Structural changes in a cryo-cooled protein crystal owing to radiation damage

Abstract

The high intensity of third‐generation X‐ray sources, along with the development of cryo‐cooling of protein crystals at temperatures around 100 K, have made it possible to extend the diffraction limit of crystals and to reduce their size. However, even with cryo‐cooled crystals, radiation damage becomes a limiting factor. So far, the radiation damage has manifested itself in the form of a loss of overall diffracted intensity and an increase in the temperature factor. The structure of a protein (myrosinase) after exposure to different doses of X‐rays in the region of 20 × 1015 photons mm−2 has been studied. The changes in the structure owing to radiation damage were analysed using Fourier difference maps and occupancy refinement for the first time. Damage was obvious in the form of breakage of disulfide bonds, decarboxylation of aspartate and glutamate residues, a loss of hydroxyl groups from tyrosine and of the methylthio group of methionine. The susceptibility to radiation damage of individual groups of the same kind varies within the protein. The quality of the model resulting from structure determination might be compromised owing to the presence of radiolysis in the crystal after an excessive radiation dose. Radiation‐induced structural changes may interfere with the interpretation of ligand‐binding studies or MAD data. The experiments reported here suggest that there is an intrinsic limit to the amount of data which can be extracted from a sample of a given size.

Monitoring Structural Transformations in Crystals. Part 15. Structural Changes in Crystals Caused by UV Radiation Despite the Lack of a Photochemical Reaction

“…An increase of a cell volume resulting from X-rays damages was described in the last decade mainly for crystals of macromolecules. For such crystals this increase is linear in most cases [17,18]. However, a non-linear dependence was also presented [14] and even a decrease of a cell volume was described [36].…”

“…However, a non-linear dependence was also presented [14] and even a decrease of a cell volume was described [36]. In the case of X-rays and crystals of macromolecules, the reasons of such observations were explained as being a result of radiochemical reactions [17], electrostatic repulsions [18,20] and internal pressure [20]. It was also said Scheme 2 The definition of the intramolecular geometrical parameters Table 2 Values of the geometrical parameters describing a Yang photocyclization that the complete explanation could not be given [19].…”

“…It should be also said that a phenomenon of an increase of cell constants caused by electromagnetic radiation is known in the crystallographic literature concerning radiation damage, however, in the case of use of X-rays [13][14][15][16][17][18][19][20] and not for UV radiation. An increase of a cell volume resulting from X-rays damages was described in the last decade mainly for crystals of macromolecules.…”

“…X-ray structure analysis has also helped to monitor the decrease of crystal diffracting power caused by X-rays [13][14][15][16][17][18][19][20] (crystal damage by bright X-ray beams is a serious problem in crystallographic studies of macromolecules [14][15][16][17][18][19][20]). However, monitoring crystal damages caused by UV radiation has been extremely rare [21].…”

Measurements of accurate x-ray scattering data of protein solutions using small stationary sample cells

“…[5][6][7][8] Radiation exposure can also induce specific chemical and structural damage to protein crystals, such as cleavage of disulfide bonds, decarboxylation of acidic residues, and increases in individual atomic B-factors. 4,7,[9][10][11] The situation is much worse in protein solutions at room temperatures without cryocooling. Aggregation of the molecules becomes a significant problem during the collection of scattering data at several concentrations, which are needed to extrapolate a scattering profile to a concentration of zero.…”