Radiation Damage In Protein Crystals Research Articles

The early history of cryo-cooling for macromolecular crystallography.

This paper recounts the first successful cryo-cooling of protein crystals that demonstrated the reduction in X-ray damage to macromolecular crystals. The project was suggested by David C. Phillips in 1965 at the Royal Institution of Great Britain and continued in 1967 at the Weizmann Institute of Science, where the first cryo-cooling experiments were performed on lysozyme crystals, and was completed in 1969 at Purdue University on lactate dehydrogenase crystals. A 1970 publication in Acta Crystallographica described the cryo-procedures, the use of cryo-protectants to prevent ice formation, the importance of fast, isotropic cryo-cooling and the collection of analytical data showing more than a tenfold decrease in radiation damage in cryo-cooled lactate dehydrogenase crystals. This was the first demonstration of any method that reduced radiation damage in protein crystals, which provided crystallographers with suitable means to employ synchrotron X-ray sources for protein-crystal analysis. Today, fifty years later, more than 90% of the crystal structures deposited in the Protein Data Bank have been cryo-cooled.

Development of an X-ray fluorescence holographic measurement system for protein crystals.

Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α2β2 tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm(3)) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

Imperfection and radiation damage in protein crystals studied with coherent radiation.

Fringes and speckles occur within diffraction spots when a crystal is illuminated with coherent radiation during X-ray diffraction. The additional information in these features provides insight into the imperfections in the crystal at the sub-micrometre scale. In addition, these features can provide more accurate intensity measurements (e.g. by model-based profile fitting), detwinning (by distinguishing the various components), phasing (by exploiting sampling of the molecular transform) and refinement (by distinguishing regions with different unit-cell parameters). In order to exploit these potential benefits, the features due to coherent diffraction have to be recorded and any change due to radiation damage properly modelled. Initial results from recording coherent diffraction at cryotemperatures from polyhedrin crystals of approximately 2 µm in size are described. These measurements allowed information about the type of crystal imperfections to be obtained at the sub-micrometre level, together with the changes due to radiation damage.

Neutron protein crystallography: A complementary tool for locating hydrogens in proteins

Neutron protein crystallography is a powerful tool for investigating protein chemistry because it directly locates hydrogen atom positions in a protein structure. The visibility of hydrogen and deuterium atoms arises from the strong interaction of neutrons with the nuclei of these isotopes. Positions can be unambiguously assigned from diffraction at resolutions typical of protein crystals. Neutrons have the additional benefit to structural biology of not inducing radiation damage in protein crystals. The same crystal could be measured multiple times for parametric studies. Here, we review the basic principles of neutron protein crystallography. The information that can be gained from a neutron structure is presented in balance with practical considerations. Methods to produce isotopically-substituted proteins and to grow large crystals are provided in the context of neutron structures reported in the literature. Available instruments for data collection and software for data processing and structure refinement are described along with technique-specific strategies including joint X-ray/neutron structure refinement. Examples are given to illustrate, ultimately, the unique scientific value of neutron protein crystal structures.

Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies.

Electron paramagnetic resonance (EPR) and online UV-visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV-visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5-0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.

Radiation damage in protein crystals is reduced with a micron-sized X-ray beam

Radiation damage is a major limitation in crystallography of biological macromolecules, even for cryocooled samples, and is particularly acute in microdiffraction. For the X-ray energies most commonly used for protein crystallography at synchrotron sources, photoelectrons are the predominant source of radiation damage. If the beam size is small relative to the photoelectron path length, then the photoelectron may escape the beam footprint, resulting in less damage in the illuminated volume. Thus, it may be possible to exploit this phenomenon to reduce radiation-induced damage during data measurement for techniques such as diffraction, spectroscopy, and imaging that use X-rays to probe both crystalline and noncrystalline biological samples. In a systematic and direct experimental demonstration of reduced radiation damage in protein crystals with small beams, damage was measured as a function of micron-sized X-ray beams of decreasing dimensions. The damage rate normalized for dose was reduced by a factor of three from the largest (15.6 μm) to the smallest (0.84 μm) X-ray beam used. Radiation-induced damage to protein crystals was also mapped parallel and perpendicular to the polarization direction of an incident 1-μm X-ray beam. Damage was greatest at the beam center and decreased monotonically to zero at a distance of about 4 μm, establishing the range of photoelectrons. The observed damage is less anisotropic than photoelectron emission probability, consistent with photoelectron trajectory simulations. These experimental results provide the basis for data collection protocols to mitigate with micron-sized X-ray beams the effects of radiation damage.

Energy dependence of site-specific radiation damage in protein crystals.

It is important to consider radiation damage to crystals caused by data collection when solving structures and critical when determining protein function, which can often depend on very subtle structural characteristics. In this study the rate of damage to specific sites in protein crystals cooled at 100 K is found to depend on the energy of the incident X-ray beam. Several lysozyme crystals were each subjected to 3-26 MGy of cumulative X-ray exposure by collecting multiple data sets from each crystal at either 9 keV or 14 keV. The integrated electron density surrounding each S atom in the structure was calculated for each data set and the change in electron density was evaluated as a function of dose at the two energies. The rate of electron density decrease per cubic Å per MGy was determined to be greater at 14 keV than at 9 keV for cysteine sulfurs involved in disulphide bridges; no statistically significant differences in the decay rates were found for methionine sulfurs. These preliminary results imply that it might be possible to minimize certain types of specific radiation damage by an appropriate choice of energy. Further experiments studying a variety of photolabile sites over a wider range of energies are needed to confirm this conclusion.

How different oxidation states of crystalline myoglobin are influenced by X-rays

X-ray induced radiation damage of protein crystals is well known to occur even at cryogenic temperatures. Redox active sites like metal sites seem especially vulnerable for these radiation-induced reductions. It is essential to know correctly the oxidation state of metal sites in protein crystal structures to be able to interpret the structure–function relation. Through previous structural studies, we have tried to characterise and understand the reactions between myoglobin and peroxides. These reaction intermediates are relevant because myoglobin is proposed to take part as scavenger of reactive oxygen species during oxidative stress, and because these intermediates are similar among the haem peroxidases and oxygenases. We have in our previous studies shown that these different myoglobin states are influenced by the X-rays used. In this study, we have in detail investigated the impact that X-rays have on these different oxidation states of myoglobin. An underlying goal has been to find a way to be able to determine mostly unreduced states. We have by using single-crystal light absorption spectroscopy found that the different oxidation states of myoglobin are to a different extent influenced by the X-rays (e.g. ferric Fe III myoglobin is faster reduced than ferryl Fe IV═O myoglobin). We observe that the higher oxidation states are not reduced to normal ferrous Fe II or ferric Fe III states, but end up in some intermediate and possibly artificial states. For ferric myoglobin, it seems that annealing of the radiation-induced/reduced state can reversibly more or less give the starting point (ferric myoglobin). Both scavengers and different dose-rates might influence to which extent the different states are affected by the X-rays. Our study shows that it is essential to do a time/dose monitoring of the influence X-rays have on each specific redox-state with spectroscopic techniques like single-crystal light absorption spectroscopy. This will determine to which extent you can collect X-ray diffraction data on your crystal before it becomes too heavily influenced/reduced by X-rays. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

Radiation damage in protein crystals examined under various conditions by different methods

Investigation of radiation damage in protein crystals has progressed in several directions over the past couple of years. There have been improvements in the basic procedures such as calibration of the incident X-ray intensity and calculation of the dose likely to be deposited in a crystal of known size and composition with this intensity. There has been increased emphasis on using additional techniques such as optical, Raman or X-ray spectroscopy to complement X-ray diffraction. Apparent discrepancies between the results of different techniques can be explained by the fact that they are sensitive to different length scales or to changes in the electronic state rather than to movement of atoms. Investigations have been carried out at room temperature as well as cryo-temperatures and, in both cases, with the introduction of potential scavenger molecules. These and other studies are leading to an overall description of the changes which can occur when a protein crystal is irradiated with X-rays at both cryo- and room temperatures. Results from crystallographic and spectroscopic radiation-damage experiments can be reconciled with other studies in the field of radiation physics and chemistry.

Room-temperature scavengers for macromolecular crystallography: increased lifetimes and modified dose dependence of the intensity decay

The advent of highly intense wiggler and undulator beamlines has reintroduced the problem of X-ray radiation damage in protein crystals even at cryogenic temperatures (100 K). Although cryocrystallography can be utilized for the majority of protein crystals, certain macromolecular crystals (e.g. of viruses) suffer large increases in mosaicity upon flash cooling and data are still collected at room temperature (293 K). An alternative mechanism to cryocooling for prolonging crystal lifetime is the use of radioprotectants. These compounds are able to scavenge the free radical species formed upon X-ray irradiation which are thought to be responsible for part of the observed damage. Three putative radioprotectants, ascorbate, 1,4-benzoquinone and 2,2,6,6-tetramethyl-4-piperidone (TEMP), were tested for their ability to prolong lysozyme crystal lifetimes at 293 K. Plots of relative summed intensity against dose were used as a metric to assess radioprotectant ability: ascorbate and 1,4-benzoquinone appear to be effective, whereas studies on TEMP were inconclusive. Ascorbate, which scavenges OH* radicals (k(OH) = 8 x 10(9) M(-1) s(-1)) and electrons with a lower rate constant (k(e-(aq)) = 3.0 x 10(8) M(-1) s(-1)), doubled the crystal dose tolerance, whereas 1,4-benzoquinone, which also scavenges both OH* radicals (k(OH) = 1.2 x 10(9) M(-1) s(-1)) and electrons (k(e-(aq)) = 1.2 x 10(10) M(-1) s(-1)), offered a ninefold increase in dose tolerance at the dose rates used. Pivotally, these preliminary results on a limited number of samples show that the two scavengers also induced a striking change in the dose dependence of the intensity decay from a first-order to a zeroth-order process.

Quantifying X-ray radiation damage in protein crystals

Quantifying X-ray radiation damage in protein crystals

Quantifying X-ray radiation damage in protein crystals at cryogenic temperatures

The dependence of radiation damage to protein crystals at cryogenic temperatures upon the X-ray absorption cross-section of the crystal has been examined. Lysozyme crystals containing varying heavy-atom concentrations were irradiated and diffraction patterns were recorded as a function of the total number of incident photons. An experimental protocol and a coefficient of sensitivity to absorbed dose, proportional to the change in relative isotropic B factor, are defined that together yield a sensitive and robust measure of damage. Radiation damage per incident photon increases linearly with the absorption coefficient of the crystal, but damage per absorbed photon is the same for all heavy-atom concentrations. Similar damage per absorbed photon is observed for crystals of three proteins with different molecular sizes and solvent contents.

How to Avoid Premature Decay of Your Macromolecular Crystal: A Quick Soak for Long Life

Radiation damage to biological samples is currently one of the major limiting factors in macromolecular X-ray crystallography, since it severely and irreversibly affects the quality of the data that can be obtained from a diffraction experiment. However, radiation damage can effectively be reduced by utilizing the electron and radical scavenging potential of certain small-molecule compounds. We propose an approach to protect macromolecular crystals prior to data collection by quick soaking with scavengers. This, in favorable cases, can more than double crystal lifetime in the X-ray beam. The approach has the potential to yield diffraction data of superior quality and hence to increase the amount of high-quality diffraction data and of structural information attainable from a single crystal.

How does Radiation Damage in Protein Crystals Depend on X-Ray Dose?

Is radiation damage to cryopreserved protein crystals strictly proportional to accumulated dose at the high-flux density of beams from undulators at third-generation synchrotron sources? The answer is “yes,” for overall damage to several different kinds of protein crystals at flux densities up to 10 15 ph/sec/mm 2 (APS beamline 19-ID). We find that, at 12 keV (1 Å wavelength), about ten absorbed photons are sufficient to “kill” a unit cell. As this corresponds to about one elastically scattered photon, each unit cell can contribute only about one photon to total Bragg diffraction. The smallest crystal that can yield a full data set to 3.5 Å resolution has a diameter of about 20 μm (100 Å unit cell).

Investigation of possible free-radical scavengers and metrics for radiation damage in protein cryocrystallography.

The introduction of highly intense wiggler and undulator beamlines has reintroduced the problem of X-ray radiation damage in protein crystals even at cryogenic temperatures. Several metrics for monitoring radiation damage are considered and unit-cell volume expansion is systematically investigated using crystals of three different types, but it is found to be too variable to be a useful metric. Radical scavengers of secondary radiation damage are investigated as possible mitigating agents. Styrene is found to be ineffective. A method of spectroscopically measuring the radiation damage with a microspectrophotometer was used and, in conjunction with crystallographic data, provided tentative but suggestive evidence for the efficacy of ascorbate as a free-radical scavenging agent in cryocooled hen egg-white lysozyme crystals.

Radiation damage of protein crystals at cryogenic temperatures between 40 K and 150 K

X-ray radiation damage of lysozyme single crystals by an intense monochromatic beam from the Advanced Photon Source is studied at cryogenic temperatures between 40 K and 150 K. The results confirm that primary radiation damage is both linearly dependent on the X-ray dose and independent of temperature. The upper limit for the primary radiation damage observed in our previous study [Teng & Moffat (2000), J. Synchrotron Rad. 7, 313-317] holds over the wider temperature range of this study. The X-ray diffraction quality of the data acquired at 40 K is superior to those at 100 K, apparently due to temperature dependence of secondary and tertiary radiation damage and to reduced thermal motion.

Primary radiation damage of protein crystals by an intense synchrotron X-ray beam

X-ray radiation damage of a lysozyme single crystal by an intense monochromatic beam from a third-generation radiation source at the Advanced Photon Source has been studied. The results show that primary radiation damage is linearly dependent on the X-ray dose even when the crystal is at cryogenic temperatures. The existence of an upper limit for the primary radiation damage was observed. Above the threshold of approximately 1 x 10(7) Gy, excessive damage of the crystal develops which is interpreted as the onset of secondary and/or tertiary radiation damage. This upper limit of X-ray dose is compared with Henderson's limit [Henderson (1990). Proc. R. Soc. London, B241, 6-8], and its implication for the amount of useful X-ray diffraction data that can be obtained for crystals of a given scattering power is also discussed.

Radiation damage in protein crystals at low temperature.

This paper describes the study of the effects of radiation damage on the quality of data collected from a protein crystal at 100 K. It is shown that radiation damage causes measurable effects in the diffraction pattern. This implies that, even at liquid nitrogen temperatures, there is a limit to the size of a crystal from which a complete data set can be collected.

Sequential radiation damage in protein crystallography.

Radiation damage in protein crystals is described in terms of a sequential process of protein disordering. A new radiation-damage model has been tested against data from several protein crystals and can describe radiation damage corresponding to loss of the original intensity in excess of 80%. The model is an extension of previous models which characterize radiation damage in terms of successive conformational transitions of the protein from an undamaged to a spatially disordered to finally an amorphous state. The proposed model provides a more-general positional characterization of the disordered protein and includes, prior to the disordered state, a new dose-dependent state in which the protein conformation resembles the undamaged protein. Comparison of this model with the best previous model shows that the proposed model provides an improved fit to radiation-damage data.

The reduction of radiation damage in protein crystals by polyethylene glycol

A simple technique has been developed to lessen radiation damage of protein crystals grown from low-ionic strength solution. The technique consists of replacing the mother liquor with solutions containing 10–20% of polyethylene glycol 4000 or 20000.