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1.
    
Experimental evidence is presented for the first time for the development and time evolution of concentration‐depletion zones around protein crystals growing in microgravity and gelled on‐ground experiments. Crystal motion and buoyancy‐driven fluid movements as a result of residual accelerations and g‐jitters are demonstrated to have an adverse effect on the stability of these depletion zones, provoking the breakdown of their radial symmetry. These findings may explain some of the controversial results reported on the quality of single crystals grown under microgravity in previous space missions.  相似文献   

2.
    
Mosaic spread values for crystals grown in microgravity were measured using synchrotron radiation. Full width at half maximum (FWHM) values for diffraction line profiles in the range 10–20′′ (arc seconds, 1′′ = 1°/3600) were observed. These values are similar to those measured for crystals grown on earth using the gel-acupuncture method. The crystals analysed are composed of from two to five domains producing peaks having widths from 5 to 15′′. The distribution of these domains is neither homogeneous (with domains of lower quality concentrated in the centre of the crystal) nor isotropic (producing peaks whose width changes depending on the observation direction). Methodological aspects are also discussed, with special consideration of the effects of mosaic spread on the data-collection procedures for high-resolution (low-intensity) reflections.  相似文献   

3.
    
The physicochemical aspects of protein crystallization in reduced‐gravity environments (µg) have been investigated with the Advanced Protein Crystallization Facility during six space missions. This review summarizes the results, dealing with the mechanisms of nucleation and crystal growth and with the quality of the crystals that were obtained under reduced gravity as well as under normal gravity on earth. Statistical analyses of the experimental data strongly support the fact that µg has a positive effect on crystallization and on crystal quality. A comparison of experiments and theories of protein crystallization in reduced‐gravity environments is presented. Recommendations for improving the performance of protein crystallization experiments in µg and on earth are discussed.  相似文献   

4.
    
Very high quality crystals of tetragonal hen egg‐white lysozyme were grown in the Advanced Protein Crystallization Facility (APCF) on board the Space Shuttle using a modified free‐interface diffusion (FID) reactor designed ad hoc to have a longer diffusion path. This design allows the performance of true counter‐diffusion experiments. Crystals were obtained under the classical chemical conditions defined 50 y ago with NaCl as a crystallizing agent and acetate pH 4.5 as a buffer. Counter‐diffusion crystallization allows a `physical' instead of chemical optimization of growth conditions: indeed, this method screens for the best supersaturation conditions in a single trial and yields crystals of very high quality. A complete diffraction data set was collected at atomic resolution from one of these crystals using synchrotron radiation at the DESY–EMBL beamlines. The overall Rmerge on intensities in the resolution range 31–0.94 Å was 5.2% and the data were 98.9% complete. Refinement was carried out with the programs CNS and SHELX97 to a final crystallographic R factor of 12.26% for 72 390 reflections. A mean standard uncertainty in the atomic positions of 0.024 Å was estimated from inversion of blocked least‐squares matrices. 22 side chains show alternate conformations and the loop 59–75 adopts in the same crystal packing two conformations that were observed for either triclinic or tetragonal lysozyme in previous high‐resolution studies. In addition to 255 water molecules, the crystallizing agent (one hexacoordinated sodium ion and five chloride anions) participates in the ordered lysozyme hydration shell.  相似文献   

5.
    
Models of apocrustacyanin C1 were refined against X‐ray data recorded on Bending Magnet 14 at the ESRF to resolutions of 1.85 and 2 Å from a space‐grown and an earth‐grown crystal, respectively, both using vapour‐diffusion crystal‐growth geometry. The space crystals were grown in the APCF on the NASA Space Shuttle. The microgravity crystal growth showed a cyclic nature attributed to Marangoni convection, thus reducing the benefits of the microgravity environment, as reported previously [Chayen et al. (1996), Q. Rev. Biophys. 29 , 227–278]. A subsequent mosaicity evaluation, also reported previously, showed only a partial improvement in the space‐grown crystals over the earth‐grown crystals [Snell et al. (1997), Acta Cryst. D 53 , 231–239], contrary to the case for lysozyme crystals grown in space with liquid–liquid diffusion, i.e. without any major motion during growth [Snell et al. (1995), Acta Cryst. D 52 , 1099–1102]. In this paper, apocrustacyanin C1 electron‐density maps from the two refined models are now compared. It is concluded that the electron‐density maps of the protein and the bound waters are found to be better overall for the structures of apocrustacyanin C1 studied from the space‐grown crystal compared with those from the earth‐grown crystal, even though both crystals were grown using vapour‐diffusion crystal‐growth geometry. The improved residues are on the surface of the protein, with two involved in or nearby crystal lattice‐forming interactions, thus linking an improved crystal‐growth mechanism to the molecular level. The structural comparison procedures developed should themselves be valuable for evaluating crystal‐growth procedures in the future.  相似文献   

6.
    
Ｐｒｏｔｅｉｎｃｒｙｓｔａｌｇｒｏｗｔｈｈａｓｂｅｅｎｏｆｇｒｅａｔｉｍｐｏｒｔａｎｃｅｉｎｅｘｐｌｏｉｔａｔｉｏｎｏｆｓｔｒｕｃｔｕｒｅｓｏｆｂｉｏｌｏｇｉｃａｌｍａｃｒｏｍｏｌｅｃｕｌｅｓ.Ｔｈｅｍｉｃｒｏｇｒａｖｉｔｙｅｎｖｉｒｏｎｍｅｎｔｉｎｓｐａｃｅｉｓａｎｉｄｅａｌｐｌａｃｅｔｏｓｔｕｄｙｔｈｅｃｏｍｐｌｉｃａｔｅｄｐｒｏｔｅｉｎｃｒｙｓｔａｌｌｉｚａｔｉｏｎｐｒｏｃｅｓｓａｎｄｔｏｇｒｏｗｇｏｏｄｑｕａｌｉｔｙｐｒｏｔｅｉｎｃｒｙｓｔａｌｓ.Ｓｉｎｃｅｔｈｅ1980ｓ,ｎｏｔｉｃｅａｂｌ…  相似文献   

7.
    
Manganese superoxide dismutase was crystallized in microgravity with 35 PCAM experiments (Protein Crystallization Apparatus for Microgravity) on the ISS (International Space Station) from 5 December 2001 to 19 April 2002. Crystals were very large in size and could easily be seen by eye. Crystals with 0.45 × 0.45 mm cross‐sections and of up to 3 mm in length were obtained in several drops: an 80‐fold increase in crystal volume compared with the largest earth‐grown crystal. A smaller crystal (0.15 × 0.30 mm in cross‐section and 1.6 mm in length) was soaked in cryoprotectant and placed in a cryoloop. Diffraction data were collected at 100 K at the BioCARS bending‐magnet beamline. The space group was C2221, with unit‐cell parameters a = 100.64, b = 107.78, c = 179.82 Å. Diffraction spots to 1.26 Å resolution were observed. Unfortunately, the high‐resolution diffraction degraded owing to radiation damage and the resolution limit for the complete data set was 1.35 Å. It is anticipated that increasing the crystal volume and diffraction limit through microgravity crystal growth will enable several types of technically challenging structure determinations.  相似文献   

8.
    
A modified capillary‐growth method is described that has substantial advantages for standard and high‐throughput protein crystal growth. Protein‐containing drops are injected into vapor‐permeable flexible X‐ray‐transparent polyester tubing. The protein concentration in the drop increases over time by water transport through the tubing wall at a rate controlled by the wall thickness and ambient relative humidity. Unlike in conventional vapor‐diffusion growth, the evaporation rate from the drop is constant over a longer time period, providing more suitable conditions for nucleation, and can be controlled by varying the tubing thickness and surrounding humidity. In situ X‐ray diffraction can be performed at room temperature or, by flash‐cooling, at low temperatures. Compared with glass capillaries or thick‐wall plastic tubing, sealing and handling the tubing and extracting crystals are much easier.  相似文献   

9.
    
(Pro‐Pro‐Gly)10 is one of the most widely studied collagen polypeptide models. Microgravity crystal growth of (Pro‐Pro‐Gly)10 was carried out in the Advanced Protein Crystallization Facility aboard the Space Shuttle Discovery during the STS‐95 mission. Crystals were successfully grown in all experiments, using both dialysis and free‐interface diffusion methods. The quality of the microgravity‐grown crystals and of ground‐grown counterparts was assessed by X‐ray synchrotron diffraction. Microgravity‐grown crystals exhibited a significant improvement in terms of dimensions and resolution limit. As previously reported, crystals were orthorhombic, space group P212121. However, the diffraction pattern showed weak reflections, never previously measured, that were consistent with new unit‐cell parameters a = 26.9, b = 26.4, c = 182.5 Å. This allowed the derivation of a new model for the arrangement of the triple‐helical molecules in the crystals.  相似文献   

10.
    
The behavior of protein crystal growth is estimated from measurements performed at both the microscopic and molecular levels. In the absence of solutal flow, it was determined that a model that balances the macromolecular flux toward the crystal surface with the flux of the crystal surface well characterizes crystal growth observed using microscopic methods. Namely, it was determined that the model provides accurate estimates for the crystal‐growth velocities upon evaluation of crystal‐growth measurements obtained in time. Growth velocities thus determined as a function of solution supersaturation were further interpreted using established deterministic models. From analyses of crystal‐growth velocities, it was found that the mode of crystal growth varies with respect to increasing solution supersaturation, possibly owing to kinetic roughening. To verify further the hypothesis of kinetic roughening, crystal growth at the molecular level was examined using atomic force microscopy (AFM). From the AFM measurements, it was found that the magnitude of surface‐height fluctuations, h(x), increases with increasing solution supersaturation. In contrast, the estimated characteristic length, ξ, decreases rapidly upon increasing solution supersaturation. It was conjectured that the magnitude of both h(x) and ξ could possibly determine the mode of crystal growth. Although the data precede any exact theory, the non‐critical divergence of h(x) and ξ with respect to increasing solution supersaturation was nevertheless preliminarily established. Moreover, approximate models to account for behavior of both h(x) and ξ are also presented.  相似文献   

11.
    
With careful experimental design, indexed synchrotron white‐beam X‐ray topographs of ribonuclease S crystals at ambient temperature could be recorded with a definition and contrast comparable to that of monochromatic beam topographs of other proteins reported in the literature. By excluding wavelengths longer than 1 Å from the white beam with a filter, a radiation dose equivalent to that required to record about 18 topographs could be tolerated without appreciable radiation damage to the samples. Bragg angles of 0.5° or less were required to select low‐index harmonically pure reflections with high intensities and extinction lengths only several times the sample's thickness. The resulting X‐ray topographs in some cases showed topographic detail and in others showed the even featureless background that has been considered characteristic of a protein crystal of low mosaicity. The ribonuclease S crystals were well ordered single crystals of a quality comparable to other protein crystals that have been studied by X‐ray topography.  相似文献   

12.
    
Crystals of bovine adenosine deaminase (ADA) grown over a two‐week period in the presence of an inhibitor (ADA complex) were found to be of low quality for X‐ray diffraction analysis. Furthermore, ADA incubated in the absence of an inhibitor (ADA native) did not form any crystals using conventional crystallization methods. A solution‐stirring technique was used to obtain high‐quality ADA complex and ADA native crystals. The crystals obtained using this technique were found to be of high quality and were shown to have high structural resolution for X‐ray diffraction analyses. The results reported here indicate that the solution‐stirring technique promotes nucleation and improves the quality of protein crystals.  相似文献   

13.
    
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 Å. 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.  相似文献   

14.
    
Crystals of insulin grown in microgravity on Space Shuttle Mission STS‐95 were extremely well ordered and unusually large (many >2 mm). The physical characteristics of six microgravity and six earth‐grown crystals were examined by X‐ray analysis employing superfine ϕ slicing and unfocused synchrotron radiation. This experimental setup allowed hundreds of reflections to be precisely examined from each crystal in a short period of time. The microgravity crystals were on average 34 times larger, had sevenfold lower mosaicity, had 54‐­fold higher reflection peak heights and diffracted to significantly higher resolution than their earth‐grown counterparts. A single mosaic domain model could account for the observed reflection profiles in microgravity crystals, whereas data from earth crystals required a model with multiple mosaic domains. This statistically significant and unbiased characterization indicates that the microgravity environment was useful for the improvement of crystal growth and the resultant diffraction quality in insulin crystals and may be similarly useful for macromolecular crystals in general.  相似文献   

15.
    
The motion of protein drops on crystallization media during routine handling is a major factor affecting the reproducibility of crystallization conditions. Drop stability can be enhanced by chemical patterning to more effectively pin the drop's contact line. As an example, a hydrophilic area is patterned on an initially flat hydrophobic glass slide. The drop remains confined to the hydrophilic area and the maximum drop size that remains stable when the slide is rotated to the vertical position increases. This simple method is readily scalable and has the potential to significantly improve the outcomes of hanging‐drop and sitting‐drop crystallization.  相似文献   

16.
    
A numerical model of the equilibration of a hanging-drop experiment has been developed and tested. To obtain accurate calculations with a given precipitant, the vapor pressure of water over water/precipitant solutions must be known for various concentrations of the precipitant. The calculations of the model are in excellent agreement with all available experimental data on hanging-drop equilibration when the necessary vapor pressures are known (ammonium sulfate and sodium chloride). By varying each of the relevant rate constants in the model, the rate-limiting step in the equilibration of a hanging drop is determined. This analysis clearly shows that the rate-limiting step is the diffusion of water vapor from the drop to the reservoir, which agrees with experimental findings. Since the diffusion of water vapor is the rate-limiting step, there is virtually no precipitant concentration gradient in the drop during equilibration. As a result, there is no gravity-induced convection owing to the equilibration. Thus, whereas gravity might have an effect during crystal growth, gravity does not affect the equilibration rate of a hanging-drop experiment to a significant extent, and the diffusion of water vapor will remain the rate-limiting step in the absence of gravity. Finally, the effects of several of the parameters, such as initial drop volume, drop-to-reservoir distance and temperature, are considered quantitatively. The equilibration rate was found to vary nearly linearly with drop volume. The equilibration rate decreases roughly by a factor of three as the temperature decreases from 293 to 276 K. This decrease in the equilibration rate is greater than would be expected when just considering the change in the diffusion coefficient of water vapor in air. This large dependence can, however, be attributed to the change in water-vapor pressure. Most surprisingly, a linear dependence on drop-to-reservoir distance is found, a result that agrees very well with experiment.  相似文献   

17.
    
In attempts to obtain protein crystals of a sufficient size for structural studies, lack of knowledge of the physicochemical properties of protein solutions and of their crystal-growth behaviour lead to a bottleneck for drug design as well as for X-ray crystallography. Most formal investigations on crystal-growth phenomena have been focused on equilibrium studies, where the protein is soluble, and on the kinetics of crystal growth, which is related to both nucleation and crystal-growth phenomena. The aim of this work is to measure the crystal-growth rate along a capillary tube used as a growing cell. These experiments were carried out using the gel-acupuncture technique [García-Ruiz et al. (1993). Mater. Res. Bull. 28 , 541–546; García-Ruiz & Moreno (1994). Acta Cryst. D 50 , 484–490; García-Ruiz & Moreno (1997). J. Cryst. Growth, 178 , 393–401]. Crystal-growth investigations took place using lysozyme and thaumatin I as standard proteins. The maximum average growth rate obtained in the lower part of the capillary tube was about 35 Å s−1 and the minimum average growing rate in the upper part of the capillary tube was about 8 Å s−1. The crystal-growth rate as a function of the supersaturation was experimentally estimated at a constant height along the capillary tube.  相似文献   

18.
    
Protein crystallization screens frequently yield salt crystals as well as protein crystals. A simple method for determining whether a crystal is composed of salt or macromolecules is suggested. A drop containing one or more crystals is transferred to a glass cover slip and the cover slip is then passed through the flame of a Bunsen burner. Macromolecule crystals are destroyed by this treatment, while salt crystals generally remain. The test can be performed after other commonly used tests such as crushing and staining.  相似文献   

19.
    
Mass transport takes place within the mesoscopic to macroscopic scale range and plays a key role in crystal growth that may affect the result of the crystallization experiment. The influence of mass transport is different depending on the crystallization technique employed, essentially because each technique reaches supersaturation in its own unique way. In the case of batch experiments, there are some complex phenomena that take place at the interface between solutions upon mixing. These transport instabilities may drastically affect the reproducibility of crystallization experiments, and different outcomes may be obtained depending on whether or not the drop is homogenized. In diffusion experiments with aqueous solutions, evaporation leads to fascinating transport phenomena. When a drop starts to evaporate, there is an increase in concentration near the interface between the drop and the air until a nucleation event eventually takes place. Upon growth, the weight of the floating crystal overcomes the surface tension and the crystal falls to the bottom of the drop. The very growth of the crystal then triggers convective flow and inhomogeneities in supersaturation values in the drop owing to buoyancy of the lighter concentration‐depleted solution surrounding the crystal. Finally, the counter‐diffusion technique works if, and only if, diffusive mass transport is assured. The technique relies on the propagation of a supersaturation wave that moves across the elongated protein chamber and is the result of the coupling of reaction (crystallization) and diffusion. The goal of this review is to convince protein crystal growers that in spite of the small volume of the typical protein crystallization setup, transport plays a key role in the crystal quality, size and phase in both screening and optimization experiments.  相似文献   

20.
将拟南芥AtCBL9基因构建入原核表达载体pET-22b(+),在大肠杆菌中得到大量表达,用亲和层析和分子筛排阻层析2种方法对表达蛋白进行纯化。SDS-PAGE和动态光散射分析结果显示,AtCBL9蛋白在体外主要以单体和二体形式存在,单体和二体为同一蛋白,并具有较高的纯度。从单体蛋白中筛选出了微晶,为AtCBL9晶体生长优化及其最终结果解析和功能阐明奠定了基础。  相似文献   

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