Kill or cure: radiation damage in cryo-cooled macromolecular crystals
Elspeth Garman
Laboratory of Molecular Biophysics, Rex Richards Building, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K.
e-mail: elspeth@biop.ox.ac.uk
1. Introduction
Macromolecular crystals commonly suffer severe radiation damage during room temperature X-ray data collection and this was characterized in a classic and systematic study as long ago as 1962 [1]. Data are now routinely collected with the sample held at around 100K, significantly reducing the diffusion rates of secondary damage products, and usually resulting in higher resolution and better quality data. In fact, over 85% of all macromolecular crystal diffraction data are now collected at or near 100K [2]. Other benefits include the facility to flash-cool crystals into cryogen and store them while they are in peak condition, and the potential for using cryocrystallography for new experiments, particularly the freeze trapping of reaction intermediates. Some disadvantages of cryocrystallography are that cryoconditions have to be established and optimized for best results, a reliable cryostat must be installed, and that the crystal mosaicity usually (but not necessarily) increases on flash-cooling.
However, even in samples which are cryocooled, observation of radiation damage has become commonplace at wiggler and undulator synchrotron beamlines. It is a limiting problem in the optimum use of these high brilliance beams, which are routinely attenuated or defocused so that a longer time is available for data collection. [Note that there is so far little convincing evidence for a dose-rate effect: i.e. a less intense beam will inflict the same damage per absorbed photon as an intense one.] Radiation damage also accounts for the increasing popularity of SAD (Single-wavelength Anomalous Dispersion) experiments, since the dispersive signal necessary for success in MAD (Multi-wavelength Anomalous Dispersion) experiments is easily obscured by non-isomorphism arising from radiation induced unit cell expansion and specific structural changes [3-5]. Unless the effects of radiation damage on a structure are understood, incorrect conclusions on biological mechanism can result due to specific damage, in particular decarboxylation of residues.
On a more positive note, exciting new experiments aimed at characterising and reducing this damage are now underway, and at utilising it for structure solution and enzymatic studies.
2. Theoretical Dose Limit
The theoretical X-ray dose limit for a crystal held at 77K to lose roughly half its diffraction power has been calculated to be approximately 2 x 10 7 Gy (J kg -1 )[6] . For a typical protein crystal, this dose is delivered by 1.6 x 10 16 photons mm -2 incident 1.54Å X-rays; equivalent to about 2.5 years on a state-of-the-art rotating anode and around 5 minutes on an undulator beamline. Experimental observation is that broadly the latter limit holds, but it must be noted that samples containing heavily absorbing atoms will reach the dose limit in a shorter time [7].
3. Factors affecting the rate of radiation damage.
Figure 1 summarises the parameters which might have an affect on the rate of radiation damage to macromolecular crystal. In order to design mitigation strategies, each variable must be investigated systematically (i.e. varying only one at a time) and for a statistically significant number of samples. Beam fluxes must be regularly calibrated, since in line monitors drift with time, and the flux will also vary with beam energy. Work is underway which should enable us to characterize the important variables in relation to radiation damage progression. These include (at various sites worldwide): a) temperature of data collection, b) crystal heating, c) dose/dose rate effects, d) wavelength dependence, e) use of scavengers, f) change in beam focusing conditions, and g) investigation of experimental dose limit, including prediction of time to the limit from absorption cross sections of crystal and solvent [7].
3. Towards controlling radiation damage at cryotemperatures.
Radiation damage control strategies are very limited. Crystals soaked in heavy atom compounds can be back soaked to remove disordered absorbers, and thought can be given to exchanging heavy components in the mother liquor.
There is currently no easy way to monitor damage on-line, so mitigation strategies are hard to test quantitatively other than by looking at the rate of specific structural damage. Unfortunately, unit cell expansion, although linear with dose, has been shown to be irreproducible in rate among different crystals of the same protein [8, 9]. The use of scavengers, although promising [8], is probably not going to result in slowing the damage rate by more than a factor 2 or 3 at the very most.
Figure 1. Diagrammatic representation of an X-ray diffraction experiment, annotated to
show the parameters to be investigated to understand radiation damage progression.
4. Correcting data for radiation damage.
Researchers are becoming increasingly aware of the effects of radiation damage on the biological conclusions being drawn from structures. In a study of the primary photoreaction of bacteriorhodopsin [10] in conjunction with an on-line spectrophotometer (350-800nm), a synchrotron X-ray beam induced half the protein to convert into an orange species during data collection, and further experiments at different radiation doses were necessary to identify the true structural changes.
Software correction procedures for extrapolating individual reflection intensities back to their zero dose level are being developed [11,12], given multiple measurements of each reflection. This strategy should enable the useful lifetime of the crystal to be extended. It is anticipated that these ideas will eventually be incorporated into the standard data processing software and high throughout pipelines.
5. Utilising radiation damage.
The specific structural damage inflicted by X-rays has been turned to advantage in elegant experiments which establish a new way of phasing macromolecular structures, suitable for use at high brilliance synchrotron beamlines. In the RIP (Radiation Induced Phasing) method, a low dose (with attenuators) data set is collected, followed by a `burn’ delivering approximately quarter to a half of the Henderson dose limit. This dose destroys the disulfide alone bonds and causes other specific damage. A second low dose dataset is then collected, and the phases obtained from finding the `heavy’ (i.e. sulphur) atom sites. [13]. Although using RIP for structure solution may yet be confined to a limited number of favourable cases, when combined with, for example, sulphur SAD, it will undoubtedly be a very useful phasing tool, as exemplified by the very recent solution of a small two domain protein, which had resisted all other methods of structure solution.
Radiation damage can also be used to induce reduction in crystals which can be used for new types of experiment. For instance, damage, in conjunction with more conventional data collection and microspectrophotometer monitoring, has been utilized to eludicate the catalytic pathway of horseradish peroxidase.redox [14].
Radiation damage at cryotemperatures has emerged very recently as a new and important area, but experiments which vary only one parameter are hard to design and lengthy to analyse. However, significant progress is being made, which may yet allow the flux of the undulator beamlines to be fully utilised at cryo-temperatures, thus overcoming the crystallographer’s current dilemma: rate of radiation damage versus diffraction intensity.
References.
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