The elucidation of the three dimensional structure of biological macromolecules has provided an important contribution to our current understanding of many basic mechanisms involved in life processes. This enormous impact largely results from the ability of X-ray crystallography to provide accurate structural details at atomic resolution that are a prerequisite for a deeper insight on the way in which bio-macromolecules interact with each other to build up supramolecular nano-machines capable of performing specialized biological functions. With the advent of high-energy synchrotron sources and the development of sophisticated software to solve X-ray and neutron crystal structures of large molecules, the crystallization step has become even more the bottleneck of a successful structure determination. This review introduces the general aspects of protein crystallization, summarizes conventional and innovative crystallization methods and focuses on the new strategies utilized to improve the success rate of experiments and increase crystal diffraction quality.
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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 that influence crystallization, such as pH, ionic strength and precipitant concentration, as well as physical parameters such as temperature, sample volume and overall methodology. It also includes the application of some unique procedures and approaches, and the addition of novel components such as detergents, ligands or other small molecules that may enhance nucleation or crystal development.
Here, an attempt is made to provide guidance on how optimization might best be applied to crystal-growth problems, and what parameters and factors might most profitably be explored to accelerate and achieve success. Keywords: X-ray diffraction; additives; crystal growth; nucleation; pH; precipitants; proteins; strategy. Schematic illustration of the successive grid search strategy for protein crystallization redrawn from….
Crystals of a hexagonal canavalin, b hexagonal Turnip yellow…. Histogram showing the number of successful protein crystallizations as a function of polyethylene…. Shown here are the electrostatic surfaces of the complementarity-defining regions of two different…. Distribution of the crystallization pH and corresponding distribution of the isoelectric point of….
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Optional text in email:. Save Cancel. Create a file for external citation management software Create file Cancel. Full-text links Cite Favorites. Abstract 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. Figures Figure 1 5 Crystals obtained from an initial…. Figure 1 13 Crystals obtained from an initial screening matrix are usually unsuitable for X-ray data….
Crystals obtained from an initial screening matrix are usually unsuitable for X-ray data collection because of insufficient size, thin plate or needle morphologies, because they grow as multi-crystals and inseparable clusters or because they display obvious defects such as cracks and fissures.
Although data of marginal quality may occasionally be obtained even from crystals such as these using, for example, synchrotron microbeams, they cannot provide the high-quality data that assure an accurate and precisely determined structure. Figure 2 5 Additional examples of protein, nucleic…. Figure 2 13 Additional examples of protein, nucleic acid and virus crystals that demand optimization once…. Additional examples of protein, nucleic acid and virus crystals that demand optimization once initial conditions have been identified from screening matrixes.
Figure 3 5 Schematic illustration of the successive…. Figure 3 13 Schematic illustration of the successive grid search strategy for protein crystallization redrawn from…. On the left, components of the grid search are displayed separately. The bottom square shows the variation in pH across the columns. The square above it shows the variation in precipitant concentration in the rows.
The combination of these two layers produces the pH versus precipitant grid that serves as the basis for the two-dimensional crystallization approach. Fixed concentrations of other reagents can be added onto this grid as indicated by the upper squares labeled 1 and 2.
The diagram on the right illustrates how solution parameters are chosen using this strategy. Broad screen experiments shown at the bottom are set up using three different precipitating agents. Tight ranges of pH and precipitant concentration are centered about the conditions in the droplet yielding crystals. Figure 4 5 Twinned crystals are observed for…. Figure 4 13 Twinned crystals are observed for a cubic canavalin, b …. These are all obvious cases of twinning, where re-entry angles are evident in a , spiral arrangements in c and d and overlapping scales in b.
Figure 5 5 Crystals of a …. Figure 5 13 Crystals of a hexagonal canavalin, b hexagonal Turnip yellow…. Crystals of a hexagonal canavalin, b hexagonal Turnip yellow mosaic virus , c prismatic hexagonal concanavalin B and d rhombohedral canavalin. The crystals in a and c exhibit severe hollows at their growth ends and the crystal in b exhibits apparent re-entry angles.
These are, however, all perfect untwinned single crystals; the abnormalities are owing to transport processes of molecules in their mother liquors. Figure 6 5 Exploring drop ratios. These different…. Figure 6 13 Exploring drop ratios. These different drop ratios are plotted to show the different…. Exploring drop ratios.
These different drop ratios are plotted to show the different initial and final protein and precipitant concentrations, as well as the unique equilibration path. Figure 7 5 Above is a plot of…. Figure 7 13 Above is a plot of the average center-to-center distances of five proteins of….
The proteins are as follows: Below is a plot of the average surface-to-surface distance for the same set of proteins as a function of protein concentration. Figure 8 5 Each curve in the diagram…. Figure 8 13 Each curve in the diagram describes the solubility as its log of a…. Even though equivalent concentrations of salts having the same valences produce the same ionic strength, the curves differ markedly, illustrating the specific ion effects that a salt imposes on a protein.
It is therefore necessary to evaluate the effects of at least several salts on the crystallization of a protein. Figure 9 5 Histogram showing the number of…. Figure 9 13 Histogram showing the number of successful protein crystallizations as a function of ammonium…. Histogram showing the number of successful protein crystallizations as a function of ammonium sulfate concentration.
Figure 10 5 Histogram showing the number of…. Figure 10 13 Histogram showing the number of successful protein crystallizations as a function of methyl…. Histogram showing the number of successful protein crystallizations as a function of methyl pentanediol MPD concentration. Figure 11 5 Histogram showing the number of…. Figure 11 13 Histogram showing the number of successful protein crystallizations as a function of polyethylene….
Histogram showing the number of successful protein crystallizations as a function of polyethylene glycol concentration. Figure 12 5 This figure shows the pH…. Figure 12 13 This figure shows the pH intervals and associated buffers over which crystals were…. This figure shows the pH intervals and associated buffers over which crystals were obtained for eight proteins. The crystallization was buffer-specific for several of the samples even though several buffers were used with overlapping pH ranges.
Furthermore, as is evident here, one protein, papain, exhibited more than one pH interval for crystallization, reflecting its multiple pH-dependent solubility minima.
Figure 13 5 Shown here are the electrostatic…. Figure 13 13 Shown here are the electrostatic surfaces of the complementarity-defining regions of two different….
Optimization of Crystallization Conditions for Biological Macromolecules
Crystallization of Biological Macromolecules