Prog. Biophys. Mol. Biol. 101 (2009) 13-25 (original) (raw)
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Crystal growth of proteins, nucleic acids, and viruses in gels
Progress in Biophysics and Molecular Biology, 2009
Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.
Journal of Crystal Growth, 1999
Quasi-planar wave re#ection pro"le and X-ray topography studies have been done to characterize the mosaicity of solution-and gel-grown crystals of three proteins, turkey egg-white (TEW) lysozyme, thaumatin, and a bacterial aspartyl-tRNA synthetase (AspRS) as well as of one virus, tomato bushy stunt virus (TBSV). These materials are representative of a large range of molecular weight, overall particle shapes, crystals habits, packings, and solvent contents. Measurements of the full-width at half-maximum (FWHM) of re#ections show that these di!erent crystals have all a weak mosaicity. Topographs display the same features as those of the well-studied hen egg-white (HEW) lysozyme crystals: misorientation generated at the seed level for TEW lysozyme or thaumatin crystals and/or strains at growth sector boundaries for AspRS crystals. No growth defects are evidenced for TBSV crystals. For the study of crystals di!racting at lower resolution (AspRS and virus), a less absorbant sample holder, which facilitates crystal positioning in the X-ray beam, has been developed. The results obtained for solution-and gel-grown crystals do not show important di!erences. However, for TEW lysozyme and thaumatin crystals, one notices a larger dispersion of results in the solution case and an overall tendency for improved reproducibility of quality for gel-grown crystals.
Advances in Crystal Growth Techniques of Biological Macromolecules
Revista de la Sociedad Química de Mexico, 2005
The structural knowledge of some biological macromolecules helps to understand their mechanisms of working and their role on health sciences, food science and even their effect on the economy. In this work, some recent solutions to crystallogenesis for structural analysis (convective transport, counter-diffusion, the challenge of membrane protein crystallization, and high throughput techniques) are described. Finally, investigations on microgravity, crystal growth under magnetic and electric fields, as well as crystal growth in mesophases (lipid membranes) and microfluidics are carefully revised.
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
Post-crystallization treatments for improving diffraction quality of protein crystals
Acta Crystallographica Section D Biological Crystallography, 2005
X-ray crystallography is the most powerful method for determining the three-dimensional structure of biological macromolecules. One of the major obstacles in the process is the production of high-quality crystals for structure determination. All too often, crystals are produced that are of poor quality and are unsuitable for diffraction studies. This review provides a compilation of post-crystallization methods that can convert poorly diffracting crystals into data-quality crystals. Protocols for annealing, dehydration, soaking and cross-linking are outlined and examples of some spectacular changes in crystal quality are provided. The protocols are easily incorporated into the structure-determination pipeline and a practical guide is provided that shows how and when to use the different post-crystallization treatments for improving crystal quality.
High Resolution Imaging as a Characterization Tool for Biological Crystals
Annals of the New York Academy of Sciences, 2004
A BSTRACT : Biomolecular crystals consist of large unit cells that form a rather flexible medium that is able to accommodate a certain degree of lattice distortion, leading to several interesting issues ranging from structural to physical properties. Several techniques, from X-ray diffraction to microscopy, have been adapted to study the structural and physical properties of biomolecular crystals systematically. The use of synchrotron-based monochromatic X-ray diffraction topography, with triple axis diffractometry and rocking curve measurements, to characterize biomolecular crystals is reviewed. Recent X-ray diffraction images from gel and solution grown lysozyme crystals are presented. Defect structures in these crystals are discussed, together with reciprocal space mapping, and compared with results obtained from crystals grown in a low gravity environment.
Acta Crystallographica Section D Biological Crystallography, 2005
A microscope for quantitative analysis of the birefringence properties of samples is introduced. The microscope is used to measure variations in the slow optical axis position (SOAP) across hen egg-white lysozyme, glucose isomerase and ®bronectin crystals. By comparing these variations with indicators of diffraction quality, it is shown that the optical properties of a protein crystal provide a non-invasive method of determining crystal diffraction quality before any X-ray data collection is attempted.
Optimization of crystallization conditions for biological macromolecules
Acta crystallographica. Section F, Structural biology communications, 2014
For the successful X-ray structure determination of macromolecules, it is first necessary to identify, usually by matrix screening, conditions that yield some sort of crystals. Initial crystals are frequently microcrystals or clusters, and often have unfavorable morphologies or yield poor diffraction intensities. It is therefore generally necessary to improve upon these initial conditions in order to obtain better crystals of sufficient quality for X-ray data collection. Even when the initial samples are suitable, often marginally, refinement of conditions is recommended in order to obtain the highest quality crystals that can be grown. The quality of an X-ray structure determination is directly correlated with the size and the perfection of the crystalline samples; thus, refinement of conditions should always be a primary component of crystal growth. The improvement process is referred to as optimization, and it entails sequential, incremental changes in the chemical parameters tha...
Growth and Characterization of High-quality Protein Crystals for X-ray Crystallography
Annals of the New York Academy of Sciences, 2009
Tetragonal hen egg white lysozyme is grown by the batch method in solution and gel media to study the influence of high magnetic fields on the quality of macromolecular crystals. The crystallographic quality of crystals grown in the absence and in the presence of 7-and 10-T fields are analyzed in terms of mosaicity and high-resolution X-ray imaging methods. Crystals grown by the batch method from solution showed a remarkable enhancement of the crystallographic quality, although the overall crystal quality was higher for gel-grown crystals than solution-grown crystals. The observed improvement in crystal quality can be attributed to the suppression of convective transport during the crystal growth process and the control of the nucleation kinetics by the use of a magnetic force.