Combining Counter-Diffusion and Microseeding to Increase the Success Rate in Protein Crystallization

Abstract

A new method of increasing the success rate in protein crystallization screening experiments by combining microseeding with counter-diffusion crystallization in capillaries (SCD) is presented. We have investigated the number of crystallization hits obtained with and without microseeding with 10 model proteins. For the cases studied, SCD generally increases the number of hits and is particularly useful when only relatively low protein concentration stocks are available, either because the stocks were prepared for, e.g., vapor diffusion experiments, or because the protein is poorly soluble. In either case, the addition of seeds becomes necessary to overcome the nucleation energy barrier so that crystal growth can take place even when the wave of supersaturation that passes along the capillary is insufficient to promote nucleation.

Introduction to protein crystallization

“…This can only be performed, however, where crystals, even poor crystals, of the protein under investigation have previously been obtained and can be manipulated to serve as seeds (Gavira et al, 2011). A common problem in macromolecular crystallization is inducing crystals to grow that have never previously been observed.…”

“…Seeding is frequently a useful technique for initiating the growth of crystals or inducing nucleation and growth at a lower level of supersaturation than might otherwise spontaneously occur. This can only be performed, however, where crystals, even poor crystals, of the protein under investigation have previously been obtained and can be manipulated to serve as seeds (Gavira et al, 2011). A common problem in macromolecular crystallization is inducing crystals to grow that have never previously been observed.…”

Capillary-Seeding Crystallization and Preliminary Crystallographic Analysis of a Solvent-Tolerant Elastase from Pseudomonas aeruginosa Strain K

“…Seeding with counter diffusion was successful in a capillary because the seeds were stable during mixing at the protein concentration in the capillary. Microseeding enhances nucleation when high protein concentrations are unavailable [19]. The crystals grew inside the capillary to 1 × 0.1 × 0.05 mm 3 using 1 M ammonium phosphate monobasic and 0.1 M sodium citrate tribasic dihydrate pH5.6 as the precipitant.…”

“…Inspired by Gavira et al [19], 10 μL of protein was filled in 0.5 mm diameter capillaries, which were dipped into 2 μL of a seeding droplet. Next, the capillary was sealed at one end with clay and another end was attached to anagarose-gel tube.…”

Choosing the Method of Crystallization to Obtain Optimal Results

“…The techniques have been miniaturised and automated to nanoscale by using robotics, special crystallisation trays, microfluidics, and micro-chips, thereby also increasing the reproducibility of the results. Moreover, combinations of techniques, such as vapour batch, gels and oils [34] as well as conbining counter-diffusion and microseeding [35,36] are increasingly being used.…”