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1.
X-ray radiation damage to cryocooled ( approximately 100 K) macromolecular crystals has emerged as a general problem, especially since the advent of third generation synchrotron undulator sources. Interest in understanding the physical and chemical phenomena behind the observed effects is growing rapidly. The specific structural damage seen in electron density maps has to be accounted for when studying intermediates, and can sometimes be related to biological function. Radiation damage induces non-isomorphism, thus hampering traditional phasing methods. However, specific damage can also be used to obtain phases. With an increased knowledge of expected crystal lifetime, beamline characteristics and types of damage, macromolecular crystallographers might soon be able to account for radiation damage in data collection, processing and phasing. 相似文献
2.
Garman E 《Current opinion in structural biology》2003,13(5):545-551
Macromolecular crystals commonly suffer rapid radiation damage during room temperature X-ray data collection. Therefore, data are now routinely collected with the sample held at around 100K, significantly reducing secondary radiation damage, and usually resulting in higher resolution and better quality data. At synchrotron sources, the frequent observation of radiation damage even at cryotemperatures has prompted the development of exciting new experiments aimed at characterising and reducing this damage, and using it for structure determination and enzymatic studies. Current research into cryotechniques seeks to understand the basic physical and chemical processes involved in flash-cooling and radiation damage, which should eventually enable the rational optimisation of cryoprotocols. 相似文献
3.
Pflugrath JW 《Methods (San Diego, Calif.)》2004,34(3):415-423
Cryocrystallography is routinely used in macromolecular crystallography laboratories. The main advantage of X-ray diffraction data collection near 100K is that crystals display much less radiation damage than seen at room temperature. Techniques and tools are described to facilitate cryoprotecting and flash-cooling crystals for data collection. 相似文献
4.
In recent years, the awareness of potential radiation damage of metal centers in protein crystals during crystallographic data collection has received increasing attention. The radiation damage can lead to radiation-induced changes and reduction of the metal sites. One of the research fields where these concerns have been comprehensively addressed is the study of the reaction intermediates of the heme peroxidase and oxygenase reaction cycles. For both the resting states and the high-valent intermediates, the X-rays used in the structure determination have given undesired side effects through radiation-induced changes to the trapped intermediates. However, X-rays have been used to generate and trap the peroxy/hydroperoxy state in crystals. In this review, the structural work and the influence of X-rays on these intermediates in myoglobin are summarized and viewed in light of analogous studies on similar intermediates in peroxidases and oxygenases. 相似文献
5.
W M Clemons D E Brodersen J P McCutcheon J L May A P Carter R J Morgan-Warren B T Wimberly V Ramakrishnan 《Journal of molecular biology》2001,310(4):827-843
We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 A resolution were used as a starting point to improve the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved less important than attention to detail in yielding crystals that diffracted beyond 3 A resolution. Despite improvements in technology and methodology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of the asymmetric unit, crystal variability and sensitivity to radiation damage. Some steps that were useful for determination of the atomic structure were: the use of anomalous scattering from the LIII edges of osmium and lutetium to obtain the necessary phasing signal; the use of tunable, third-generation synchrotron sources to obtain data of reasonable quality at high resolution; collection of derivative data precisely about a mirror plane to preserve small anomalous differences between Bijvoet mates despite extensive radiation damage and multi-crystal scaling; the pre-screening of crystals to ensure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; pre-incubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction with biochemical data on protein-RNA interactions, to map out the architecture of the 30 S subunit prior to the construction of a detailed atomic-resolution model. 相似文献
6.
An estimated half of all proteins contain a metal, with these being essential for a tremendous variety of biological functions. X-ray crystallography is the major method for obtaining structures at high resolution of these metalloproteins, but there are considerable challenges to obtain intact structures due to the effects of radiation damage. Serial crystallography offers the prospect of determining low-dose synchrotron or effectively damage free XFEL structures at room temperature and enables time-resolved or dose-resolved approaches. Complementary spectroscopic data can validate redox and or ligand states within metalloprotein crystals. In this opinion, we discuss developments in the application of serial crystallographic approaches to metalloproteins and comment on future directions. 相似文献
7.
The flash-cooling of crystals in macromolecular crystallography has become commonplace. The procedure makes it possible to collect data from much smaller specimens than was the case in the past Also, flash-cooled crystals are much less prone to radiation damage than their room-temperature counterparts, allowing data to be accumulated over extended periods of time. Notwithstanding the attractiveness of the technique, it does have potential disadvantages. First, better methods need to be developed to prevent damage to crystals on freezing. There is also a risk that structures determined at low temperature may suggest conclusions based on aspects of the structure that are not necessarily relevant at room temperature. 相似文献
8.
Ionizing radiation damages DNA and also induces oxidative stress, which can affect the function of proteins involved in DNA
repair, thereby causing repair of DNA damage to become less efficient. We previously developed a mathematical model of this
potentially synergistic relationship and applied it to γ-ray exposure data on the radiation-resistant prokaryote Deinococcus radiodurans. Here, we investigate the effects of radiation quality on these processes by applying the model to data on exposures of D. radiodurans to heavy ions with linear energy transfer (LET) of 18.5–11,300 keV/μm. The model adequately describes these data using three
parameters combinations: radiogenic DNA damage induction, repair protein inactivation and cellular repair capacity. Although
statistical uncertainties around best-fit parameter estimates are substantial, the behaviors of model parameters are consistent
with current knowledge of LET effects: inactivation cross-sections for both DNA and proteins increase with increasing LET;
DNA damage yield per unit of radiation dose also increases with LET; protein damage per unit dose tends to decrease with LET;
DNA and especially protein damage yields are reduced when cells are irradiated in the dry state. These results suggest that
synergism between oxidative stress and DNA damage may play an important role not only during γ-ray exposure, but during high-LET
radiation exposure as well. 相似文献
9.
Jimin Wang 《Protein science : a publication of the Protein Society》2016,25(9):1585-1592
It is common knowledge that macromolecular crystals are damaged by the X‐rays they are exposed to during conventional data collection. One of the claims made about the crystallographic data collection now being collected using X‐ray free‐electron lasers (XFEL) is that they are unaffected by radiation damage. XFEL data sets are assembled by merging data obtained from a very large number of crystals, each of which is exposed to a single femtosecond pulse of radiation, the duration of which is so short that diffraction occurs before the damage done to the crystal has time to become manifest, i.e. “diffraction‐before‐destruction.” However, recent theoretical studies have shown that many of the elemental electronic processes that ultimately result in the destruction of such crystals occur during a single pulse. It is predicted that the amplitudes of atomic scattering factor could be reduced by as much as 75% within the first 5 femtoseconds of such pulses, and that different atoms will respond in different ways. Experimental evidence is provided here that these predictions are correct. 相似文献
10.
High resolution protein crystallography using synchrotron radiation is one of the most powerful tools in modern biology. Improvements in resolution have arisen from the use of X-ray beamlines with higher brightness and flux and the development of advanced detectors. However, it is increasingly recognised that the benefits brought by these advances have an associated cost, namely deleterious effects of X-ray radiation on the sample (radiation damage). In particular, X-ray induced reduction and damage to redox centres has been shown to occur much more rapidly than other radiation damage effects, such as loss of resolution or damage to disulphide bridges. Selection of an appropriate combination of in-situ single crystal spectroscopies during crystallographic experiments, such as UV-visible absorption and X-ray absorption spectroscopy (XAFS), allows for effective monitoring of redox states in protein crystals in parallel with structure determination. Such approaches are also essential in cases where catalytic intermediate species are generated by exposure to the X-ray beam. In this article, we provide a number of examples in which multiple single crystal spectroscopies have been key to understanding the redox status of Fe and Cu centres in crystal structures. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State. 相似文献
11.
The peculiarities of radiation response in animals at low environmental temperatures are analyzed in the context of radiation
safety of the Arctic/Northern wildlife. The paper includes a data review on radiation effects in cold environments based on
international and Russian publications since 1948, which forms a supplement to the EPIC and FREDERICA data collections. In
homoiothermic and heterothermic animals, imbalances in thermoregulation caused by ionizing radiation are discussed, which
increase energy loss of animals, and decrease their fitness to the Arctic/Northern climate. In poikilothermic animals, both
radiation damage and recovery are temperature dependant, their rates being slow in the cold environment. At low temperatures,
radiation damage of biological tissues is conserved in hidden form; when the temperature of poikilothermic animal rises to
a normal level, radiation injury is developed rapidly similar to acute dose response. Additionally, a mathematical model is
described, demonstrating the combined effects of chronic radiation exposures and seasonal temperature variations on a fish
population. Computer simulations show that at the same level of irradiation, the overall radiation damage to Arctic/Northern
poikilothermic fish is higher than that to the fish from warm climate. Considering the peculiarities of radiation effects
in the cold climate, the Arctic/Northern fauna might be expected to be more vulnerable to chronic radiation stress compared
to temperate fauna. In the case of acute radiation exposure during winter periods, hibernation of heterothermic and cooling
of poikilothermic animals may provide temporary protection from acute radiation effects. 相似文献
12.
Clustered damage sites other than double-strand breaks (DSBs) have the potential to contribute to deleterious effects of ionizing radiation, such as cell killing and mutagenesis. In the companion article (Semenenko et al., Radiat. Res. 164, 180-193, 2005), a general Monte Carlo framework to simulate key steps in the base and nucleotide excision repair of DNA damage other than DSBs is proposed. In this article, model predictions are compared to measured data for selected low-and high-LET radiations. The Monte Carlo model reproduces experimental observations for the formation of enzymatic DSBs in Escherichia coli and cells of two Chinese hamster cell lines (V79 and xrs5). Comparisons of model predictions with experimental values for low-LET radiation suggest that an inhibition of DNA backbone incision at the sites of base damage by opposing strand breaks is active over longer distances between the damaged base and the strand break in hamster cells (8 bp) compared to E. coli (3 bp). Model estimates for the induction of point mutations in the human hypoxanthine guanine phosphoribosyl transferase (HPRT) gene by ionizing radiation are of the same order of magnitude as the measured mutation frequencies. Trends in the mutation frequency for low- and high-LET radiation are predicted correctly by the model. The agreement between selected experimental data sets and simulation results provides some confidence in postulated mechanisms for excision repair of DNA damage other than DSBs and suggests that the proposed Monte Carlo scheme is useful for predicting repair outcomes. 相似文献
13.
Electron paramagnetic resonance study of radiation damage in photosynthetic reaction center crystals
Electron paramagnetic resonance (EPR) was used to simultaneously study radiation-induced cofactor reduction and damaging radical formation in single crystals of the bacterial reaction center (RC). Crystals of Fe-removed/Zn-replaced RC protein from Rhodobacter ( R.) sphaeroides R26 were irradiated with varied radiation doses at cryogenic temperature and analyzed for radiation-induced free radical formation and alteration of light-induced photosynthetic electron transfer activity using high-field (HF) D-band (130 GHz) and X-band (9.5 GHz) EPR spectroscopies. These analyses show that the formation of radiation-induced free radicals saturated at doses 1 order of magnitude smaller than the amount of radiation at which protein crystals lose their diffraction quality, while light-induced RC activity was found to be lost at radiation doses at least 1 order of magnitude lower than the dose at which radiation-induced radicals exhibited saturation. HF D-band EPR spectra provide direct evidence for radiation-induced reduction of the quinones and possibly other cofactors. These results demonstrate that substantial radiation damage is likely to have occurred during X-ray diffraction data collection used for photosynthetic RC structure determination. Thus, both radiation-induced loss of photochemical activity in RC crystals and reduction of the quinones are important factors that must be considered when correlating spectroscopic and crystallographic measurements of quinone site structures. 相似文献
14.
Fleck CM Schöllnberger H Kottbauer MM Dockal T Prüfert U 《Mathematical biosciences》1999,155(1):13-44
A new model (Random Coincidence Model--Radiation Adapted (RCM-RA)) is proposed which explains a possible pseudo threshold for stochastic radiation effects. It describes the formation of cancer in the case of multistep fixation of lesions in the critical regions of tumor associated genes such as proto-oncogenes or tumor-suppressor genes. The RCM-RA contains two different possibilities of DNA damage to complementary nucleotides. The damage may be caused either by radiation or by natural processes such as cellular radicals or thermal damage or by chemical cytotoxins. The model is based on the premise that radiation initially is bionegative, damaging organisms at their different levels of organization. The radiation, however, also induces various cellular radioprotective mechanisms which decrease the damage by natural processes. Considering both effects together, the theory explains apparent thresholds in the dose-response relation for radiation carcinogenesis without contradiction to the classical assumption that radiation is predominantly bionegative at doses typically found in occupational exposures. 相似文献
15.
Eugenia Pechkova Shailesh Tripathi Raimond B.G. Ravelli Sean McSweeney Claudio Nicolini 《Journal of structural biology》2009,168(3):409-418
A detailed analysis of structural and intensity changes induced by X-ray radiation is presented for two types of proteinase K crystals: crystal grown by classical hanging drop method and those grown by Langmuir–Blodgett (LB) nanotemplate. The comparison of various parameters (e.g. intensity per sigma ratio, unit-cell volume, number of unique reflections, B-factors) and electron density maps as a function of radiation dose, demonstrates that crystals, grown by the LB nanotemplate method, appear to be more resistant against radiation damage than crystals grown by the classical hanging drop method. 相似文献
16.
Electron microscopy was applied to thin crystals of yeast tRNAPhe. The crystals embedded in glucose yield Bragg reflections with a spacing smaller than 4 A. The measurement of radiation damage rate demonstrates that they are 4 to 14 times less susceptible to electron exposures than protein crystals embedded in glucose. 相似文献
17.
Vives i Batlle J 《Radiation and environmental biophysics》2012,51(3):225-243
In the present paper, a two-age-class group, logistic growth model for generic populations of non-human biota is described in order to assess non-stochastic effects of low linear energy-transfer radiation using three endpoints: repairable radiation damage, impairment of reproductive ability and, at higher radiation dose rates, mortality. This model represents mathematically the exchange between two life stages considering fecundity, growth and mortality. Radiation effects are modeled with a built-in self-recovery pool whereupon individuals can repair themselves. In acute effects mode, the repairing pool becomes depleted due to radiation and the model tends to lethality mode. A base calibration of the model's two free parameters is possible assuming that in acute mode 50% of the individuals die on 30 days when a radiation dose equal to the LD(50/30) is applied during that period. The model, which requires 10 species-dependent life-history parameters, was applied to fish and mammals. Its use in the derivation of dose-rate screening values for the protection of non-human biota from the effects of ionizing radiation is demonstrated through several applications. First, results of model testing with radiation effects data for fish populations from the EPIC project show the predictive capability of the model in a practical case. Secondly, the model was further verified with FREDERICA radiation effects data for mice and voles. Then, consolidated predictions for mouse, rabbit, dog and deer were generated for use in a population model comparison made within the IAEA EMRAS II project. Taken together, model predictions suggest that radiation effects are more harmful for larger organisms that generate lower numbers of offspring. For small mammal and fish populations, dose rates that are below 0.02 Gy day(-1) are not fatal; in contrast, for large mammals, chronic exposure at this level is predicted to be harmful. At low exposure rates similar to the ERICA screening dose rate of 2.4 × 10(-4) Gy day(-1), long-term effects on the survivability of populations are negligible, supporting the appropriateness of this value for radiological assessments to wildlife. 相似文献
18.
Bill Pedrini Ching-Ju Tsai Guido Capitani Celestino Padeste Mark S. Hunter Nadia A. Zatsepin Anton Barty W. Henry Benner Sébastien Boutet Geoffrey K. Feld Stefan P. Hau-Riege Richard A. Kirian Christopher Kupitz Marc Messerschmitt John I. Ogren Tommaso Pardini Brent Segelke Garth J. Williams John C. H. Spence Rafael Abela Matthew Coleman James E. Evans Gebhard F. X. Schertler Matthias Frank Xiao-Dan Li 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2014,369(1647)
Membrane proteins arranged as two-dimensional crystals in the lipid environment provide close-to-physiological structural information, which is essential for understanding the molecular mechanisms of protein function. Previously, X-ray diffraction from individual two-dimensional crystals did not represent a suitable investigational tool because of radiation damage. The recent availability of ultrashort pulses from X-ray free-electron lasers (XFELs) has now provided a means to outrun the damage. Here, we report on measurements performed at the Linac Coherent Light Source XFEL on bacteriorhodopsin two-dimensional crystals mounted on a solid support and kept at room temperature. By merging data from about a dozen single crystal diffraction images, we unambiguously identified the diffraction peaks to a resolution of 7 Å, thus improving the observable resolution with respect to that achievable from a single pattern alone. This indicates that a larger dataset will allow for reliable quantification of peak intensities, and in turn a corresponding increase in the resolution. The presented results pave the way for further XFEL studies on two-dimensional crystals, which may include pump–probe experiments at subpicosecond time resolution. 相似文献
19.
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 A 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 A resolution has a diameter of about 20 microm (100 A unit cell). 相似文献
20.
The majority of 3D structures of macromolecules are currently determined by macromolecular crystallography, which employs the diffraction of X-rays on single crystals. However, during diffraction experiments, the X-rays can damage the protein crystals by ionization processes, especially when powerful X-ray sources at synchrotron facilities are used. This process of radiation damage generates photo-electrons that can get trapped in protein moieties. The 3D structure derived from such experiments can differ remarkably from the structure of the native molecule. Recently, the crystal structures of different oxidation states of horseradish peroxidase and nickel-containing superoxide dismutase were determined using crystallographic redox titration performed during the exposure of the crystals to the incident X-ray beam. Previous crystallographic analyses have not shown the distinct structures of the active sites associated with the redox state of the structural features of these enzymes. These new studies show that, for protein moieties that are susceptible to radiation damage and prone to reduction by photo-electrons, care is required in both the design of the diffraction experiment and the analysis and interpretation. 相似文献