International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 10.3, pp. 256-261
https://doi.org/10.1107/97809553602060000829

Chapter 10.3. Radiation damage

E. F. Garmana*

aDepartment of Biochemistry, South Parks Road, Oxford, OX1 3QU, England
Correspondence e-mail: elspeth.garman@bioch.ox.ac.uk

Radiation damage and its effects on diffraction data and the biological information obtained from macromolecular structures has re-emerged as a concern for crystallographers as ever more intense synchrotron-produced X-ray beams become available. Cryocrystallographic techniques, where crystals are cooled to around 100 K, are no longer enough, and there is also a resurgence of interest in room-temperature data collection where damage is swift. The current status of our knowledge and understanding of radiation damage in macromolecular crystallography is summarized.

10.3.1. Introduction

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Radiation damage to macromolecular crystalline samples during the experiment is a problem inherent in using ionizing radiation to obtain diffraction patterns, and has presented a challenge to protein crystallography (MX) since the beginning of the field, often necessitating the use of many crystals to assemble a complete data set. The damage manifests itself as a decrease in diffraction intensity caused by the destruction of crystallinity. This in turn is a result of the absorption in the sample of photons in the beam by either the photoelectric effect (total absorption of the photon and ejection of an inner-shell electron) or Compton scattering (inelastic scattering of the photon which escapes, following a varying amount of energy loss to an atomic electron). At the incident energies used for MX, the former effect has a much higher cross section and dominates the absorption, accounting for over 90% of the energy deposited by the beam. Each photoelectron has enough energy to subsequently induce up to 500 ionization events, which in turn can result in the formation of radical species in the crystal. In protein crystals, the presence of anything between 20 and 80% solvent means that the radiolysis of water and other components of the solvent is an important contributor to the creation of these species. Some of the energy deposited by the beam by these processes is converted into heat and induces a temperature rise in the sample. The diffracted photons are scattered elastically and thus do not contribute to the damage.

The usage of the terms `primary', `secondary' and `tertiary' damage is dependent on the context. The definitions adopted here are as follows: primary damage is the ionization of an atom due to photoelectric absorption or Compton scattering, whereas secondary damage is the formation of up to 500 lower-energy secondary electrons per primary absorption event. These have a temperature-independent continuous slowing down approximation range and induce further ionization and excitation events. The mobilities of the subsequent carriers of energy are reduced at lower temperatures, while the primary photoelectron has a temperature-independent mean track length of a few micrometres (for 12 keV photons) (O'Neill et al., 2002[link]). Tertiary damage is the effect on the crystal lattice and other mechanical consequences of the energy loss in the sample. Damage can also be classified as direct if the primary absorption event occurs at an atom in the protein molecule, or indirect if the radiation is absorbed by the surrounding solvent and the reactive species formed subsequently interact with the protein.

The universal metric against which the decay indicators of a crystal are conveniently measured is the absorbed dose, defined as the energy absorbed per unit mass of the sample (1 Gy = 1 J kg−1). To calculate this requires knowledge of the sample size and composition (i.e. the number of each atom type in the unit cell) so that its absorption coefficients can be computed, and detailed information about the incident beam [energy, size, shape and flux (in photons s−1)].

The earliest investigation of radiation damage in MX was carried out at room temperature (RT) by Blake & Phillips (1962[link]), who concluded that the damage was proportional to the absorbed dose and that the observed form of the decay with dose could be described by a first-order exponential function. They deduced that a single 8 keV X-ray photon absorbed by the crystal disrupted around 70 protein molecules, and disordered a further 90 for doses up to about 20 Mrad (0.2 MGy, since 1 Gy = 1 J kg−1 = 100 rad), above which the decay of the undamaged part of the crystal was faster than expected from an exponential law, due perhaps to some sort of cumulative effect. They also suggested that specific structural damage was suffered by the protein molecule; these conclusions were reached without knowledge of either the sequence or three-dimensional structure of the protein.

Despite reports from the early days of synchrotron use that crystals had longer lifetimes at higher dose rates at RT (e.g. Helliwell, 1988[link]), this was only systematically investigated recently, when an inverse dose-rate effect was measured in-house between dose rates of 6 and 10 Gy s−1, the higher rate giving four times the dose tolerance (i.e. four times the dose required to halve the total diffraction intensity, D1/2) for chicken egg-white lysozyme crystals (Southworth-Davies et al., 2007[link]). For irradiation at a dose rate of 2800 Gy s−1 at a synchrotron at RT, ten times the dose tolerance has been recorded (Barker et al., 2009[link]).

Up until the 1990s, MX data were collected at RT, where the recommended practice was to monitor the intensity, I0, of a particular reflection as the experiment proceeded, and to discard the crystal once the intensity had dropped to 0.85I0, or at the very worst, 0.70I0 if the particular crystals were in very short supply (Blundell & Johnson, 1976[link]).

10.3.2. Cryocrystallography as a mitigation strategy

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The search for strategies to reduce the rate of radiation damage in MX has resulted in the development over the last 20 years of cryocrystallographic techiques, whereby a crystal held by surface tension in a fibre, Mylar or polyimide loop is flash-cooled either into liquid nitrogen (77 K) or gaseous nitrogen at around 100 K (Garman, 1999[link]; Garman & Schneider, 1997[link]; see also Chapter 10.1[link] ). If the cooling is swift enough, the solvent around and within the crystal is present as amorphous solid, and does not form crystalline ice which would interfere with the diffraction and degrade the crystal order. To achieve this, cryoprotectant agents are added to the mother liquor, and optimum results can be obtained if the concentration of cryoprotectant results in equal shrinkage of the solvent channels and unit cell when the crystal is cooled (Juers & Matthews, 2001[link]). At 100 K, nearly all the radical species in the crystal are immobilized and so, although the primary radiation damage is unchanged and the resulting electrons of higher energy, and perhaps holes, are still mobile, other radicals that are subsequently formed (e.g. hydroxyl and other oxygen-centred radicals) have significantly lower mobility and thus their potential to inflict damage is quenched.

Widespread use of these techniques seemed to overcome most radiation-damage problems, since cryocooling resulted in a vastly extended dose tolerance (around a factor of 70; Nave & Garman, 2005[link]) for most macromolecular crystals. However, with the advent of more intense second- and third-generation synchrotron X-ray beams in the 1990s, observations of radiation damage to samples, even when held at 100 K, became common.

10.3.3. Characteristics of radiation damage at cryotemperatures

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Systematic studies on this phenomenon have identified two separate indicators of damage as a function of dose: global (Fig. 10.3.3.1[link]) and specific (Fig. 10.3.3.2[link]) damage. The former results in a loss of the measured reflection intensities particularly at high resolution, expansion of the unit-cell volume, increasing values of the measure of the internal consistency of the data which quantifies the difference between reflection intensities that should ideally be the same (Rmerge), an increase in both the scaling B factors for the data and the atomic B factors of the refined structure, rotation of the molecule within the unit cell, and often (but not always) an increase in mosaicity. Visible changes in the samples are also observed (see Fig. 10.3.3.3[link]). On warming following irradiation, bubbles of gas, thought to be hydrogen or CO2, are emitted. Of more direct relevance to the biological interpretation of structures, specific structural damage to particular covalent bonds is observed to occur in a reproducible order in many proteins: first disulfide bridges are broken, then glutamates and aspartates are decarboxylated, tyrosine residues lose their hydroxyl group, and subsequently the carbon–sulfur bonds in methionines are cleaved (Burmeister, 2000[link]; Ravelli & McSweeney, 2000[link]; Weik et al., 2000[link]). Enzyme mechanisms can involve susceptible residues, so special care is required when interpreting structures that may have been modified by X-ray damage during the data collection. Metalloproteins are particularly vulnerable to partial reduction during the diffraction experiment and may not be in their native state by the end of the data collection (Carugo & Djinović Carugo, 2005[link]). This specific damage often occurs well before there is any obvious degradation of the diffraction pattern.

[Figure 10.3.3.1]

Figure 10.3.3.1 | top | pdf |

Global radiation-damage indicators as a function of dose for four holoferritin crystals (Owen et al., 2006[link]): (a) unit-cell volume, (b) Wilson B factor, (c) Rmerge and (d) I(mean)/I0(mean).

[Figure 10.3.3.2]

Figure 10.3.3.2 | top | pdf |

Specific structural damage inflicted on a cryocooled crystal of apoferritin during sequential data sets collected at ID14–4, ESRF: (a) 2FoFc map of Glu63 contoured at 0.2 e Å−3 after a dose of 2.5 MGy and (b) after 50 MGy. (c) 2FoFc map of Met96 contoured at 0.2 e Å−3 after a dose of 2.5 MGy and (d) after 50 MGy, showing loss of electron density around the disordered atoms (Garman & Owen, 2006[link]).

[Figure 10.3.3.3]

Figure 10.3.3.3 | top | pdf |

Photograph of a 400 µm neuraminidase crystal (subtype N9 from avian influenza isolated from a Noddy Tern) that has been irradiated on ID14–4 at the ESRF at 100 K and then allowed to warm to room temperature. The three black marks are from the 100 × 100 µm beam, the dis­colouration being an indication of radiation damage.

In addition, as the diffraction experiment proceeds, the growing specific structural damage combined with the gradual increase in unit-cell volume and possible movement of the protein molecule within the unit cell induces creeping non-isomorphism on three simultaneous fronts. MAD (multiple-wavelength anomalous dispersion) structure solution thus becomes problematic, since by the time the third wavelength is collected, the cell and atomic structure can have changed such that the reflection intensities are significantly altered and this effect can obscure the anomalous signal required for structure solution.

The global effects at 100 K are thought to be dose-rate independent up to the flux densities currently used (1015 photons s−1 mm−2) (Sliz et al., 2003[link]). Another study con­curred with this finding, but indicated that there could be a second-order dose-rate effect based upon analysis of difference electron-density maps, specific damage being slightly more severe at higher dose rates (Leiros et al., 2006[link]). Conversely, however, Owen et al. (2006[link]) reported a small (10%) reduction in D1/2 for a dose-rate increase from 0.4 × 104 to 4.0 × 104 Gy s−1.

Radiation damage thus ultimately results in lower-resolution structures, failed MAD structure solutions and sometimes the inaccurate interpretation of biological results if no control experiments are carried out to account for radiation-damage artefacts (Ravelli & Garman, 2006[link]). It is thus an issue to be taken seriously by the structural biologist.

10.3.4. Understanding radiation damage

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In order to monitor and understand radiation damage, it is vital to have reliable metrics to allow systematic and comparative studies. Several different global measures extracted from the data are currently being utilized: [I/σ(I)]/[I0/σ(I0)], where I0 is the initial intensity of a data set or of a set of reflections, I(mean)/I0(mean), the `decay R factor', Rd, a pairwise R factor between identical and symmetry-related reflections occurring on different diffraction images, which is plotted against the difference in dose, ΔD, between the images in which the reflections were observed (Diederichs, 2006[link]), isotropic B factor (Brel), which was found to be linearly dependent on dose, and a coefficient of sensitivity (SAD = ΔBrelD2, where ΔBrel/8π2 is the change in relative isotropic B factor and ΔD is the change in dose). The latter coefficient relates the increase in mean-squared atomic displacements to the dose, and was found to be a robust measure for radiation damage (Kmetko et al., 2006[link]). Unit-cell volume is not used, since it has been found to be an unreliable indicator (Murray & Garman, 2002[link]; Ravelli et al., 2002[link]). Some of the above metrics are more appropriate than others for use in particular circumstances, e.g. if maximizing the resolution, trying to minimize radiation-damage-induced artefacts, or carrying out SAD (single-wavelength anomalous dispersion)/MAD structure solution. Care must be exercised in the calculation of electron-density maps extracted from sequential data sets, since the maps can be biased by the refinement, and so usually the phases from the refined structure obtained from the first data set are used for the map calculations of structures from the subsequent data sets.

There is postulated to be a universal `dose limit' for biological samples, beyond which they cannot be expected to survive intact and are destroyed by the energy deposited in them. From analogy with observations in electron microscopy (for 100 keV electrons), a dose limit of D1/2 = 20 MGy for a cryocooled (77 K) protein crystal was calculated by Henderson (1990[link]). More recently Owen et al. (2006[link]) have published an experimental study of the dose limit for cryocooled (100 K) protein crystals, in which it was found that the total intensity of diffraction had a linear dependence on absorbed dose (as opposed to the exponential dependence at room temperature). The dose required to halve the total intensity of the diffraction pattern was 43 MGy, but an upper limit of 30 MGy was suggested (I = 0.7I0), beyond which point the biological information obtained from the experiment might be compromised. Coincidentally, this is the value of intensity loss previously recommended as the absolute maximum allowable for individual reflections during room-temperature data collection (Blundell & Johnson, 1976[link]).

For systematic radiation-damage experiments and also for the 30 MGy experimental dose limit to be a useful predictive experimental tool, the dose absorbed by the crystal must be calculated. This can be conveniently computed for MX using the program RADDOSE (Murray et al., 2004[link]; Paithankar et al., 2009[link]), which requires information on the crystal, protein, cell and mM concentration of mother liquor, as well as the beam characteristics, and the duration and number of the exposures. The output gives the time available before the experimental dose limit is reached and the predicted temperature rise in the crystal using a simple isothermal `lumped model'. Version 3 of RADDOSE takes into account the possible escape from the sample of fluorescence X-rays from the decay of excited atoms (only significant for atoms with atomic number greater than 20) following photoelectron emission, and also the energy deposited in the crystal by Compton scattering (non-negligible only above incident X-ray energies of 20 keV). If the crystal is bigger than the beam the program overestimates the dose, since the rotation of the crystal during the experiment is not taken into account and new un-irradiated material will be brought in with time, thus decreasing the dose.

To account for the observed damage effects at 100 K, information must be gleaned from the radiation chemistry literature. Several analyses of the mobility of the various species formed upon X-ray exposure have been carried out and they help to explain the effectiveness of cryocooling for MX. Protons are only known to become mobile in amorphous ice above [\sim]115 K (Fisher & Devlin, 1995[link]) and although [^{\bullet}{\rm OH}] radicals are trapped at 77 K in an aqueous glass of 6 M CsF (Becker et al., 1994[link]) and are inferred to be trapped in ice at 100 K but are thought to be mobile above 130 K and thus able to recombine (Symons, 1999[link]), they have also been reported to be mobile at 77 K in a glass of DNA (Lange & Httermann, 1995[link]). According to electron spin resonance measurements (Jones et al., 1987[link]), at 77 K positive holes in proteins are rapidly trapped, forming amido radicals on the protein backbone chain, whereas the electrons produced by inelastic interactions have significant mobility. Rao et al. (1983[link]) showed that electrons added to proteins at 77 K are able to move efficiently until they encounter S—S bonds, where they are trapped.

Complementary methods are increasingly being employed for the observation of radiation damage in MX and specially designed instruments are being constructed on synchrotron beamlines to facilitate measurements performed simultaneously with X-ray diffraction, including UV–vis microspectroscopy (McGeehan et al., 2009[link]), UV–vis fluorescence, X-ray spectroscopy (Yano et al., 2005[link]) and Raman spectroscopy (Owen et al., 2009[link]). Optical peaks at 400 nm and between 550 and 600 nm are signatures of the formation of the disulfide radical anion, [R{\rm SS}R^{\bullet}] (Fig. 10.3.4.1[link]) (Weik et al., 2002[link]), and the hydrated electron, respectively. The reduction of metal sites by the X-ray beam can also be observed online from their change in optical absorption (Hough et al., 2008[link]).

[Figure 10.3.4.1]

Figure 10.3.4.1 | top | pdf |

Microspectrophotometer absorption spectra of native and ascorbate-soaked hen egg-white lysozyme crystals at 100 K, showing the disulfide radical 400 nm peak formed on irradiation by a synchrotron X-ray beam, and the suppression of this peak on the addition of ascorbate, which has an absorption peak at 350 nm (Murray & Garman, 2002[link]).

10.3.5. Mitigating and correcting for radiation damage

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Various experimental parameters have been investigated to find ways of reducing the rate of radiation damage at cryotemperatures, including further reducing the temperature, changing the incident wavelength and adding radical scavengers.

Various studies have investigated the rate of global and specific damage infliction with temperature variation, and there is as yet no consensus on this issue. At 40 K, global indicators improved little (Teng & Moffat, 2002[link]), but a study at 8 K found a 25% increase in dose tolerance (Chinte et al., 2007[link]). Another investigation, which also monitored global indicators, reported no improvement for insulin crystal dose tolerance at 15 K, but a 23% improvement for holoferritin, which has a large iron core (Meents et al., 2007[link]). However, significant protection against specific damage (a factor of 30 at 40 K compared to 110 K) has been observed as monitored by the intensity of the photoreduced peak in the X-ray absorption spectrum at the iron K edge on an iron-containing metalloprotein (Corbett et al., 2007[link]).

At current fluxes (4 × 1014 photons s−1 mm−2), heating of the sample by the beam is predicted to be no more than 15 K (Mhaisekar et al., 2005[link]), so nitrogen cooling to 100 K is adequate to avoid the movement of species other than the already mobile electrons and perhaps positive holes (Jones et al., 1987[link]).

Despite much anecdotal evidence that longer-wavelength incident radiation causes swifter damage than shorter-wavelength incident radiation, systematic studies of this relationship (Shimizu et al., 2007[link]; Weiss et al., 2005[link]) have shown no detectable change in the damage rate with dose over the range 6.2 keV (2 Å) to 33 keV (0.275 Å). Although the absorption is higher at longer wavelengths, the probability of diffraction is also higher, and the ratio of scattering to dose is not predicted to change by more than 20% for a typical protein crystal (no heavy atoms) over the range of energies currently used in MX (7–20 keV).

Two scavengers have been tested at RT in the past (styrene and polyethylene glycol), but neither was found to be very effective and they have not been widely utilized. However, recently, 0.5 M ascorbate and 0.5 M 1,4-benzoquinone were reported to give an increase by factors of two and nine, respectively, in D1/2 at RT. Most interestingly, for both scavengers, the dose dependence of the intensity decay was modified to a linear relationship rather than exhibiting the exponential intensity decay of the native crystal (Barker et al., 2009[link]). The decay of the total diffraction intensity observed at 100 K is predominantly linear (Fig. 10.3.3.1[link]), and this result indicates that the radiation chemistry of the degradation of the crystalline order has been modified by the scavengers in a similar way as it is by temperature if cryocooled.

At 100 K, several scavengers have now been tested: styrene was reported to be ineffective but ascorbate reduced both global and specific damage (Murray & Garman, 2002[link]). Nicotinic acid and 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) have been found to be effective (Kauffmann et al., 2006[link]). A large number of potential scavengers were screened using an online microspectrophotometer (McGeehan et al., 2009[link]) for their ability to quench the formation of the disulfide radical anion 400 nm peak, and only ascorbate, 1,4-benzoquinone and TEMP (2,2,6,6,-tetramethyl-4-piperidone) eradicated it (Southworth-Davies & Garman, 2007[link]). Thus, from the research carried out so far at 100 K, it seems that scavengers deserve further study but are not going to give a large improvement in dose tolerance and do not seem to be as potentially effective as they are at RT.

In addition to these experimental strategies, software development is underway to correct data for radiation damage `after the event'. These methods include zero-dose extrapolation, which uses multiple measurements of the same reflection to estimate its probable intensity at the beginning of the experiment (Blake & Phillips, 1962[link]; Diederichs et al., 2003[link]), and the occupancy refinement of heavy-atom sites as a function of dose (Schiltz et al., 2004[link]).

10.3.6. Using radiation damage

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The specific structural damage inflicted by radiation can be utilized for phasing, for example RIP, radiation-damage-induced phasing (Ravelli et al., 2003[link]), whereby a low-dose data collection (little specific damage) is followed by a `burn', inflicted either by the X-ray beam or by a UV laser (Nanao & Ravelli, 2006[link]), and then another low-dose data collection (disulfides broken and the relevant sulfur atoms thus disordered). This method has also been combined with anomalous scattering (RIPAS) (Zwart et al., 2004[link]).

Radiation damage can also be used to elucidate biological function and mechanism, and there are increasing numbers of such reports. For instance, the `back-door' escape route of product clearance in acetylcholinesterase was identified using the X-ray beam to radiocleave a non-hydrolysable substrate analogue bound in the enzymatic site and collecting diffraction data sets at 100 and 150 K, the dynamic changes involved becoming visible at the higher temperature (Colletier et al., 2008[link]).

Other work exploiting radiation damage includes an inventive data-collection regime on a series of crystals utilizing the reduction of metal centres by the X-ray beam to track the resulting conformational changes, which shed light on the catalytic pathway of horseradish peroxidase (Berglund et al., 2002[link]).

10.3.7. Open questions

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There are still many areas where systematic investigations are required to improve our understanding of the radiation chemistry within an irradiated protein crystal held at either room temperature or at various cryotemperatures, so that better strategies for minimizing damage can be developed. For instance, regarding specific damage, the varying susceptibility of the same type of residue within a protein is not yet understood: there appears to be no correlation with solvent accessibility (Fioravanti et al., 2007[link]). On the data-collection side, there is as yet no compelling evidence to warrant the routine use of helium cryostats (and the associated expense) apart from the case of metalloproteins, and there exists an urgent need to establish the best experimental strategies and optimum crystal sizes to allow the most advantageous use of newly available microbeams at synchrotrons.

The most useful contribution to be made by MX radiation-damage research is in identifying concrete experimental protocols for everyday use on synchrotron beamlines so that researchers can ensure that they obtain the maximum possible amount of high-quality data from their crystals. This would firstly facilitate structure solution, and secondly avoid compromising the biological information extracted from the structure once obtained.

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