High Resolution Protein Crystals Using an Efficient Convection-Free Geometry

Abstract

Macromolecular crystallography is the most direct and accurate approach to determine the three-dimensional structure of biological macromolecules. The growth of high quality single crystals, yielding diffraction to the highest X-ray resolution, remains a bottleneck in this methodology. Here we show that through a modification of the batch crystallization method, an entirely convection-free crystallization environment is achieved, which enhances the purity and crystallinity of protein crystals. This is accomplished by using an upside-down geometry, where crystals grow at the “ceiling” of a growth-cell completely filled with the crystallization solution. The “ceiling crystals” experience the same diffusion-limited conditions as in space microgravity experiments. The new method was tested on bovine insulin and two hen egg-white lysozyme polymorphs. In all cases, ceiling crystals diffracted X-rays to resolution limits beyond that for other methods using similar crystallization conditions without further optimization. In addition, we demonstrate that the ceiling crystallization method leads to crystals with much lower impurity incorporation.

Technique for High-Quality Protein Crystal Growth by Control of Subgrain Formation under an External Electric Field

“…However, the growth of high-quality single crystals of proteins is difficult, which is an impediment to accurate elucidation of the 3D structures of protein molecules. Therefore, the establishment of crystallization techniques to obtain high-quality single crystals of proteins has been intensively pursued using magnetic fields [1][2][3][4][5][6][7][8], microgravity [9][10][11][12][13][14][15][16], electric fields [17][18][19][20][21], solution flow [22][23][24][25] and gel as a growth host media [26][27][28][29][30][31][32]. In this article, the improvement of the crystal quality of tetragonal hen egg white (HEW) lysozyme crystals as a model protein is demonstrated by applying an external electric field.…”

A practical kit for micro-scale application of the ceiling crystallisation method

“…1 So, in this section we only present a few crystals to show that the new ceiling crystallisation kit works well and gives good output.…”

“…1 In these setups, ceiling crystals heterogeneously nucleate on the cover slips. 15 Because solution evaporation must not occur during ceiling crystallisation, tight sealing is required to avoid any solution or air leakage out of or into the growth cell at the cover slip-vial interface.…”

Promising approaches to crystallization of macromolecules suppressing the convective mass transport to the growing crystal

“…This method was developed 50 in 2008 and was later improved. 51 The schematic representation of ceiling crystallization is shown in Fig. 2.…”