Real‐space analysis of radiation‐induced specific changes with independent component analysis

A method of analysis is presented that allows for the separation of specific radiation‐induced changes into distinct components in real space. The method relies on independent component analysis (ICA) and can be effectively applied to electron density maps and other types of maps, provided that they can be represented as sets of numbers on a grid. Here, for glucose isomerase crystals, ICA was used in a proof‐of‐concept analysis to separate temperature‐dependent and temperature‐independent components of specific radiation‐induced changes for data sets acquired from multiple crystals across multiple temperatures. ICA identified two components, with the temperature‐independent component being responsible for the majority of specific radiation‐induced changes at temperatures below 130 K. The patterns of specific temperature‐independent radiation‐induced changes suggest a contribution from the tunnelling of electron holes as a possible explanation. In the second case, where a group of 22 data sets was collected on a single thaumatin crystal, ICA was used in another type of analysis to separate specific radiation‐induced effects happening on different exposure‐level scales. Here, ICA identified two components of specific radiation‐induced changes that likely result from radiation‐induced chemical reactions progressing with different rates at different locations in the structure. In addition, ICA unexpectedly identified the radiation‐damage state corresponding to reduced disulfide bridges rather than the zero‐dose extrapolated state as the highest contrast structure. The application of ICA to the analysis of specific radiation‐induced changes in real space and the data pre‐processing for ICA that relies on singular value decomposition, which was used previously in data space to validate a two‐component physical model of X‐ray radiation‐induced changes, are discussed in detail. This work lays a foundation for a better understanding of protein‐specific radiation chemistries and provides a framework for analysing effects of specific radiation damage in crystallographic and cryo‐EM experiments.     

Raman spectroscopic study on sodium hyaluronate: an effect of proton and γ irradiation

Raman spectroscopy was applied to the analysis of structural changes in lyophilised sodium hyaluronate after proton and γ irradiation (0.5, 5, 50, 100, 200 and 600 Gy). Characteristic Raman bands of the polysaccharide were sensitive to irradiation. Significant damage was observed at doses of 50 Gy or higher. The spectral changes confirmed radiation‐induced loss of native solution conformation, destruction of primary structure, fragmentation, cross‐linking and elimination of functional groups. Differences in the effects of proton and γ radiation on sodium hyaluronate are discussed. Copyright © 2010 John Wiley & Sons, Ltd.     

Letter to the editor

Abstract

According to a molecular dynamics (MD) technique, radiation effects of an amorphous Pd₈₀Si₂₀ alloy induced by N ion were simulated in order to study its structural change observed experimentally before and after irradiation. This experimental structural change was well reproduced by the isobaric MD simulation and by a proposed model. Simultaneously, the formation process of radiation-induced damage was investigated in the simulation.     

Effects of soft X‐ray radiation damage on paraffin‐embedded rat tissues supported on ultralene: a chemical perspective

Radiation damage is an important aspect to be considered when analysing biological samples with X‐ray techniques as it can induce chemical and structural changes in the specimens. This work aims to provide new insights into the soft X‐ray induced radiation damage of the complete sample, including not only the biological tissue itself but also the substrate and embedding medium, and the tissue fixation procedure. Sample preparation and handling involves an unavoidable interaction with the sample matrix and could play an important role in the radiation‐damage mechanism. To understand the influence of sample preparation and handling on radiation damage, the effects of soft X‐ray exposure at different doses on ultralene, paraffin and on paraffin‐embedded rat tissues were studied using Fourier‐transform infrared (FTIR) microspectroscopy and X‐ray microscopy. Tissues were preserved with three different commonly used fixatives: formalin, glutaraldehyde and Karnovsky. FTIR results showed that ultralene and paraffin undergo a dose‐dependent degradation of their vibrational profiles, consistent with radiation‐induced oxidative damage. In addition, formalin fixative has been shown to improve the preservation of the secondary structure of proteins in tissues compared with both glutaraldehyde and Karnovsky fixation. However, conclusive considerations cannot be drawn on the optimal fixation protocol because of the interference introduced by both substrate and embedding medium in the spectral regions specific to tissue lipids, nucleic acids and carbohydrates. Notably, despite the detected alterations affecting the chemical architecture of the sample as a whole, composed of tissue, substrate and embedding medium, the structural morphology of the tissues at the micrometre scale is essentially preserved even at the highest exposure dose.     

Icosahedral-Cluster-Reinforced Structures in Irradiated Metallic Materials

A nonequilibrium state has been discovered which is induced by ion irradiation in metallic materials (solid solutions of Fe–Ni, Fe–Cr–Ni, Ni–Cr, Cu–Ni, Fe–Cr, and V–Ti–Cr systems and in pure metals Zr and Ti) at high levels of radiation damage, and the features of this state are considered. In the region of existence of this state, both the ion and the electron subsystems of the metal show highly anomalous properties. Moreover, the occurrence of this state is accompanied by substantial diffraction effects – X-ray line splitting – and, as indicated by electron microscopy, by the formation of a cluster structure. Simulation by the methods of molecular dynamics suggests that the clusters observed are atomic groups of icosahedral (quintuple) symmetry formed in the neighborhood of radiation vacancies. These clusters reinforce the matrix, and this should result in substantial changes in strength and electronic properties of the material. The results of the computer simulation agree with the observed diffraction effects.     

Raman spectroscopic study of serum albumins: an effect of proton‐ and γ‐irradiation

Raman spectroscopy was applied to analyse structural changes in serum albumins (bovine serum albumin, BSA; human serum albumin, HSA) following proton and γ‐irradiation (0.5, 5 and 50 Gy). Characteristic Raman bands of both polypeptide backbone and amino acid residues were sensitive to irradiation. Significant damage of HSA/BSA was observed only at the highest dose (50 Gy). Raman spectra confirmed radiation‐induced denaturation, destruction of helical structures and aggregation of serum albumins. The differences in the dose‐dependent effects of proton and γ‐radiation on studied proteins are discussed. Copyright © 2007 John Wiley & Sons, Ltd.     

EELS characterization of radiolytic products in frozen samples

Electron energy loss spectroscopy (EELS) was used to obtain information about the radiation chemistry of frozen aqueous specimens in the electron microscope by observing the hydrogen and oxygen K-edges. Measurements on frozen solutions of 30% hydrogen peroxide revealed the presence of molecular oxygen identified by a distinct 531-eV peak at the O K-edge even for electron doses below 100 e/nm2. The molecular oxygen content of irradiated H?O? solution was determined by least squares fitting of O K-edge reference spectra from water and gas-phase oxygen. It was found that the fraction of molecular oxygen to water oxygen was in the range 0.03-0.05. EELS from pure frozen water showed no features attributable to molecular oxygen or molecular hydrogen (K edge at ~13 eV) even at high electron doses above 10? e/nm2. Spectra from frozen sucrose and protein solutions and their mixtures, however, did show evolution of a molecular hydrogen peak at ~13 eV for doses above 10? e/nm2, consistent with previous measurements and indicative of hydrogen bubble formation. Molecular oxygen was not observed in any of the frozen solutions of organic compounds indicating that oxygen is not a major product of free radical decay, in contrast to molecular hydrogen formation.     

Light flux density threshold at which protein denaturation is induced by synchrotron radiation circular dichroism beamlines

New high‐flux synchrotron radiation circular dichroism (SRCD) beamlines are providing important information for structural biology, but can potentially cause denaturation of the protein samples under investigation. This effect has been studied at the new CD1 dedicated SRCD beamline at ISA in Denmark, where radiation‐induced thermal damage effects were observed, depending not only on the radiation flux but also on the focal spot size of the light. Comparisons with similar studies at other SRCD facilities worldwide has lead to the estimation of a flux density threshold under which SRCD beamlines should be operated when samples are to be exposed to low‐wavelength vacuum ultraviolet radiation for extended periods of time.     

Mechanical modifications in dense polyethylene induced by energetic electron beams

Effects of radiation damage in ultra high molecular weight polyethylene are investigated by using energetic electron beams. Special attention is devoted to the mechanical characterization of the polymer, before and after the modification induced by 5 MeV electrons, as a function of the absorbed dose. Elastic modules, ultimate tensile and compressive strengths and roughness and hardness, have been measured in pristine and electron irradiated polymers. Infrared spectroscopy, scanning electron microscopy and differential scanning calorimetry are employed in order to investigate the microscopic modifications induced by the electron energy release to the polymeric chains. Results indicate that the polymer loses hydrogen and becomes rich in carbon content. Cross-linking effects are responsible for the higher mechanical resistance, fragility and hardness of the polymer submitted to a high absorbed dose.     

Raman spectroscopic study of calf thymus DNA: an effect of proton‐ and γ‐irradiation

Raman spectroscopy was applied to analyse structural changes in calf thymus double‐stranded deoxyribonucleic acid (dsDNA) after proton‐ and γ‐irradiation (0.5, 5 and 50 Gy). Characteristic Raman bands of phosphodiester linkages, nucleic bases and deoxyribose moieties were sensitive to irradiation. A significant damage of the macromolecules was observed only at the highest dose (50 Gy) of both types of radiations. Spectral changes confirmed a radiation‐induced alteration of the native structure of dsDNA. Nucleic bases, especially pyrimidines, were the most sensitive to radiation, while some alterations in the sugar–phosphate backbone were also detected. The differences in the dose‐dependent effects of proton vs γ‐irradiation on studied biomolecule are discussed. Copyright © 2007 John Wiley & Sons, Ltd.     

Radiation damage to protein specimens from electron beam imaging and diffraction: a mini‐review of anti‐damage approaches,with special reference to synchrotron X‐ray crystallography

Recent research progress using X‐ray cryo‐crystallography with the photon beams from third‐generation synchrotron sources has resulted in recognition that this intense radiation commonly damages protein samples even when they are held at 100 K. Other structural biologists examining thin protein crystals or single particle specimens encounter similar radiation damage problems during electron diffraction and imaging, but have developed some effective countermeasures. The aim of this concise review is to examine whether analogous approaches can be utilized to alleviate the X‐ray radiation damage problem in synchrotron macromolecular crystallography. The critical discussion of this question is preceded by presentation of background material on modern technical procedures with electron beam instruments using 300–400 kV accelerating voltage, low‐dose exposures for data recording, and protection of protein specimens by cryogenic cooling; these practical approaches to dealing with electron radiation damage currently permit best resolution levels of 6 Å (0.6 nm) for single particle specimens, and of 1.9 Å for two‐dimensional membrane protein crystals. Final determination of the potential effectiveness and practical value of using such new or unconventional ideas will necessitate showing, by experimental testing, that these produce significantly improved protection of three‐dimensional protein crystals during synchrotron X‐ray diffraction.     

Evidence for a cubic-to-icosahedral transition of quasi-free Pd-H-clusters controlled by the hydrogen content

An in situ synchrotron radiation study of quasi-free five nanometer-sized palladium clusters during hydrogen absorption is combined with molecular dynamics simulations to investigate the structural development. In the diffraction patterns, strong intensity changes are found that provide evidence for a structural phase transformation that is significantly different from the α--Pd-H bulk phase transition. The structural transition is reversible and driven by the hydrogen concentration. The intensity changes are consistent with a cubic-to-icosahedral structural phase transition obtained in molecular dynamical simulations using embedded-atom-method potentials. Received 15 October 2001 and Received in final form 7 February 2002     

Electron attachment to uracil: effective destruction at subexcitation energies

We demonstrate that electrons at energies below the threshold for electronic excitation (<3 eV) effectively decompose gas phase uracil generating a mobile hydrogen radical and the corresponding closed shell uracil fragment anion (U-H)(-). The reaction is energetically driven by the large electron affinity of the (U-H) radical. This observation has significant consequences for the molecular picture of radiation damage, i.e., genotoxic effects or damage of living cells due to the secondary component of high energy radiation.     

Influence of irradiation conditions on the retention of hydrogen isotopes in structural materials

The influence of irradiation conditions on the retention of hydrogen isotopes in structural materials (austenitic steel) under heating is considered. The specimens under study were irradiated either in a reactor or by bombarding them with hydrogen-isotope ions of variable fluence and energy at accelerators. An investigation of irradiated specimens with an EM-300 transition electron microscope was accompanied by studying the kinetics of hydrogen release from samples with a high-vacuum mass spectrometer. Also, the kinetics of hydrogen-isotope release from specimens of structural materials treated with a deuterium plasma was studied. It was found that, under the effect of irradiation, the materials being studied develop radiation defects, which appear to be efficient traps for hydrogen atoms, retaining them up to rather high temperatures (650 K). It is also shown that blisters formed in the materials treated with a hydrogen plasma contain both molecular hydrogen and hydrocarbons—in particular, methane.     

Mutagenic effect of accelerated heavy ions on bacterial cells

The heavy ion accelerators of the Joint Institute for Nuclear Research were used to study the regularities and mechanisms of formation of different types of mutations in prokaryote cells. The induction of direct (lac⁻, ton B⁻, col B) mutations for Esherichia coli cells and reverse his⁻ → His⁺ mutations of Salmonella typhimurium, Bacillus subtilis cells under the action of radiation in a wide range of linear energy transfer (LET) was studied. The regularities of formation of gene and structural (tonB trp-) mutations for Esherichia coli bacteria under the action of accelerated heavy ions were studied. It was demonstrated that the rate of gene mutations as a function of the dose under the action of Γ rays and accelerated heavy ions is described by linear-quadratic functions. For structural mutations, linear “dose-effect” dependences are typical. The quadratic character of mutagenesis dose curves is determined by the “interaction” of two independent “hitting” events in the course of SOS repair of genetic structures. The conclusion made was that gene mutations under the action of accelerated heavy ions are induced by δ electron regions of charged particle tracks. The methods of SOS chromotest, SOS lux test, and λ prophage induction were used to study the regularities of SOS response of cells under the action of radiations in a wide LET range. The following proposition was substantiated: the molecular basis for formation of gene mutations are cluster single-strand DNA breaks, and that for structural mutations, double-strand DNA breaks. It was found out that the LET dependence of the relative biological efficiency of accelerated ions is described by curves with a local maximum. It was demonstrated that the biological efficiency of ionizing radiations with different physical characteristics on cells with different genotype, estimated by the lethal action, induction of gene and deletion mutations, precision excision of transposons, is determined by the specific features of energy transfer of the radiations that affect the character of induced DNA damage, and the efficiency inducible and constitutive cell repair systems. The growth of relative biological efficiency of heavy charged particles is determined by the growth of the damage yield of the DNA participating in the formation of radiation-induced effects, and higher efficiency of inducible repair systems. It was established that the LET value (L _max) for which the maximum (according to the applied irradiation criteria) coefficients of relative biological efficiency are observed varies depending on the character of the registered radiation induced effect. It was demonstrated that for gene mutations and induction of precision excision of mobile elements the values of L _max are realized in a LET range of ≈20 keV/μm. For lethal effects of irradiation and induction of deletion mutations the value of L _max is ≈ 100 and 50 keV/μm, respectively. The differences in the L _max for the studied radiation gene effectis are determined by the different type of DNA damage participating in the mutation process. A molecular model of the formation of gene mutations in Escherichia coli cells under the action of ionizing radiation was proposed. Basic DNA radiation damage and main repair ways were considered in the framework of this model. The basis is the idea of the decisive role of mutagenic, error-prone, branch of SOS repair in fixing premutation DNA damage into point mutations. It was demonstrated that the central mechanism in this process is the formation of an inducible multi-enzymatic complex including the DNA polymerase V (Umu C), RecA-protease, SSB proteins, subunits of DNA polymerase III, performing erroneous DNA synthesis on the damaged matrix. A mathematical model of induction of gene mutations under ultraviolet cell irradiation was developed based on the molecular model.     

THz Radiation Generation via the Interaction of Ultra‐short Laser Pulses with the Molecular Hydrogen Plasma

In this paper, a two dimensional Particle In Cell‐Monte Carlo Collision simulation scheme is used to examine the THz generation via the interaction of high intensity ultra‐short laser pulses with an underdense molecular hydrogen plasma slab. The influences of plasma density, laser pulse duration and its intensity on the induced plasma current density and the subsequent effects on the generated THz signal characteristics are studied. It is observed that the induced current density in the plasma medium and THz spectral intensity are increased at the higher laser pulse intensities, laser pulse durations and plasma densities. Moreover, the generated THz electric field amplitude is reduced at the higher laser pulse durations. A wider frequency range for the generated THz signal is shown at the lower laser pulse durations and higher plasma densities. Additionally, it is found that the induced current density in hydrogen plasma medium is the dominant factor influencing the generation of THz pulse radiation. (© 2016 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The many faces of radiation‐induced changes

During diffraction experiments even cryo‐cooled protein crystals can be significantly damaged due to chemical and physical changes induced by absorbed X‐ray photons. The character and scale of the observed effects depend strongly on the temperature and the composition of crystals. The absorption of radiation energy results in incremental regular changes to the crystal structure, making its impact on the process of solving the structure strongly correlated with other experimental variables. An understanding of all the dependencies is still limited and does not allow for a precise prediction of the outcome of a particular diffraction experiment. Results are presented of diffraction experiments performed under different experimental conditions. The influence of temperature and crystal composition on different characteristics of radiation damage is analyzed. The observed effects are discussed in terms of their impact on data processing and phasing procedures.     

Radiation damage reveals promising interaction position

High‐resolution structural data of protein inhibitor complexes are the key to rational drug design. Synchrotron radiation allows for atomic resolutions but is frequently accompanied by radiation damage to protein complexes. In this study a human aldose reductase mutant complexed with a bromine‐substituted inhibitor was determined to atomic resolution [Protein Data Bank (PDB) code 3onc ]. Though the radiation dose was moderate, a selective disruption of a bromine–inhibitor bond during the experiment was observed while the protein appears unaffected. A covalent bond to bromine is cleaved and the displaced atom is not scattered throughout the crystal but can most likely be assigned as a bromide to an additional difference electron density peak observed in the structure. The bromide relocates to an adjacent unoccupied site where promising interactions to protein residues stabilize its position. These findings were verified by a second similar structure determined with considerably higher radiation dose (PDB code 3onb ).     

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.     

Effective scavenging at cryotemperatures: further increasing the dose tolerance of protein crystals

The rate of radiation damage to macromolecular crystals at both room temperature and 100 K has previously been shown to be reduced by the use of certain radical scavengers. Here the effects of sodium nitrate, an electron scavenger, are investigated at 100 K. For sodium nitrate at a concentration of 0.5 M in chicken egg‐white lysozyme crystals, the dose tolerance is increased by a factor of two as judged from the global damage parameters, and no specific structural damage to the disulfide bonds is seen until the dose is greatly in excess (more than a factor of five) of the value at which damage appears in electron density maps derived from a scavenger‐free crystal. In the electron density maps, ordered nitrate ions adjacent to the disulfide bonds are seen to lose an O atom, and appear to protect the disulfide bonds. In addition, results reinforcing previous reports on the effectiveness of ascorbate are presented. The mechanisms of action of both scavengers in the crystalline environment are elucidated.