Plastic-embedded protein crystals

Abstract

Rapid vitrification followed by the replacement of the vitrified water by a solvent (freeze substitution) and then resin is a widely used procedure for preparing biological samples for electron microscopy. The resulting plasticembedded samples permit convenient room-temperature sectioning (microtomy) and can yield well preserved cellular structures. Here this procedure has been applied to crystalline protein samples, and it is shown that it is possible to freeze-substitute vitrified crystals while preserving some of their original diffraction properties. The plastic-embedded crystals were used to collect a series of complete room-temperature data sets at a powerful macromolecular crystallography synchrotron beamline. Whereas one normally observes specific damage to disulfide bonds upon X-ray radiation, no such damage was seen for the plastic-embedded sample. The X-ray diffraction data allowed an initial atomic analysis to be made of the effects of freeze-substitution and plastic embedding on biological samples.

Progress in research into radiation damage in cryo-cooled macromolecular crystals

“…One of these is in specimen preparation, and Ravelli et al (2007) describe first attempts to embed a protein crystal in plastic using techniques developed for electron microscopy samples.…”

Preservation of high resolution protein structure by cryo-electron microscopy of vitreous sections

“…These studies generally showed high resolution detail (up to ∼1 nm) in the uncut block, but much worse preservation in the subsequent sections, suggesting that cutting is a major source of damage [7–9]. Moreover, a recent X-ray diffraction study demonstrated reflections to better than 6 Å from crystals of lysozyme embedded in plastic, thus showing that embedded protein can be very well preserved and again consistent with the idea of damage due to cutting [10].…”

Data storage based on photochromic and photoconvertible fluorescent proteins

“…Whatever the process involved, having crystals that easily photoconvert from green to red in dried conditions could potentially be a great advantage in terms of robustness of the biological matter used as optical data storage media. Since there would be no need to maintain and control the humidity in the sample, we could even envisage preserving and embedding single crystals inside a plastic polymer matrix as it has been already shown to be possible (Ravelli et al, 2007).…”