Abstract
A technique is described for generating large well diffracting crystals from conditions that yield microcrystals. Crystallization using this technique is both rapid (crystals appear in <1 h) and robust (48 out of 48 co-crystallized with a fragment library, compared with 26 out of 48 using conventional hanging drop). Agarose gel is used to exclude nucleation inducing elements from the remaining crystallization cocktail. The chemicals in the crystallization cocktail are partitioned into high concentration components (presumed to induce aggregation by reducing water activity) and low concentration nucleation agents (presumed to induce nucleation through direct interaction). The nucleation agents are then combined with 2% agarose gel and deposited on the crystallization shelf of a conventional vapor diffusion plate. The remaining components are mixed with the protein and placed in contact with the agarose drop. This technique yielded well diffracting crystals of lysozyme, cubic insulin, proteinase k, and ferritin (ferritin crystals diffracted to 1.43 Å). The crystals grew rapidly, reaching large size in less than one hour (maximum size was achieved in 1–12 h). This technique is not suitable for poorly expressing proteins because small protein volumes diffuse out of the agarose gel too quickly. However, it is a useful technique for situations where crystals must grow rapidly (such as educational applications and preparation of beamline test specimens) and in situations where crystals must grow robustly (such as co-crystallization with a fragment library).
Highlights
It is convenient to have a robust crystallization protocol that yields high quality protein crystals even when the mother liquor is perturbed, for example by addition of chemicals from a fragment library [1,2]
L is the predicted length of each side of the crystal, Lc is an adjustable constant that corresponds to the asymptotic value, t is the time over which crystallization has occurred, τ is an adjustable constant that corresponds to the time needed for the crystal to reach 12 Lc, and Lmax is an adjustable constant which indicates the upper limit for crystal size
We found simple and effective partitions of the crystallization cocktail for four test proteins, with some of the chemicals sequestered in an agar pellet and some present in the protein drop
Summary
It is convenient to have a robust crystallization protocol that yields high quality protein crystals even when the mother liquor is perturbed, for example by addition of chemicals from a fragment library [1,2]. We present a general technique for reliably and rapidly growing protein crystals by identifying elements that induce nucleation [3] and excluding these chemicals from the remaining crystallization solution by sequestering them in agarose gel [4]. This technique combines a vapor-diffusion approach to crystal growth [5] with a free-diffusion approach to nucleation [6]. A pellet of gelled nucleation inducing chemicals is deposited on the crystallization shelf. The solution of protein and precipitant is placed in contact with the pellet composed of gelled nucleation inducing chemicals
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