-10 mg per time series (~30 time points).
In chaotic/turbulent mixing, chaotic/turbulent flow breaks the solution into eddies small enough for reactants to diffuse rapidly. Mixing can be much more rapid than in laminar flow mixers, but requires much higher flow rates. In its current iteration, the BioCAT mixer can access time regimes as low as ~80 µs and a complete experiment can be performed with 10-100 mg of sample.
Currently experiments are collaborations with beamline staff, and users are encouraged to discuss possible experiments with the SAXS scientific contact.
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 scatterless in-vacuum JJ x-ray slits are used as the collimating beam slits, and a two sets 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 active beamstop which uses indirect detection on a photodiode.