Protein crystal growth on board Shenzhou 3: a concerted effort improves crystal diffraction quality and facilitates structure determination.

The crystallization of 16 proteins was carried out using 60 wells on board Shenzhou 3 in 2002. Although the mission was only 7 days, careful and concerted planning at all stages made it possible to obtain crystals of improved quality compared to their ground controls for some of the proteins. Significantly improved resolutions were obtained from diffracted crystals of 4 proteins. A complete data set from a space crystal of the PEP carboxykinase yielded significantly higher resolution (1.46A vs. 1.87A), I/sigma (22.4 vs. 15.5), and a lower average temperature factor (29.2A(2) vs. 42.9A(2)) than the best ground-based control crystal. The 3-D structure of the enzyme is well improved with significant ligand density. It has been postulated that the reduced convection and absence of macromolecule sedimentation under microgravity have advantages/benefits for protein crystal growth. Improvements in experimental design for protein crystal growth in microgravity are ongoing.

Crystal structure of human epidermal growth factor and its dimerization.

Epidermal growth factor (EGF) is a typical growth-stimulating peptide and functions by binding to specific cell-surface receptors and inducing dimerization of the receptors. Little is known about the molecular mechanism of EGF-induced dimerization of EGF receptors. The crystal structure of human EGF has been determined at pH 8.1. There are two human EGF molecules A and B in the asymmetric unit of the crystals, which form a potential dimer. Importantly, a number of residues known to be indispensable for EGF binding to its receptor are involved in the interface between the two EGF molecules, suggesting a crucial role of EGF dimerization in the EGF-induced dimerization of receptors. In addition, the crystal structure of EGF shares the main features of the NMR structure of mouse EGF determined at pH 2.0, but structural comparisons between different models have revealed new detailed features and properties of the EGF structure.

Molecular-replacement studies of Trichosanthes kirilowii lectin 1: a structure belonging to the family of type 2 ribosome-inactivating proteins.

Trichosanthes kirilowii lectin 1 (TKL-1) isolated from the tuber of T. kirilowii consists of two chains, each with a molecular weight of about 30 kDa. It has immunological properties which are similar to some ribosome-inactivating proteins (RIPs). TKL-1 was crystallized in space group P2(1)2(1)2(1) and diffraction data were collected to 2.7 A resolution. The molecular-replacement method was applied to solve the structure, using different chains of ricin, abrin-a and trichosanthin as search models. A set of consistent solutions was further verified by R(omit) profile analysis. In addition, the spatial arrangement of the two chains of TKL-1 is identical to that of type 2 RIPs.

Crystallization and preliminary X-ray diffraction studies of human epidermal growth factor.

Human epidermal growth factor (hEGF), a 6.2 kDa protein of 53 amino acids with three internal disulfide bridges, has been crystallized by the hanging-drop method. hEGF crystallizes in space group P3(1)21 (or P3(2)21) using MgCl(2) as precipitant, with unit-cell parameters a = b = 61.4, c = 87.0 A. Another type of crystal, obtained using NaCl as precipitant, belongs to a tetragonal point group and has unit-cell dimensions a = b = 102.5, c = 166.6 A. The trigonal crystals with the smaller unit cell diffract X-rays better and a native data set from a single crystal has been collected to 3.0 A resolution.

Bound-solvent structures for microgravity-, ground control-, gel- and microbatch-grown hen egg-white lysozyme crystals at 1.8 A resolution.

A number of methods can be used to improve the stability of the protein crystal-growth environment, including growth in microgravity without an air-liquid phase boundary, growth in gels and growth under oil ('microbatch'). In this study, X-ray data has been collected from and structures refined for crystals of hen egg-white lysozyme (HEWL) grown using four different methods, liquid-liquid dialysis on Earth and in microgravity using the European Space Agency's (ESA) Advanced Protein Crystallization Facility (APCF) on board the NASA Space Shuttle Life and Microgravity Spacelab (LMS) mission (STS-78), crystallization in agarose gel using a tube liquid-gel diffusion method and crystallization in microbatch under oil. A comparison of the overall quality of the X-ray data, the protein structures and especially the bound-water structures has been carried out at 1.8 A. The lysozyme protein structures corresponding to these four different crystallization methods remain similar. A small improvement in the bound-solvent structure is seen in lysozyme crystals grown in microgravity by liquid-liquid dialysis, which has a more stable fluid physics state in microgravity, and is consistent with a better formed protein crystal in microgravity.

Protein crystal growth in microgravity using a liquid/liquid diffusion method.

Crystallization of proteins by liquid liquid diffusion method was performed in microgravity using the MDA Minilab aboard the US Space Shuttle. Three proteins, namely lysozyme, trichosanthin, and a new lechin, were crystallized in the space experiment. In contrast to the results of space experiments with a tube-like vapor diffusion method, the crystallization conditions for growing better crystals in space are remarkably different from the conditions optimized on earth. This may be due to difficulties in ground optimization, which are caused by gravity-dependent phenomena, in particular the specific convective flow occurring with liquid liquid diffusion.

The 2 A crystal structure of subtilisin E with PMSF inhibitor.

Using enzyme prepared by the DNA recombination technique, subtilisin E from Bacillus subtilis was crystallized in space group P2(1)2(1)2(1) with two molecules in an asymmetric unit. The crystal structure of PMSF-inhibited subtilisin E was solved by molecular replacement followed by refinement with the X-PLOR program. This resulted in the 2.0 A structure of subtilisin E with an R-factor of 0.191 for 8-2 A data and r.m.s. deviations from ideal values of 0.021 A and 2.294 for bond lengths and bond angles respectively. The PMSF group covalently bound to Ser221 appeared very clearly in the electron density map. Except for the active site disturbed by PMSF binding, the structural features of subtilisin E are almost the same as in other subtilisins. The calcium-binding sites are different in detail in the two independent molecules of subtilisin E. Based on the structure, the remarkably enhanced heat stability of mutant N118S of subtilisin E is discussed. It is very likely that there is an additional water molecule in the mutant structure, which is hydrogen bonded to side chains of Ser118 and its neighbouring residues Lys27 and Asp120.

Protein crystal growth in microgravity.

Protein crystal growth is quite important for the determination of protein structures which are essential to the understanding of life at molecular level as well as to the development of molecular biotechnology. The microgravity environment of space is an ideal place to study the complicated protein crystallization and to grow good-quality protein crystals. A number of crystal-growth experiments of 10 different proteins were carried out in August, 1992 on the Chinese re-entry satellite FSW-2 in space using a tube crystallization equipment made in China. A total of 25 samples from 6 proteins produced crystals, and the effects of microgravity on protein crystal growth were observed, especially for an acidic phospholipase A2 and henegg-white lysozyme which gave better crystals in space than earth-grown crystals in ground control experiments. The results have shown that the microgravity in space favors the improvement of the size, perfection, morphology and internal order of the grown protein crystals.

Protein crystallization in space.

The microgravity environment of space is an ideal place to study the complicated protein crystallization process and to grow good-quality protein crystals. A series of crystal growth experiments of 10 different proteins was carried out in space on a Chinese re-entry satellite FSW-2 in August, 1992. The experiments were performed for about two weeks at a temperature of 18.5 +/- 0.5 degrees C using a tube-like crystallization apparatus made in the Shanghai Institute of Technical Physics, Academia Sinica. More than half of 48 samples from 6 proteins produced crystals, and the effects of microgravity on protein crystal growth were observed, especially for hen-egg white lysozyme and an acidic phospholipase A2 from the venom of Agkistrodon halys Pallas. Analyses of the crystallization of these two enzymes in this mission showed that the microgravity environment in space may be beneficial to improve size, external perfection, morphology, internal order, and nucleation of protein crystals. Some of these positive microgravity effects were also demonstrated by the growth of protein crystals in gelled solution with the above two enzymes. A structural analysis of the tetragonal lysozyme crystal grown in space is in progress.

The crystal structure of silver carp insulin at medium resolution.

It is an important way of surveying the structure-function relationship of insulin to study insulins from different species. Based on the structure model of an orthorhombic crystal obtained by the molecular replacement method, the crystallographic refinement of a hexamer of silver carp insulin in an asymmetric unit has been carried out with 2.8 A resolution data using the restrained least-squares method. The comparisons of insulin structures have shown that the six silver carp insulin molecules have very similar but not identical three-dimensional structures which are similar to the known 2 Zn pig insulin structure but remarkably different in some local conformations.