experiments preliminary to the design of an X-ray-excited optical
luminescence (XEOL)-based chemical mapping tool we have used X-ray
micro (4.5 × 5.2 μm) and macro (1 × 6 mm) beams with
similar total fluxes to assess the effects of a high flux density
beam of X-rays at energies close to an absorption edge on inorganic
surfaces in air. The near surface composition of corroded cupreous
alloys was analyzed using parallel X-ray and optical photoemission
channels to collect X-ray absorption near-edge structure (XANES) data
at the Cu K edge. The X-ray fluorescence channel is characteristic
of the composition averages over several micrometers into the surface,
whereas the optical channel is surface specific to about 200 nm. While
the X-ray fluorescence data were mostly insensitive to the X-ray dose,
the XEOL-XANES data from the microbeam showed significant dose-dependent
changes to the superficial region, including surface cleaning, changes
in the oxidation state of the copper, and destruction of surface compounds
responsible for pre-edge fluorescence or phosphorescence in the visible.
In one case, there was evidence that the lead phase in a bronze had
melted. Conversely, data from the macrobeam were stable over several
hours. Apart from localized heating effects, the microbeam damage
is probably associated with the O3 loading of the surface
and increased reaction rate with atmospheric water vapor.
An online UV–visible microspectrophotometer has been developed for the macromolecular crystallography beamline at SPring-8. Details of this spectrophotometer are reported.
Measurement of the UV–visible absorption spectrum is a convenient technique for detecting chemical changes of proteins, and it is therefore useful to combine spectroscopy and diffraction studies. An online microspectrophotometer for the UV–visible region was developed and installed on the macromolecular crystallography beamline, BL38B1, at SPring-8. This spectrophotometer is equipped with a difference dispersive double monochromator, a mercury–xenon lamp as the light source, and a photomultiplier as the detector. The optical path is mostly constructed using mirrors, in order to obtain high brightness in the UV region, and the confocal optics are assembled using a cross-slit diaphragm like an iris to eliminate stray light. This system can measure optical densities up to a maximum of 4.0. To study the effect of radiation damage, preliminary measurements of glucose isomerase and thaumatin crystals were conducted in the UV region. Spectral changes dependent on X-ray dose were observed at around 280 nm, suggesting that structural changes involving Trp or Tyr residues occurred in the protein crystal. In the case of the thaumatin crystal, a broad peak around 400 nm was also generated after X-ray irradiation, suggesting the cleavage of a disulfide bond. Dose-dependent spectral changes were also observed in cryo-solutions alone, and these changes differed with the composition of the cryo-solution. These responses in the UV region are informative regarding the state of the sample; consequently, this device might be useful for X-ray crystallography.
UV–visible spectroscopy; protein crystallography; radiation damage; microspectroscopy; SPring-8
A portable and readily aligned online microspectrophotometer that can be easily installed on macromolecular crystallography beamlines is described. It allows measurement of the spectral characteristics of macromolecular crystals prior, during, and after the X-ray diffraction experiment.
X-rays can produce a high concentration of radicals within cryo-cooled macromolecular crystals. Some radicals have large extinction coefficients in the visible (VIS) range of the electromagnetic spectrum, and can be observed optically and spectrally. An online microspectrophotometer with high temporal resolution has been constructed that is capable of measuring UV/VIS absorption spectra (200–1100 nm) during X-ray data collection. The typical X-ray-induced blue colour that is characteristic of a wide range of cryo-conditions has been identified as trapped solvated electrons. Disulphide-containing proteins are shown to form disulphide radicals at millimolar concentrations, with absorption maxima around 400 nm. The solvated electrons and the disulphide radicals seem to have a lifetime in the range of seconds up to minutes at 100 K. The temperature dependence of the kinetics of X-ray-induced radical formation is different for the solvated electrons compared with the disulphide radicals. The online microspectrophotometer provides a technique complementary to X-ray diffraction for analysing and characterizing intermediates and redox states of proteins and enzymes.
radiation damage; macromolecular crystallography; online microspectrophotometry
Data on the rapid reduction of haem proteins in the X-ray beam at synchrotron sources are presented. The use of single-crystal spectroscopy to detect these changes and their implication for diffraction data collection from oxidized species is also discussed.
The structural information and functional insight obtained from X-ray crystallography can be enhanced by the use of complementary spectroscopies. Here the information that can be obtained from spectroscopic methods commonly used in conjunction with X-ray crystallography and best-practice single-crystal UV-Vis absorption data collection are briefly reviewed. Using data collected with the in situ system at the Swiss Light Source, the time and dose scales of low-dose X-ray-induced radiation damage and solvated electron generation in metalloproteins at 100 K are investigated. The effect of dose rate on these scales is also discussed.
macromolecular crystallography; single-crystal microspectrophotometry; radiation damage; myoglobin; cytochrome c
XAC1151, a small heat-shock protein from X. axonopodis pv. citri belonging to the α-crystallin family, was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 Å resolution using a synchrotron-radiation source.
The hspA gene (XAC1151) from Xanthomonas axonopodis pv. citri encodes a protein of 158 amino acids that belongs to the small heat-shock protein (sHSP) family of proteins. These proteins function as molecular chaperones by preventing protein aggregation. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 Å resolution using a synchrotron-radiation source. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 128.7, c = 55.3 Å. The crystal structure was solved by molecular-replacement methods. Structure refinement is in progress.
XAC1151; small heat-shock proteins; α-crystallins; Xanthomonas axonopodis
A comparison of X-ray diffraction and radiographic techniques for the location and characterization of protein crystals is demonstrated on membrane protein crystals mounted within lipid cubic phase material.
The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.
membrane proteins; lipid cubic phase; microradiography; microtomography
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 μs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
(170.7160) Ultrafast technology; (170.7440) X-ray imaging; (140.3450) Laser-induced chemistry; (140.7090) Ultrafast lasers; (170.0170) Medical optics and biotechnology
X-ray Luminescence CT (XLCT) is a hybrid imaging modality combining x-ray and optical imaging in which x-ray luminescent nanophosphors (NPs) are used as emissive imaging probes. NPs are easily excited using common CT energy x-ray beams, and the NP luminescence is efficiently collected using sensitive light based detection systems. XLCT can be recognized as a close analog to fluorescence diffuse optical tomography (FDOT). However, XLCT has remarkable advantages over FDOT due to the substantial excitation penetration depths provided by x-rays relative to laser light sources, long term photo-stability of NPs, and the ability to tune NP emission within the NIR spectral window. Since XCLT uses an x-ray pencil beam excitation, the emitted light can be measured and back-projected along the x-ray path during reconstruction, where the size of the X-ray pencil beam determines the resolution for XLCT. In addition, no background signal competes with NP luminescence (i.e., no auto fluorescence) in XLCT. Currently, no small animal XLCT system has been proposed or tested. This paper investigates an XLCT system built and integrated with a dual source micro-CT system. Two novel sampling paradigms that result in more efficient scanning are proposed and tested via simulations. Our preliminary experimental results in phantoms indicate that a basic CT-like reconstruction is able to recover a map of the NP locations and differences in NP concentrations. With the proposed dual source system and faster scanning approaches, XLCT has the potential to revolutionize molecular imaging in preclinical studies.
Micro-CT; dual energy; small animal imaging; nanophosphor
An α/β-type small, acid-soluble spore protein (SASP) from Bacillus subtilis, a major source of DNA protection against damaging effects in spores, was crystallized in a functionally relevant complex with a double-stranded DNA. This report provides insights into initial characterization of the complex and its structure elucidation.
An engineered variant of an α/β-type small acid-soluble spore protein (SASP) from Bacillus subtilis was crystallized in a complex with a ten-base-pair double-stranded DNA by the hanging-drop vapor-diffusion method using ammonium sulfate as a precipitating agent. Crystals grew at 281 K using sodium cacodylate buffer pH 5.5 and these crystals diffracted X-rays to beyond 2.4 Å resolution using synchrotron radiation. The crystallized complex contains two or three SASP molecules bound to one DNA molecule. The crystals belong to the hexagonal space group P6122 or P6522, with unit-cell parameters a = b = 87.0, c = 145.4 Å, α = β = 90.0, γ = 120.0°. Diffraction data were 96.6% complete to 2.4 Å resolution, with an R
sym of 8.5%. Structure solution by the multiwavelength/single-wavelength anomalous dispersion method using isomorphous crystals of selenomethionine-labeled protein is in progress.
small acid-soluble spore protein; spore resistance; DNA; Bacillus subtilis
X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded1-3. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction ‘snapshots’ are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source4. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes5. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (~200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes6. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.
Analysis of a series of diffraction data sets measured from several native as well as nicotinic acid-soaked crystals of trypsin suggests that this potential scavenger does not have any statistically significant effect on the amount of radiation damage incurred in the crystals on X-ray irradiation at 100 K.
Analysis of a series of diffraction data sets measured from four native as well as four nicotinic acid-soaked crystals of trypsin at 100 K shows a high variability in radiation-sensitivity among individual crystals for both nicotinic acid-soaked and native crystals. The level of radiation-sensitivity and the extent of its variability is statistically indistinguishable between the two conditions. This suggests that this potential scavenger does not have any statistically significant effect on the amount of radiation damage incurred in the crystals on X-ray irradiation. This is in contrast to previous results [Kauffmann et al. (2006 ▶), Structure, 14, 1099–1105] where only one crystal specimen was used for each condition (native and nicotinic acid-soaked).
protein crystallography; radiation damage; scavengers; nicotinic acid
Home-based soft X-ray time-resolved scattering experiments with nanosecond time resolution (10 ns) and nanometer spatial resolution were carried out at a table top soft X-ray plasma source (2.2–5.2 nm). The investigated system was the lyotropic liquid crystal C16E7/paraffin/glycerol/formamide/IR 5. Usually, major changes in physical, chemical, and/or optical properties of the sample occur as a result of structural changes and shrinking morphology. Here, these effects occur as a consequence of the energy absorption in the sample upon optical laser excitation in the IR regime. The liquid crystal shows changes in the structural response within few hundred nanoseconds showing a time decay of 182 ns. A decrease of the Bragg peak diffracted intensity of 30% and a coherent macroscopic movement of the Bragg reflection are found as a response to the optical pump. The Bragg reflection movement is established to be isotropic and diffusion controlled (1 μs). Structural processes are analyzed in the Patterson analysis framework of the time-varying diffraction peaks revealing that the inter-lamellar distance increases by 2.7 Å resulting in an elongation of the coherently expanding lamella crystallite. The present studies emphasize the possibility of applying TR-SXRD techniques for studying the mechanical dynamics of nanosystems.
liquid crystal; soft X-rays; time resolved X-ray scattering
Radiation-induced decay of crystal diffraction and additional specific chemical changes of macromolecules forming the crystal lattice are currently two of the main limiting factors in the acquisition of macromolecular diffraction data and macromolecular structure determination. Data-processing and phasing protocols are discussed in the context of radiation-induced changes.
In macromolecular crystallography, the acquisition of a complete set of diffraction intensities typically involves a high cumulative dose of X-ray radiation. In the process of data acquisition, the irradiated crystal lattice undergoes a broad range of chemical and physical changes. These result in the gradual decay of diffraction intensities, accompanied by changes in the macroscopic organization of crystal lattice order and by localized changes in electron density that, owing to complex radiation chemistry, are specific for a particular macromolecule. The decay of diffraction intensities is a well defined physical process that is fully correctable during scaling and merging analysis and therefore, while limiting the amount of diffraction, it has no other impact on phasing procedures. Specific chemical changes, which are variable even between different crystal forms of the same macromolecule, are more difficult to predict, describe and correct in data. Appearing during the process of data collection, they result in gradual changes in structure factors and therefore have profound consequences in phasing procedures. Examples of various combinations of radiation-induced changes are presented and various considerations pertinent to the determination of the best strategies for handling diffraction data analysis in representative situations are discussed.
radiation-induced specific changes; relative B factor; scaling B factor; experimental phasing; synchrotron radiation
Cerenkov luminescence imaging (CLI) has been successfully utilized in various fields of preclinical studies; however, CLI is challenging due to its weak luminescent intensity and insufficient penetration capability. Here, we report the design and synthesis of a type of rare-earth microparticles (REMPs), which can be dually excited by Cerenkov luminescence (CL) resulting from the decay of radionuclides to enhance CLI in terms of intensity and penetration. Methods: Yb3+- and Er3+- codoped hexagonal NaYF4 hollow microtubes were synthesized via a hydrothermal route. The phase, morphology, and emission spectrum were confirmed for these REMPs by power X-ray diffraction (XRD), scanning electron microscopy (SEM), and spectrophotometry, respectively. A commercial CCD camera equipped with a series of optical filters was employed to quantify the intensity and spectrum of CLI from radionuclides. The enhancement of penetration was investigated by imaging studies of nylon phantoms and nude mouse pseudotumor models. Results: the REMPs could be dually excited by CL at the wavelengths of 520 and 980 nm, and the emission peaks overlaid at 660 nm. This strategy approximately doubled the overall detectable intensity of CLI and extended its maximum penetration in nylon phantoms from 5 to 15 mm. The penetration study in living animals yielded similar results. Conclusions: this study demonstrated that CL can dually excite REMPs and that the overlaid emissions in the range of 660 nm could significantly enhance the penetration and intensity of CL. The proposed enhanced CLI strategy may have promising applications in the future.
The effect of the X-ray dose on room-temperature time-resolved Laue data is discussed.
Protein X-ray structures are determined with ionizing radiation that damages the protein at high X-ray doses. As a result, diffraction patterns deteriorate with the increased absorbed dose. Several strategies such as sample freezing or scavenging of X-ray-generated free radicals are currently employed to minimize this damage. However, little is known about how the absorbed X-ray dose affects time-resolved Laue data collected at physiological temperatures where the protein is fully functional in the crystal, and how the kinetic analysis of such data depends on the absorbed dose. Here, direct evidence for the impact of radiation damage on the function of a protein is presented using time-resolved macromolecular crystallography. The effect of radiation damage on the kinetic analysis of time-resolved X-ray data is also explored.
radiation damage; X-ray dose; room temperature; time-resolved crystallography; Laue crystallography
X-ray free-electron lasers (X-FELs) produce X-ray pulses with extremely brilliant peak intensity and ultrashort pulse duration. It has been proposed that radiation damage can be “outrun” by using an ultra intense and short X-FEL pulse that passes a biological sample before the onset of significant radiation damage. The concept of “diffraction-before-destruction” has been demonstrated recently at the Linac Coherent Light Source, the first operational hard X-ray FEL, for protein nanocrystals and giant virus particles. The continuous diffraction patterns from single particles allow solving the classical “phase problem” by the oversampling method with iterative algorithms. If enough data are collected from many identical copies of a (biological) particle, its three-dimensional structure can be reconstructed. We review the current status and future prospects of serial femtosecond crystallography (SFX) and single-particle coherent diffraction imaging (CDI) with X-FELs.
The purpose of this work was to study the effects of crystal structure on the solid-state photoluminescence of the trihydrate phases of ampicillin and amoxicillin, and to contrast these spectra with analogous spectra obtained on the molecules dissolved in a solution phase. The polymorphic identity of the analytes was established using x-ray powder diffraction and Fourier transform infrared absorption spectroscopy, and the solid-state luminescence spectra obtained under ambient conditions. It was found that the solid-state excitation and emission spectra of ampicillin trihydrate and amoxicillin trihydrate were dominated by energy transfer and exciton effects, which were manifested as decreases in the energy of the excitation and emission bands of the solid-state systems relative to those of the free molecule in solution. The photoluminescence data revealed that in spite of the known structural similarity of ampicillin trihydrate and amoxicillin trihydrate, the magnitude of the Davydov splitting, and the degree of band energy shifting differed between the 2 systems. This finding indicates that the small differences in crystal structure existing between the 2 compounds leads to measurable differences in the patterns of energy transfer.
polymorphism; fluorescence spectroscopy; energy transfer; exciton splitting
The crystallization and preliminary X-ray analysis of isomaltase is reported.
Isomaltase from Saccharomyces cerevisiae is an oligo-1,6-glucosidase that preferentially hydrolyzes isomaltose, with little activity towards isomaltotriose or longer oligosaccharides. An amino-acid sequence analysis of the isomaltase revealed that it belongs to glucoside hydrolase family 13. Recombinant isomaltase was purified and crystallized by the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant. The crystals belonged to space group C2, with unit-cell parameters a = 95.67, b = 115.42, c = 61.77 Å, β = 91.17°. X-ray diffraction data were collected to 1.35 Å resolution from a single crystal on a synchrotron-radiation source.
Synchrotron radiation (SR) X-ray has great potential for its applications in both diagnosis and treatment of diseases, due to its characteristic properties including coherence, collimation, monochromaticity, and exceptional brightness. Great advances have been made regarding potential medical applications of SR X-ray in recent years, particularly with the development of the third generation of SR light sources. However, multiple studies have also suggested damaging effects of SR X-ray on biological samples ranging from protein crystals to cells and biological tissues. It has become increasingly important to conduct comprehensive studies on two closely related topics regarding SR X-ray in medical applications: The safety issues regarding the medical applications of SR X-ray and the fundamental mechanisms underlying the interactions between SR X-ray and biological tissues. In this article, we attempted to provide an overview of the literatures regarding these two increasingly significant topics. We also proposed our hypothesis that there are significant differences between the biological tissue-damaging mechanisms of SR X-ray and those of normal X-ray, due to the characteristic properties of SR X-ray such as high dose rate. Future studies are warranted to test this hypothesis, which may profoundly improve our understanding regarding the fundamental mechanisms underlying the interactions between light and matter. These studies would also constitute an essential basis for establishing the safety standard for the medical applications of SR X-ray.
Synchrotron radiation; X-ray; tissue damage; ROS; radiation safety
Optically stimulated luminescence (OSL) properties of dental enamel are discussed with a view to the development of an in-vivo dose assessment technique for medical triage following a radiological/nuclear accident or terrorist event. In the OSL technique, past radiation exposure is assessed by stimulating the sample with light of one wavelength and monitoring the luminescence at another wavelength under the assumption that the luminescence originates from the recombination of radiation-induced charges trapped at metastable defects in the enamel and that the intensity of the luminescence signal is in proportion to the absorbed radiation dose. Several primary findings emerged from this research: (a) sensitivities varied considerably between different teeth and also between fragments of the same tooth, (b) OSL signals were found to decay rapidly during the first 12 hours after irradiation and slower afterwards, (c) the fading rate of the luminescence signal varied between fragments, (d) blue light stimulation yields greater sensitivity than infra-red stimulation, while the OSL signal obtained with a high-intensity pulsed green-light laser was found to be not correlated with the radiation dose. Significant challenges remain to developing a practical in-vivo technique including the development of calibration procedures and lowering minimum detectable doses.
Biodosimetry; optically stimulated luminescence; OSL; radiation; retrospective dosimetry; tooth enamel
The expression, purification and crystallization of the collagen-binding region of the E. rhusiopathiae surface protein RspB is described. The crystals diffracted to 2.2 Å resolution using synchrotron radiation.
RspB is a surface adhesin of Erysipelothrix rhusiopathiae. A recombinant form of the collagen-binding region of this protein, RspB(31–348), has been overexpressed in Escherichia coli in native and selenomethionine-derivative forms and purified using affinity and gel-permeation chromatography. Thin plate-like crystals were obtained by the hanging-drop vapour-diffusion method using the same condition for both forms. The native crystals diffracted to a resolution of 2.5 Å using an in-house X-ray source, while the selenomethionine-derivative crystals diffracted to a resolution of 2.2 Å using synchrotron radiation. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 46.19, b = 66.65, c = 101.72 Å, β = 94.11°.
RspB; Erysipelothrix rhusiopathiae; collagen binding
A retrospective analysis of radiation damage behaviour in a statistically significant number of real-life datasets is presented, in order to gauge the importance of the complications not yet measured or rigorously evaluated in current experiments, and the challenges that remain before radiation damage can be considered a problem solved in practice.
The radiation damage behaviour in 43 datasets of 34 different proteins collected over a year was examined, in order to gauge the reliability of decay metrics in practical situations, and to assess how these datasets, optimized only empirically for decay, would have benefited from the precise and automatic prediction of decay now possible with the programs RADDOSE [Murray, Garman & Ravelli (2004 ▶). J. Appl. Cryst.
37, 513–522] and BEST [Bourenkov & Popov (2010 ▶). Acta Cryst. D66, 409–419]. The results indicate that in routine practice the diffraction experiment is not yet characterized well enough to support such precise predictions, as these depend fundamentally on three interrelated variables which cannot yet be determined robustly and practically: the flux density distribution of the beam; the exact crystal volume; the sensitivity of the crystal to dose. The former two are not satisfactorily approximated from typical beamline information such as nominal beam size and transmission, or two-dimensional images of the beam and crystal; the discrepancies are particularly marked when using microfocus beams (<20 µm). Empirically monitoring decay with the dataset scaling B factor (Bourenkov & Popov, 2010 ▶) appears more robust but is complicated by anisotropic and/or low-resolution diffraction. These observations serve to delineate the challenges, scientific and logistic, that remain to be addressed if tools for managing radiation damage in practical data collection are to be conveniently robust enough to be useful in real time.
radiation damage; data collection; strategy; beamline software; datasets
The new version MS2 of the in situ on-axis micro-spectrophotometer at the macromolecular crystallography beamline X10SA of the Swiss Light Source supports the concurrent acquisition of Raman, resonance Raman, fluorescence and UV/Vis absorption spectra along with diffraction data.
The combination of X-ray diffraction experiments with optical methods such as Raman, UV/Vis absorption and fluorescence spectroscopy greatly enhances and complements the specificity of the obtained information. The upgraded version of the in situ on-axis micro-spectrophotometer, MS2, at the macromolecular crystallography beamline X10SA of the Swiss Light Source is presented. The instrument newly supports Raman and resonance Raman spectroscopy, in addition to the previously available UV/Vis absorption and fluorescence modes. With the recent upgrades of the spectral bandwidth, instrument stability, detection efficiency and control software, the application range of the instrument and its ease of operation were greatly improved. Its on-axis geometry with collinear X-ray and optical axes to ensure optimal control of the overlap of sample volumes probed by each technique is still unique amongst comparable facilities worldwide and the instrument has now been in general user operation for over two years.
macromolecular crystallography; single-crystal spectroscopy; micro-spectrophotometry; complementary techniques; Raman spectroscopy
Selenomethionine-substituted IDH was crystallized using the microbatch method. The crystals diffracted to beyond 2.0 Å resolution using synchrotron radiation.
Inositol dehydrogenase (IDH) is an enzyme that catalyses the NAD+-dependent oxidation of myo-inositol to scyllo-inosose. The enzyme has been purified to homogeneity by means of Ni2+-affinity chromatography and was crystallized in both native and selenomethionine (SeMet) labelled forms using the microbatch method. SAD X-ray diffraction data were collected to 2.0 Å resolution from a SeMet-labelled crystal at the Advanced Photon Source (APS) and a MAD data set was collected to 1.75 Å resolution at the Canadian Light Source (CLS); this is the first reported anomalous diffraction experiment from the CLS. The crystals belong to space group I222 and contain one molecule per asymmetric unit.
inositol dehydrogenases; Bacillus subtilis