Growth of shaped single crystals of proteins (original) (raw)
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Acta Crystallographica Section D Biological Crystallography, 1999
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. D50, 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 ...
Growth kinetics of protein single crystals in the gel acupuncture technique
Journal of Crystal Growth, 1997
The growth of single crystals of tetragonal HEW lysozyme and thaumatin I into glass capillaries was monitored by time lapse video-microscopy. The crystals were obtained by unidirectional transport of the precipitating agent through capillaries of internal diameter ranging from 0.2 to 1.2 mm, using the gel acupuncture technique. For crystals growing from true protein solutions, the measured average growth rates varies with capillary diameter from 1.7 to 3.7 Å/s for thaumatin and from 2.8 to 22 Å/s for lysozyme. The measured average growth rates for crystals growing into gelled protein solutions were 1.8 Å/s for thaumatin and 2.5 Å/s for lysozyme. The trend in the variation of the growth rate with time is similar and suggests that, for capillaries with internal radius lower than 0.8 mm, diffusion dominates the global mass transport control. However, the existence of convection rolls near the crystal-solution interface and close to zones with high density gradient cannot be discarded.
Crystal quality of lysozyme single crystals grown by the gel acupuncture method
Materials Research Bulletin, 1993
Lysozyme single crystals up to 2.0 mm in size have been grown from solution into capillaries which are previously punctuated into a gel layer through which the precipitant agent diffuse. These large single crystals show an excellent optical quality and it is demonstrated by in situ X-ray diffraction that they diffract up to 1.8-1.9 A.
Investigations on gravity influence upon protein crystallization by the gel acupuncture technique
Journal of Crystal Growth, 1999
Most investigations on biological macromolecules are important for the knowledge of the functions in living organisms. Nowadays it is well known that the three-dimensional structure of proteins is obtained either by NMR or X-ray crystallographic methods. The crucial part in the latter is the availability of high-quality crystals in order to perform structural characterization. Nevertheless, there are some approaches to overcome this problem from the statistical [Jancarik and Kim, J. Appl. Crystallogr. 24 (1991) 409] and physico-chemical point of view [Rie`s-Kautt and Ducruix, Methods in Enzymology 276 Part A, Ch. 3, 1997, p. 23]. Once the crystals are obtained, the following part of the research must be focused on growing the crystal in order to have an appropriate size for X-ray analysis. There are additional advances in the methods for crystallizing, growing and determining what kind of biophysical or biochemical parameters have to be taken into account in order to obtain a high quality protein crystal, these advances have been already published elsewhere [Ducruix and Giege´, Crystallization of Nucleic Acids and Proteins. A Practical Approach, IRL Press, Oxford, 1991; McPherson, The Preparation and Analysis of Protein Crystals, Wiley, New York, 1982]. In order to evaluate these parameters, we have developed a new technique, called the gel acupuncture technique for crystallizing proteins inside an X-ray capillary tube as well as for trying to study the "in situ" crystal growth phenomena [Garcı´a-Ruiz et al., Mater. Res. Bull. 28 (1993) 541; Garcı´a-Ruiz and Moreno, Acta Crystallogr. D 50 (1994) 484]. In this work, we present our recent investigations on the influence of the gravity vector upon protein crystallization. Three proteins were chosen in order to test this possible influence, taking into account the size of each: satellite tobacco mosaic virus (1000 kDa) and two proteins of "low molecular weight", thaumatin I (22 kDa) and concanavalin A (200 kda). The experimental set-up was the same for the gel acupuncture technique [Garcı´a-Ruiz et al., Mater. Res. Bull. 28 (1993) 541] and the experiments were carried out ranging from 0°(parallel) to 180°(anti-parallel) to the gravity vector to check its influence on protein crystallization. Thus, the counter-diffusion phenomena in protein crystallization were evaluated from parallel, 45°, 90°up to anti-parallel transport of the protein molecules to the gravity vector. Finally, we discussed why the molecular weight of the protein system should be taken into account in order to avoid a sedimentation 0022-0248/99/$ -see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 -0 2 4 8 ( 9 8 ) 0 0 8 3 5 -5 phenomena and how the crystal growth technique is determined in order to keep the same diffusion pattern throughout the experiment.
Combination of oils and gels for enhancing the growth of protein crystals
Journal of Applied Crystallography, 2002
This note focuses on two different ways of enhancing the use of gels in protein crystallization by applying oils to the trials. Using a ®ve-channel motorized syringe setup, crystals were grown in gelled microbatch drops under oil and compared with those grown under similar conditions in standard microbatch drops. The advantage of this technique over existing gel techniques is that numerous trials can be dispensed automatically, while consuming very small quantities of protein. The application of oil to improve the gel acupuncture technique was also investigated; crystal growth in the presence of an oil barrier was slower than in its absence, giving rise in each case to a single large crystal with no precipitation nor smaller crystals in the capillary.
Protein crystal quality studies using rod-shaped crystals
Journal of Crystal Growth, 1996
Lysozyme single crystals were grown into X-ray capillaries to a size larger than the capillary diameter thus filling it. The two ends of the same crystal grow at different rates, the difference being at least one order of magnitude. These rod-shaped crystals allow ideal diffraction experiments to test crystal quality as a function of the growth rate. In situ X-ray diffraction experiments were carded out using the capillary where the crystal grew. Oscillation pictures yield different values of the maximum resolution level, ranging from 2.5 to 1.2 A for the opposite ends of the crystal suggesting a large influence of growth rate on protein crystal quality. * Corresponding
Materials Research Bulletin, 1998
Large tetragonal hen egg white lysozyme single crystals (up to 16 mm 3 ) can be obtained by the counter-diffusion method, using high concentration silica gels. The protein crystal lattice is able to incorporate large amounts of silica while still maintaining its short-range crystallographic order. The crystal morphology is controlled by the concentration of the silica gel, which can reduce surface energy anisotropy to such an extent that spherical single crystals can be obtained as growth forms. The mechanical properties and the stability of the crystals against dehydration are improved by the incorporated hydrophilic silica polymeric network. This makes it possible to record full diffraction data sets with a resolution better than 1.5 Å from crystals glued to glass fibers. Such reinforcement of the crystals facilitates their handling at ambient conditions and opens new possibilities for the measurement of physical properties of large biological macromolecules as well as for their technological applications.
Protein crystal quality in diffusive environments and its evaluation
Journal of crystal growth, 2003
We have analyzed the crystal quality along a capillary by a precise protocol that comprises the study of tetragonal lysozyme cylindrical crystals that fill the capillary diameter (i.e. rods), the careful definition of the diffraction parameters and the use of a single software for the data reduction in order to avoid any bias in the comparison of the quality of different data sets. Our results cannot be explained on the basis of the different redundancy of the data sets and they demonstrate that the gel acupuncture method promotes a gradient of supersaturation along the capillary that yields in the same experiment crystals of increasing quality as a function of the position. However, despite being single crystals, rods have regions that show different crystal quality because they grew at different supersaturations. Our data are in agreement with the existence of a relation between length of the c-axis and crystal quality reported by other groups, but a deeper analysis of the cell parameters reveals the existence of a significant linear relation (R ¼ 0:87) with the c/a-axis ratio. This result points to the hypothesis of an ideal unit cell that yields the best crystals in terms of I=sðIÞ: r