Abstract
High-throughput imaging of protein crystallization experiments with ultraviolet (UV) light has recently become commercially available and can enable crystallographers to differentiate between crystals of protein and those of salt, as the visualization of protein crystals is based on intrinsic tryptophan fluorescence. Unfortunately, UV imaging is not a panacea, as some protein crystals will not fluoresce under UV excitation and some salt crystals are UV-fluorescently active. As a new technology, there is little experience within the general community on how to use this technology effectively and what caveats to look out for. Here, an attempt is made to identify some of the common problems that may arise using UV-imaging technology by examining test proteins, common crystallization reagents and a range of proteins by assessing their UV-Vis absorbance spectra. Some pointers are offered as to which systems may not be appropriate for this methodology.
Highlights
The primary technique used to generate three-dimensional atomiclevel structural information from biomacromolecules, X-ray crystallography, requires the production of suitable crystals for diffraction analysis
A bright fluorescent spot does not immediately mean that a crystal has been located, as other phases of the protein can lead to local high concentrations; in particular, we find that collapsed bubbles can lead to features in a UV image akin to those of crystals
A similar process can occur over the surface of the crystallization drop itself; as the drop equilibrates, the Proteins may form absorption layers (PALs) becomes visible as a wrinkled skin
Summary
The primary technique used to generate three-dimensional atomiclevel structural information from biomacromolecules, X-ray crystallography, requires the production of suitable crystals for diffraction analysis. The production of crystals is laborious and, even more discouragingly, highly stochastic (Newman et al, 2012). The rate of false negatives in identifying protein crystals using the human eye reaches 20% (Cumbaa & Jurisica, 2010), and given a success rate of less than 1% overall (Newman et al, 2012), this rate of false negatives becomes intolerable. A false negative may be a result of the loose arrangement of protein molecules in a crystal lattice (Matthews, 1968), which incorporates large solvent channels. The solvent channels can create protein crystals with a refractive index close to that of the mother liquor in which they grew, rendering them invisible under visible light. False positives, where objects in crystallization trials are interpreted as protein crystals despite not being so, are less problematic, but many a crystallographer has wasted significant resources following such spurious leads
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