Welcome to the Sample preparation for routine and advanced structural biology online seminar!

Hosts: Patrick Shaw Stewart and Stefan Kolek

Abstract:

Using phase diagrams with microseeding to prepare crystal samples for advanced data collection techniques

P. D. Shaw Stewart1, S. A. Kolek1, J. Stubbs2, P. F. M. Baldock1

1Douglas Instruments Ltd, East Garston, Hungerford RG17 7HD, United Kingdom, 2School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom

Serial data collection and microED techniques typically require “slurries” of tiny, well-ordered crystals [1, 2]. Neutron diffraction requires very large single crystals. Making samples for these techniques is often a complex process requiring many rounds of optimization. To guide them in this task, protein crystallizers often keep a notional phase diagram in mind, which has four zones: an undersaturated zone where protein always remains in solution, a metastable zone where crystals will grow when seeds are added, a crystal nucleation zone where crystals appear spontaneously, and a protein precipitation zone. However, the shape of real-life phase diagrams can vary, making the interpretation of experimental results difficult. For example, some protein-precipitant systems completely lack nucleation or metastable zones. It is therefore very helpful to determine the phase diagrams of individual target proteins experimentally. Douglas Instruments, in collaboration with the University of Southampton, has introduced a simple and rapid method of generating custom phase diagrams using just 15 – 60 µL of protein [Fig. 1]. The approach uses the microbatch-under-oil method to avoid concentrating the sample drop (as would occur in a vapor diffusion setup), and by carrying out the same procedure with and without a seedstock, the metastable zone can be identified [3]. Moreover, advanced methods often require relatively large sample volumes, and microbatch can be scaled up easily to 50 µL or larger “batches” using robotics. We present case studies where phase diagrams were used to increase control and crystal quality for routine and advanced data collection.

Figure 1. The rapid determination of a protein’s phase diagram using a microbatch-under-oil format. Blue circles indicate the conditions that were tested. Images of the wells are shown in conditions of interest. All points on the accessible phase diagram can be reached by mixing the three ingredients shown: protein stock (red circle), precipitant or cocktail stock (green circle) and a diluent, normally water (cyan circle). To find the border of the metastable zone (dotted line) the experiment was repeated with the addition of a seed-stock (results not shown).

[1] Stubbs, J., Hornsey, T., Hanrahan, L.B. Esteban, R. Bolton, M. Maly, S. Basu, J. Orlans, D. de Sanctis, J. Shim, Shaw Stewart, P. D., A.M. Orville, I. Tews and West, J. (2024). IUCrJ 11.

[2] Tremlett, C.J., Stubbs, J., Stuart, W.S., Shaw Stewart, P.D., West, J., Orville, A.M., Tews, I. and Harmer, N.J. (2025). IUCrJ 12.

[3] D'Arcy, A., Villard, F., Marsh, M. (2007). Acta Cryst. D63(4):550-4.