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The recently completed commissioning of an upgraded undulator-based beamline at BioCARS, a national user facility for macromolecular time-resolved X-ray crystallography at the Advanced Photon Source, is described.

BioCARS, a NIH-supported national user facility for macromolecular time-resolved X-ray crystallography at the Advanced Photon Source (APS), has recently completed commissioning of an upgraded undulator-based beamline optimized for single-shot laser-pump X-ray-probe measurements with time resolution as short as 100 ps. The source consists of two in-line undulators with periods of 23 and 27 mm that together provide high-flux pink-beam capability at 12 keV as well as first-harmonic coverage from 6.8 to 19 keV. A high-heat-load chopper reduces the average power load on downstream components, thereby preserving the surface figure of a Kirkpatrick–Baez mirror system capable of focusing the X-ray beam to a spot size of 90 µm horizontal by 20 µm vertical. A high-speed chopper isolates single X-ray pulses at 1 kHz in both hybrid and 24-bunch modes of the APS storage ring. In hybrid mode each isolated X-ray pulse delivers up to ∼4 × 1010 photons to the sample, thereby achieving a time-averaged flux approaching that of fourth-generation X-FEL sources. A new high-power picosecond laser system delivers pulses tunable over the wavelength range 450–2000 nm. These pulses are synchronized to the storage-ring RF clock with long-term stability better than 10 ps RMS. Monochromatic experimental capability with Biosafety Level 3 certification has been retained.

A fast, user-friendly and easily extensible beamline-control system based on a combination of Java Eclipse RCP and EPICS and featuring a user interface similar to that of the SSRL BluIce has been developed at the GM/CA-CAT macromolecular crystallography beamlines in Sector 23 of the Advanced Photon Source.

The trio of macromolecular crystallography beamlines constructed by the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) in Sector 23 of the Advanced Photon Source (APS) have been in growing demand owing to their outstanding beam quality and capacity to measure data from crystals of only a few micrometres in size. To take full advantage of the state-of-the-art mechanical and optical design of these beamlines, a significant effort has been devoted to designing fast, convenient, intuitive and robust beamline controls that could easily accommodate new beamline developments. The GM/CA-CAT beamline controls are based on the power of EPICS for distributed hardware control, the rich Java graphical user interface of Eclipse RCP and the task-oriented philosophy as well as the look and feel of the successful SSRL BluIce graphical user interface for crystallography. These beamline controls feature a minimum number of software layers, the wide use of plug-ins that can be written in any language and unified motion controls that allow on-the-fly scanning and optimization of any beamline com­ponent. This paper describes the ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce, discusses the solutions aimed at streamlining and speeding up operations and gives an overview of the tools that are provided by this new open-source control system for facilitating crystallo­graphic experiments, especially in the field of microcrystallo­graphy.

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.

The improvement of the X-ray beam quality achieved on ID14-4 by the installation of new X-ray optical elements is described.

ID14-4 at the ESRF is the first tunable undulator-based macromolecular crystallography beamline that can celebrate a decade of user service. During this time ID14-4 has not only been instrumental in the determination of the structures of biologically important molecules but has also contributed significantly to the development of various instruments, novel data collection schemes and pioneering radiation damage studies on biological samples. Here, the evolution of ID14-4 over the last decade is presented, and some of the major improvements that were carried out in order to maintain its status as one of the most productive macromolecular crystallography beamlines are highlighted. The experimental hutch has been upgraded to accommodate a high-precision diffractometer, a sample changer and a large CCD detector. More recently, the optical hutch has been refurbished in order to improve the X-ray beam quality on ID14-4 and to incorporate the most modern and robust optical elements used at other ESRF beamlines. These new optical elements will be described and their effect on beam stability discussed. These studies may be useful in the design, construction and maintenance of future X-ray beamlines for macromolecular crystallography and indeed other applications, such as those planned for the ESRF upgrade.

At the Photon Factory macromolecular crystallography beamlines, two new functions, remote monitoring and diffraction image evaluation, have been developed and installed on the beamline controlling system STARS (simple transmission and retrieval system).

Owing to recent advances in high-throughput technology in macromolecular crystallography beamlines, such as high-brilliant X-ray sources, high-speed readout detectors and robotics, the number of samples that can be examined in a single visit to the beamline has increased dramatically. In order to make these experiments more efficient, two functions, remote monitoring and diffraction image evaluation, have been implemented in the macromolecular crystallography beamlines at the Photon Factory (PF). Remote monitoring allows scientists to participate in the experiment by watching from their laboratories, without having to come to the beamline. Diffraction image evaluation makes experiments easier, especially when using the sample exchange robot. To implement these two functions, two independent clients have been developed that work specifically for remote monitoring and diffraction image evaluation. In the macromolecular crystallography beamlines at PF, beamline control is performed using STARS (simple transmission and retrieval system). The system adopts a client–server style in which client programs communicate with each other through a server process using the STARS protocol. This is an advantage of the extension of the system; implementation of these new functions required few modifications of the existing system.

Below 195 K the bacteriorhodopsin photocycle could not be adequately described with exponential kinetics, but required distributed kinetics, previously found in hemoglobin and myoglobin at temperatures below the vitrification point of the surrounding solvent. The aim of this study is to explore which factors cause the switch from this low-temperature regime to the conventional kinetics observed at ambient temperature. The photocycle was monitored by time-resolved FTIR between 180 and 280 K, using the D96N mutant. Depending on the temperature, decay and temporal redistribution of two or three intermediates (L, M, and N) were observed. Above ∼245 K an abrupt change in the kinetic behavior of the photocycle takes place. It does not affect the intermediates present but greatly accelerates their decay. Below ∼240 K a kinetic pattern with partial decay not explainable by conventional kinetics, but suggesting distributed kinetics, was dominant, while above ∼250 K there were no significant deviations from exponential behavior. The ∼245 K critical point is ≥10 K below the freezing point of interbilayer water, and we were unable to correlate it with any FTIR-detectable transition of the lipids. Therefore, we attribute the change from distributed to conventional kinetics to a thermodynamic phase transition in the protein. Most probably, it is related to the freezing/thawing of internal fluctuations of the protein, known as the dynamic phase transition, although in bacteriorhodopsin the latter is usually believed to take place at least 15 K below the observed critical temperature of ∼245 K.

The dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed.

X-ray crystallography provides structural details of biological macromolecules. Whereas routine data are collected close to 100 K in order to mitigate radiation damage, more exotic temperature-controlled experiments in a broader temperature range from 15 K to room temperature can provide both dynamical and structural insights. Here, the dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed. Experimental strategies of kinetic crystallography are discussed that have allowed the generation and trapping of macromolecular intermediate states by combining reaction initiation in the crystalline state with appropriate temperature profiles. A particular focus is on recruiting X-ray-induced changes for reaction initiation, thus unveiling useful aspects of radiation damage, which otherwise has to be minimized in macromolecular crystallography.

The ultimate goal of synchrotron data collection is to obtain the best possible data from the best available crystals, and the combination of automation and remote access at Stanford Synchrotron Radiation Lightsource (SSRL) has revolutionized the way in which scientists achieve this goal. This has also seen a change in the way novice crystallographers are trained in the use of the beamlines, and a wide range of remote tools and hands-on workshops are now offered by SSRL to facilitate the education of the next generation of protein crystallographers.

For the past five years, the Structural Molecular Biology group at the Stanford Synchrotron Radiation Lightsource (SSRL) has provided general users of the facility with fully remote access to the macromolecular crystallography beamlines. This was made possible by implementing fully automated beamlines with a flexible control system and an intuitive user interface, and by the development of the robust and efficient Stanford automated mounting robotic sample-changing system. The ability to control a synchrotron beamline remotely from the comfort of the home laboratory has set a new paradigm for the collection of high-quality X-ray diffraction data and has fostered new collaborative research, whereby a number of remote users from different institutions can be connected at the same time to the SSRL beamlines. The use of remote access has revolutionized the way in which scientists interact with synchrotron beamlines and collect diffraction data, and has also triggered a shift in the way crystallography students are introduced to synchrotron data collection and trained in the best methods for collecting high-quality data. SSRL provides expert crystallographic and engineering staff, state-of-the-art crystallography beamlines, and a number of accessible tools to facilitate data collection and in-house remote training, and encourages the use of these facilities for education, training, outreach and collaborative research.

The PHENIX software for macromolecular structure determination is described.

Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. How­ever, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallo­graphic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.

The Protein Crystallography Station user facility at Los Alamos National Laboratory not only offers open access to a high-performance neutron beamline, but also actively supports and develops new methods in protein expression, deuteration, purification, robotic crystallization and the synthesis of substrates with stable isotopes and provides assistance with data-reduction and structure-refinement software and comprehensive neutron structure analysis.

The Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center is a high-performance beamline that forms the core of a capability for neutron macromolecular structure and function determination. Neutron diffraction is a powerful technique for locating H atoms and can therefore provide unique information about how biological macro­molecules function and interact with each other and smaller molecules. Users of the PCS have access to neutron beam time, deuteration facilities, the expression of proteins and the synthesis of substrates with stable isotopes and also support for data reduction and structure analysis. The beamline exploits the pulsed nature of spallation neutrons and a large electronic detector in order to collect wavelength-resolved Laue patterns using all available neutrons in the white beam. The PCS user facility is described and highlights from the user program are presented.

X-ray crystallography is a critical tool in the study of biological systems. It is able to provide information that has been a prerequisite to understanding the fundamentals of life. It is also a method that is central to the development of new therapeutics for human disease. Significant time and effort are required to determine and optimize many macromolecular structures because of the need for manual interpretation of complex numerical data, often using many different software packages, and the repeated use of interactive three-dimensional graphics. The Phenix software package has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on automation. This has required the development of new algorithms that minimize or eliminate subjective input in favour of built-in expert-systems knowledge, the automation of procedures that are traditionally performed by hand, and the development of a computational framework that allows a tight integration between the algorithms. The application of automated methods is particularly appropriate in the field of structural proteomics, where high throughput is desired. Features in Phenix for the automation of experimental phasing with subsequent model building, molecular replacement, structure refinement and validation are described and examples given of running Phenix from both the command line and graphical user interface.

Photoactive Yellow Protein (PYP), a phototaxis photoreceptor from Ectothiorhodospira halophila, is a small water-soluble protein that iscrystallisable and excellently photo-stable. It can be activated with light(λmax= 446 nm), to enter a series of transientintermediates that jointly form the photocycle of this photosensor protein.The most stable of these transient states is the signalling state forphototaxis, pB.The spatial structure of the ground state of PYP, pG and the spectralproperties of the photocycle intermediates have been very well resolved.Owing to its excellent chemical- and photochemical stability, also the spatialstructure of its photocycle intermediates has been characterised with X-raydiffraction and multinuclear NMR spectroscopy. Surprisingly, the resultsobtained showed that their structure is dependent on the molecular contextin which they are formed. Therefore, a large range of diffraction-,scattering- and spectroscopic techniques is now being employed to resolvein detail the dynamical changes of the structure of PYP while it progressesthrough its photocycle. This approach has led to considerable progress,although some techniques still result in mutually inconsistent conclusionsregarding aspects of the structure of particular intermediates.Recently, significant progress has also been made with simulations withmolecular dynamics analyses of the initial events that occur in PYP uponphoto activation. The great challenge in this field is to eventually obtainagreement between predicted dynamical alterations in PYP structure, asobtained with the MD approach and the actually measured dynamicalchanges in its structure as evolving during photocycle progression.

Accurate measurement of photon flux from an X-ray source is a parameter required to calculate the dose absorbed by a sample. The development of a model for determining the photon flux incident on pin diodes, and experiments to test this model, are described for incident energies between 4 and 18 keV used in macromolecular crystallography.

Accurate measurement of photon flux from an X-ray source, a parameter required to calculate the dose absorbed by the sample, is not yet routinely available at macromolecular crystallography beamlines. The development of a model for determining the photon flux incident on pin diodes is described here, and has been tested on the macromolecular crystallography beamlines at both the Swiss Light Source, Villigen, Switzerland, and the Advanced Light Source, Berkeley, USA, at energies between 4 and 18 keV. These experiments have shown that a simple model based on energy deposition in silicon is sufficient for determining the flux incident on high-quality silicon pin diodes. The derivation and validation of this model is presented, and a web-based tool for the use of the macromolecular crystallography and wider synchrotron community is introduced.

Myoglobin, a small globular heme protein that binds gaseous ligands such asO2, CO and NO reversibly at the heme iron, provides an excellent modelsystem for studying structural and dynamic aspects of protein reactions. Flashphotolysis experiments, performed over wide ranges in time and temperature, reveal a complex ligand binding reaction with multiple kinetic intermediates, resulting from protein relaxation and movements of the ligand within the protein. Our recent studies of carbonmonoxy-myoglobin (MbCO) mutant L29W, using time-resolved infrared spectroscopy in combination with x-ray crystallography, have correlated kinetic intermediates with photoproduct structures that are characterized by the CO residing in different internal protein cavities, so-called xenon holes. Here we have used Fourier transform infrared temperature derivative spectroscopy (FTIR-TDS) to further examine the role of internal cavities in the dynamics. Different cavities can be accessed by the CO ligands at different temperatures, and characteristic infrared absorption spectra have been obtained for the different locations of the CO ligand within the protein, enabling us to monitor ligand migration through the protein as well as conformational changes of the protein.

The folding mechanism of the β-sheet protein CspA, the major cold shock protein of Escherichia coli, was previously reported to be a concerted, two-state process. We have reexamined the folding of CspA using multiple spectroscopic probes of the equilibrium transition and laser-induced temperature jump (T-jump) to achieve better time resolution of the kinetics. Equilibrium temperature-dependent Fourier transform infrared (1634 cm–1) and tryptophan fluorescence measurements reveal probe-dependent thermal transitions with midpoints (Tm) of 66 ± 1 and 61 ± 1 °C, respectively. Singular-value decomposition analysis with global fitting of the temperature-dependent infrared (IR) difference spectra reveals two spectral components with distinct melting transitions with different midpoints. T-Jump relaxation measurements of CspA probed by IR and fluorescence spectroscopy show probe-dependent multiexponential kinetics characteristic of non-two-state folding. The frequency-dependent IR transients all show biphasic relaxation with average time constants of 50 ± 7 and 225 ± 25 μs at a Tf of 77 °C and almost equal amplitudes. Similar biphasic kinetics are observed using Trp fluorescence of the wild-type protein and the Y42W and T68W mutants, with comparable lifetimes. All of these observations support a model for the folding of CspA through a compact intermediate state. The transient IR and fluorescence spectra are consistent with a diffuse intermediate having β-turns and substantial β-sheet structure. The loop β3–β4 structure is likely not folded in the intermediate state, allowing substantial solvent penetration into the barrel structure.

The MiNaXS (P03) beamline of the new third-generation synchrotron radiation source PETRA III (DESY, Germany) has been designed to perform small-, ultra-small- and wide-angle X-ray scattering in both transmission and grazing-incidence geometries. The high photon flux available at the beamline enables time-resolved investigations of kinetic phenomena with a time resolution below 100 ms. The microfocus endstation started user operation in May 2011.

The P03 beamline, also called the microfocus and nanofocus X-ray scattering (MiNaXS) beamline, exploits the excellent photon beam properties of the low-emittance source PETRA III to provide a microfocused/nanofocused beam with ultra-high intensity for time-resolved X-ray scattering experiments. The beamline has been designed to perform X-ray scattering in both transmission and reflection geometries. The microfocus endstation started user operation in May 2011 ▶. An overview of the beamline status and of some representative results highlighting the performance of the microfocus endstation at MiNaXS are given.

Two sample-scanning features have been implemented for the macromolecular crystallography beamlines at APS sector 23: automated diffraction-based rastering employing multiple polygon-shaped two-dimensional grids overlaid on a sample to locate and center small and invisible crystals or to find the best-diffracting regions in a larger crystal, and automated data collection along a three-dimensional vector to mitigate the effects of radiation damage.

Automated scanning capabilities have been added to the data acquisition software, JBluIce-EPICS, at the National Institute of General Medical Sciences and the National Cancer Institute Collaborative Access Team (GM/CA CAT) at the Advanced Photon Source. A ‘raster’ feature enables sample centering via diffraction scanning over two-dimensional grids of simple rectangular or complex polygonal shape. The feature is used to locate crystals that are optically invisible owing to their small size or are visually obfuscated owing to properties of the sample mount. The raster feature is also used to identify the best-diffracting regions of large inhomogeneous crystals. Low-dose diffraction images taken at grid positions are automatically processed in real time to provide a quick quality ranking of potential data-collection sites. A ‘vector collect’ feature mitigates the effects of radiation damage by scanning the sample along a user-defined three-dimensional vector during data collection to maximize the use of the crystal volume and the quality of the collected data. These features are integrated into the JBluIce-EPICS data acquisition software developed at GM/CA CAT where they are used in combination with a robust mini-beam of rapidly changeable diameter from 5 µm to 20 µm. The powerful software–hardware combination is being applied to challenging problems in structural biology.

Background

In kinetic crystallography, the usually static method of X-ray diffraction is expanded to allow time-resolved analysis of conformational rearrangements in protein structures. To achieve this, reactions have to be triggered within the protein crystals of interest, and optical spectroscopy can be used to monitor the reaction state. For this approach, a modified form of H-Ras p21 was designed which allows reaction initiation and fluorescence readout of the initiated GTPase reaction within the crystalline state. Rearrangements within the crystallized protein due to the progressing reaction and associated heterogeneity in the protein conformations have to be considered in the subsequent refinement processes.

Results

X-ray diffraction experiments on H-Ras p21 in different states along the reaction pathway provide detailed information about the kinetics and mechanism of the GTPase reaction. In addition, a very high data quality of up to 1.0 Å resolution allowed distinguishing two discrete subconformations of H-Ras p21, expanding the knowledge about the intrinsic flexibility of Ras-like proteins, which is important for their function. In a complex of H-Ras•GppNHp (guanosine-5'-(β,γ-imido)-triphosphate), a second Mg2+ ion was found to be coordinated to the γ-phosphate group of GppNHp, which positions the hydrolytically active water molecule very close to the attacked γ-phosphorous atom.

Conclusion

For the structural analysis of very high-resolution data we have used a new 'two-chain-isotropic-refinement' strategy. This refinement provides an alternative and easy to interpret strategy to reflect the conformational variability within crystal structures of biological macromolecules. The presented fluorescent form of H-Ras p21 will be advantageous for fluorescence studies on H-Ras p21 in which the use of fluorescent nucleotides is not feasible.

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.

Summary

Since the structure determination of bacteriorhodopsin in 1990, much progress has been made in the further development and use of electron crystallography. In this review, we provide a concise overview of the new developments in electron crystallography concerning 2D crystallization, data collection and data processing. Based on electron crystallographic work on bacteriorhodopsin, the acetylcholine receptor and aquaporins, we highlight the unique advantages and future perspectives of electron crystallography for the structural study of membrane proteins. These advantages include the visualization of membrane proteins in their native environment without detergent-induced artifacts, the trapping of different states in a reaction pathway by time-resolved experiments, the study of non-specific protein-lipid interactions and the characterization of the charge state of individual residues in membrane proteins.

Beamline ID23-2, the first dedicated and highly automated high-throughput monochromatic macromolecular crystallography microfocus beamline, is described.

The first phase of the ESRF beamline ID23 to be constructed was ID23-1, a tunable MAD-capable beamline which opened to users in early 2004. The second phase of the beamline to be constructed is ID23-2, a monochromatic microfocus beamline dedicated to macromolecular crystallography experiments. Beamline ID23-2 makes use of well characterized optical elements: a single-bounce silicon (111) monochromator and two mirrors in Kirkpatrick–Baez geometry to focus the X-ray beam. A major design goal of the ID23-2 beamline is to provide a reliable, easy-to-use and routine microfocus beam. ID23-2 started operation in November 2005, as the first beamline dedicated to microfocus macromolecular crystallography. The beamline has taken the standard automated ESRF macromolecular crystallography environment (both hardware and software), allowing users of ID23-2 to be rapidly familiar with the microfocus environment. This paper describes the beamline design, the special considerations taken into account given the microfocus beam, and summarizes the results of the first years of the beamline operation.

On-the-fly adaptive edge scanning and shuttle on-the-fly rastering fluorescence techniques have been developed to improve the efficiency of macromolecular crystallography beamlines.

This paper reports on several developments of X-ray fluorescence techniques for macromolecular crystallography recently implemented at the National Institute of General Medical Sciences and National Cancer Institute beamlines at the Advanced Photon Source. These include (i) three-band on-the-fly energy scanning around absorption edges with adaptive positioning of the fine-step band calculated from a coarse pass; (ii) on-the-fly X-ray fluorescence rastering over rectangular domains for locating small and invisible crystals with a shuttle-scanning option for increased speed; (iii) fluorescence rastering over user-specified multi-segmented polygons; and (iv) automatic signal optimization for reduced radiation damage of samples.

The catalytic decomposition of hydrogen peroxide by Cu(II) complexes with polymers bearing L-alanine (PAla) and glycylglycine (PGlygly) in their side chain was studied in alkaline aqueous media. The reactions were of pseudo-first order with respect to [H2O2] and [L-Cu(II)] (L stands for PAla or PGlygly) and the reaction rate was increased with pH increase. The energies of activation for the reactions were determined at pH 8.8, in a temperature range of 293–308 K. A suitable mechanism is proposed to account for the kinetic data, which involves the Cu(II)/Cu(I) redox pair, as has been demonstrated by ESR spectroscopy. The trend in catalytic efficiency is in the order PGlygly>PAla, due to differences in modes of complexation and in the conformation of the macromolecular ligands.

Complementary techniques greatly aid the interpretation of macromolecule structures to yield functional information, and can also help to track radiation-induced changes. A new on-axis spectrometer being integrated into the macromolecular crystallography beamlines of the Swiss Light Source is presented.

X-ray crystallography at third-generation synchrotron sources permits tremendous insight into the three-dimensional structure of macromolecules. Additional information is, however, often required to aid the transition from structure to function. In situ spectroscopic methods such as UV–Vis absorption and (resonance) Raman can provide this, and can also provide a means of detecting X-ray-induced changes. Here, preliminary results are introduced from an on-axis UV–Vis absorption and Raman multimode spectrometer currently being integrated into the beamline environment at X10SA of the Swiss Light Source. The continuing development of the spectrometer is also outlined.