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Diffraction-based structural methods contribute a large fraction of the biomolecular structural models available, providing a critical understanding of macromolecular architecture. These methods require crystallization of the target molecule, which remains a primary bottleneck in crystal-based structure determination. The National High-Throughput Crystallization Center at Hauptman–Woodward Medical Research Institute has focused on overcoming obstacles to crystallization through a combination of robotics-enabled high-throughput screening and advanced imaging to increase the success of finding crystallization conditions. This paper will describe the lessons learned from over 20 years of operation of our high-throughput crystallization services. The current experimental pipelines, instrumentation, imaging capabilities and software for image viewing and crystal scoring are detailed. New developments in the field and opportunities for further improvements in biomolecular crystallization are reflected on.

A system engineered to produce a large-scale structural change from X-ray-induced di­sulfide bond cleavage allows residue-specific changes to be interpreted with SAXS. This system is used to investigate how radical scavengers reduce bond breakage during data collection at 10°C with the results providing insight into mitigation strategies for crystallographic and solution experiments., X-ray-based techniques are a powerful tool in structural biology but the radiation-induced chemistry that results can be detrimental and may mask an accurate structural understanding. In the crystallographic case, cryocooling has been employed as a successful mitigation strategy but also has its limitations including the trapping of non-biological structural states. Crystallographic and solution studies performed at physiological temperatures can reveal otherwise hidden but relevant conformations, but are limited by their increased susceptibility to radiation damage. In this case, chemical additives that scavenge the species generated by radiation can mitigate damage but are not always successful and the mechanisms are often unclear. Using a protein designed to undergo a large-scale structural change from breakage of a di­sulfide bond, radiation damage can be monitored with small-angle X-ray scattering. Using this, we have quantitatively evaluated how three scavengers commonly used in crystallographic experiments – sodium nitrate, cysteine, and ascorbic acid – perform in solution at 10°C. Sodium nitrate was the most effective scavenger and completely inhibited fragmentation of the di­sulfide bond at a lower concentration (500 µM) compared with cysteine (∼5 mM) while ascorbic acid performed best at 5 mM but could only reduce fragmentation by ∼75% after a total accumulated dose of 792 Gy. The relative effectiveness of each scavenger matches their reported affinities for solvated electrons. Saturating concentrations of each scavenger shifted fragmentation from first order to a zeroth-order process, perhaps indicating the direct contribution of photoabsorption. The SAXS-based method can detect damage at X-ray doses far lower than those accessible crystallographically, thereby providing a detailed picture of scavenger processes. The solution results are also in close agreement with what is known about scavenger performance and mechanism in a crystallographic setting and suggest that a link can be made between the damage phenomenon in the two scenarios. Therefore, our engineered approach might provide a platform for more systematic and comprehensive screening of radioprotectants that can directly inform mitigation strategies for both solution and crystallographic experiments, while also clarifying fundamental radiation damage mechanisms.

In 2005 we reviewed microgravity for macromolecular crystallization, four years after the final flight of the Space Shuttle Orbiter, and five years before the first commercial flight to the Interna...

Understanding protein folding is crucial for protein sciences. The conformational spaces and energy landscapes of cold (unfolded) protein states, as well as the associated transitions, are hardly explored. Furthermore, it is not known how structure relates to the cooperativity of cold transitions, if cold and heat unfolded states are thermodynamically similar, and if cold states play important roles for protein function. We created the cold unfolding 4-helix bundle DCUB1 with a de novo designed bipartite hydrophilic/hydrophobic core featuring a hydrogen bond network which extends across the bundle in order to study the relative importance of hydrophobic versus hydrophilic protein-water interactions for cold unfolding. Structural and thermodynamic characterization resulted in the discovery of a complex energy landscape for cold transitions, while the heat unfolded state is a random coil. Below ∼0 °C, the core of DCUB1 disintegrates in a largely cooperative manner, while a near-native helical content is retained. The resulting cold core-unfolded state is compact and features extensive internal dynamics. Below -5 °C, two additional cold transitions are seen, that is, (i) the formation of a water-mediated, compact, and highly dynamic dimer, and (ii) the onset of cold helix unfolding decoupled from cold core unfolding. Our results suggest that cold unfolding is initiated by the intrusion of water into the hydrophilic core network and that cooperativity can be tuned by varying the number of core hydrogen bond networks. Protein design has proven to be invaluable to explore the energy landscapes of cold states and to robustly test related theories.

Dissociation of transforming growth factor beta-1 (TGFβ-1) from the inhibitory protein latency-associated peptide (LAP) can occur from low doses of X-ray irradiation of the LAP–TGFβ-1 complex, resulting in the activation of TGFβ-1, and can have health-related consequences. Using the tools and knowledge developed in the study of radiation damage in the crystallographic setting, small-angle X-ray scattering (SAXS) and complementary techniques suggest an activation process that is initiated but not driven by the initial X-ray exposure. LAP is revealed to be extended when not bound to TGFβ-1 and has a different structural conformation compared to the bound state. These studies pave the way for the structural understanding of systems impacted at therapeutic X-ray doses and show the potential impact of radiation damage studies beyond their original intent.

Many biological systems across scales of size and complexity exhibit a time-varying complex network structure that emerges and self-organizes as a result of interactions with the environment. Network interactions optimize some intrinsic cost functions that are unknown and involve for example energy efficiency, robustness, resilience, and frailty. A wide range of networks exist in biology, from gene regulatory networks important for organismal development, protein interaction networks that govern physiology and metabolism, and neural networks that store and convey information to networks of microbes that form microbiomes within hosts, animal contact networks that underlie social systems, and networks of populations on the landscape connected by migration. Increasing availability of extensive (big) data is amplifying our ability to quantify biological networks. Similarly, theoretical methods that describe network structure and dynamics are being developed. Beyond static networks representing snapshots of biological systems, collections of longitudinal data series can help either at defining and characterizing network dynamics over time or analyzing the dynamics constrained to networked architectures. Moreover, due to interactions with the environment and other biological systems, a biological network may not be fully observable. Also, subnetworks may emerge and disappear as a result of the need for the biological system to cope with for example invaders or new information flows. The confluence of these developments renders tractable the question of how the structure of biological networks predicts and controls network dynamics. In particular, there may be structural features that result in homeostatic networks with specific higher-order statistics (e.g., multifractal spectrum), which maintain stability over time through robustness and/or resilience to perturbation. Alternative, plastic networks may respond to perturbation by (adaptive to catastrophic) shifts in structure. Here, we explore the opportunity for discovering universal laws connecting the structure of biological networks with their function, positioning them on the spectrum of time-evolving network structure, that is, dynamics of networks, from highly stable to exquisitely sensitive to perturbation. If such general laws exist, they could transform our ability to predict the response of biological systems to perturbations—an increasingly urgent priority in the face of anthropogenic changes to the environment that affect life across the gamut of organizational scales.

The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2., Graphical abstract, Durdağı et al. represent radiation damage-free high-resolution SARS-CoV-2 main protease SFX structures obtained at near-physiological temperature and performed MD simulation of apo-form proteins and three known main protease inhibitors. The structures reveal alternate conformation, while MD simulation indicates asymmetric behavior of the protein, which is invaluable information for immediate drug-repurposing studies.

The COVID19 pandemic has resulted in 25+ million reported infections and nearly 850.000 deaths. Research to identify effective therapies for COVID19 includes: i) designing a vaccine as future protection; ii) structure-based drug design; and iii) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determined two apo structures of Severe Acute Respiratory Syndrome CoronaVirus-2 main protease at ambienttemperature by Serial Femtosecond X-ray crystallography. We employed detailed molecular simulations of selected known main protease inhibitors with the structures and compared binding modes and energies. The combined structural biology and molecular modeling studies not only reveal the dynamics of small molecules targeting main protease but will also provide invaluable opportunities for drug repurposing and structure-based drug design studies against SARS-CoV-2.One Sentence SummaryRadiation-damage-free high-resolution SARS-CoV-2 main protease SFX structures obtained at near-physiological-temperature offer invaluable information for immediate drug-repurposing studies for the treatment of COVID19.

Metalloproteins comprise over one-third of proteins, with approximately half of all enzymes requiring metal to function. Accurate identification of these metal atoms and their environment is a prerequisite to understanding biological mechanism. Using ion beam analysis through particle induced X-ray emission (PIXE), we have quantitatively identified the metal atoms in 30 previously structurally characterized proteins using minimal sample volume and a high-throughput approach. Over half of these metals had been misidentified in the deposited structural models. Some of the PIXE detected metals not seen in the models were explainable as artifacts from promiscuous crystallization reagents. For others, using the correct metal improved the structural models. For multinuclear sites, anomalous diffraction signals enabled the positioning of the correct metals to reveal previously obscured biological information. PIXE is insensitive to the chemical environment, but coupled with experimental diffraction data deposited alongside the structural model it enables validation and potential remediation of metalloprotein models, improving structural and, more importantly, mechanistic knowledge.

Analysis of crystallographic, solution scattering and biochemical experiments suggests a mechanism for the sequestration of transforming growth factor β-1 by latency-associated peptide (LAP) that includes a structural change in LAP from an open to a closed conformation. This mechanism contains several potential regulatory features that are exploitable for therapeutic development., Transforming growth factor β-1 (TGFβ-1) is a secreted signalling protein that directs many cellular processes and is an attractive target for the treatment of several diseases. The primary endogenous activity regulatory mechanism for TGFβ-1 is sequestration by its pro-peptide, latency-associated peptide (LAP), which sterically prohibits receptor binding by caging TGFβ-1. As such, recombinant LAP is promising as a protein-based therapeutic for modulating TGFβ-1 activity; however, the mechanism of binding is incompletely understood. Comparison of the crystal structure of unbound LAP (solved here to 3.5 Å resolution) with that of the bound complex shows that LAP is in a more open and extended conformation when unbound to TGFβ-1. Analysis suggests a mechanism of binding TGFβ-1 through a large-scale conformational change that includes contraction of the inter-monomer interface and caging by the ‘straight-jacket’ domain that may occur in partnership through a loop-to-helix transition in the core jelly-roll fold. This conformational change does not appear to include a repositioning of the integrin-binding motif as previously proposed. X-ray scattering-based modelling supports this mechanism and reveals possible orientations and ensembles in solution. Although native LAP is heavily glycosylated, solution scattering experiments show that the overall folding and flexibility of unbound LAP are not influenced by glycan modification. The combination of crystallography, solution scattering and biochemical experiments reported here provide insight into the mechanism of LAP sequestration of TGFβ-1 that is of fundamental importance for therapeutic development.

Xylose isomerase (XI) is an industrially important metalloprotein studied for decades. Its reaction mechanism has been postulated to involve movement of the catalytic metal cofactor to several different conformations. Here, a dose-dependent approach was used to investigate the radiation damage effects on XI and their potential influence on the reaction mechanism interpreted from the X-ray derived structures. Radiation damage is still one of the major challenges for X-ray diffraction experiments and causes both global and site-specific damage. In this study, consecutive high-resolution data sets from a single XI crystal from the same wedge were collected at 100 K and the progression of radiation damage was tracked over increasing dose (0.13–3.88 MGy). The catalytic metal and its surrounding amino acid environment experience a build-up of free radicals, and the results show radiation-damage-induced structural perturbations ranging from an absolute metal positional shift to specific residue motions in the active site. The apparent metal movement is an artefact of global damage and the resulting unit-cell expansion, but residue motion appears to be driven by the dose. Understanding and identifying radiation-induced damage is an important factor in accurately interpreting the biological conclusions being drawn.

Haptic interfaces have become common in consumer electronics. They enable easy interaction and information entry without the use of a mouse or keyboard. The work presented here illustrates the application of a haptic interface to crystallization screening in order to provide a natural means for visualizing and selecting results. By linking this to a cloud-based database and web-based application program interface, the same application shifts the approach from `point and click' to `touch and share', where results can be selected, annotated and discussed collaboratively. In the crystallographic application, given a suitable crystallization plate, beamline and robotic end effector, the resulting information can be used to close the loop between screening and X-ray analysis, allowing a direct and efficient `screen to beam' approach. The application is not limited to the area of crystallization screening; `touch and share' can be used by any information-rich scientific analysis and geographically distributed collaboration.

A significant problem in biological X-ray crystallography is the radiation chemistry caused by the incident X-ray beam. This produces both global and site-specific damage. Site specific damage can misdirect the biological interpretation of the structural models produced. Cryo-cooling crystals has been successful in mitigating damage but not eliminating it altogether; however, cryo-cooling can be difficult in some cases and has also been shown to limit functionally relevant protein conformations. The doses used for X-ray crystallography are typically in the kilo-gray to mega-gray range. While disulfide bonds are among the most significantly affected species in proteins in the crystalline state at both cryogenic and higher temperatures, there is limited information on their response to low X-ray doses in solution, the details of which might inform biomedical applications of X-rays. In this work we engineered a protein that dimerizes through a susceptible disulfide bond to relate the radiation damage processes seen in cryo-cooled crystals to those closer to physiologic conditions. This approach enables a low-resolution technique, small angle X-ray scattering (SAXS), to detect and monitor a residue specific process. A dose dependent fragmentation of the engineered protein was seen that can be explained by a dimer to monomer transition through disulfide bond cleavage. This supports the crystallographically derived mechanism and demonstrates that results obtained crystallographically can be usefully extrapolated to physiologic conditions. Fragmentation was influenced by pH and the conformation of the dimer, providing information on mechanism and pointing to future routes for investigation and potential mitigation. The novel engineered protein approach to generate a large-scale change through a site-specific interaction represents a promising tool for advancing radiation damage studies under solution conditions.

In this chapter we illustrate the biological applications of small angle scattering. We cover examples of contrast matching has been used in the neutron case and with SAXS,. time-resolved studies that have helped reveal mechanism and how SAXS or SANS as a solution technique can access information missing from other studies, in particular residues that may be crystallographically undefined. We show examples of puting known structural information in context, and following large-scale functional changes or show when distinct populations co-exist in solution. The list of examples provided is not exhaustive and should be seen as a glimpse of the potential of biological mechanisms where SAXS or SANS can provide unique information complementary to other methods and help define function and mechanism.

This chapter summarizes the book, describes publications that provide complementary information, and sets the tone for the future.

Small angle neutron scattering (SANS) is a specialized application of solution scattering limited by source availability and intensity. While not routinely used for structural studies of biological samples in general, it does have unique characteristics that make it attractive to determining the individual positions of components of complexes. This is due to the scattering properties of hydrogen and deuterium allowing the technique of contrast matching. SANS is highly complementary to SAXS and provides unique information not available by other techniques. This chapter discussed SANS, instrumentation, and application.

SAXS instrumentation is available for the laboratory, at the synchrotron, and at X-ray free electron laser sources. This chapter deals with SAXS instrumentation. It covers laboratory systems, synchrotron beamlines and newer sources. Multiple synchrotron facilities have SAXS beamlines and the ability to perform SAXS studies on free electron laser sources is growing. Sample handling has adopted automation and in some cases microfluidics to reduce sample volume requirements. Sensitive detectors efficient data collection software and rapid analysis allow real time decisions to be made during data collection. Specialized apparatus enables time resolved studies. Each component is described.

This chapter provides a detailed example of SAXS data analysis from a well behaving system. After collecting the SAXS data, several data analysis procedures are illustrated to ensure the data were of sufficient quality. Many of these steps are performed by comparing multiple concentrations from a dilution series, demonstrating the importance of this step in the data collection procedure for ensuring high quality data. Finally, real space modeling and shape reconstructions are shown to determine the oligomeric state as a tetramer, and identify the correct oligomeric assembly from crystal packing predictions.

There are more specialized applications of SAXS and SANS which require specific experimental considerations. This chapter covers size exclusion chromatography which has proven to be useful to study both soluble and membrane bound proteins allowing the study of samples that show time and concentration dependent dynamics. It also describes iime-resolved techniques for SAXS and in a few cases, SANS. Finally, with improved X-ray sources, detectors, sample handling, and compute power, the ability to perform SAXS data in high-throughput is available. This is discussed in enabling the use of SAXS to study protein interactions, map macromolecular conformation, and rapidly characterize samples amongst other applications.

The field of application for solution scattering is broader than we can cover in detail and with new instrumentation, that field is only growing. XFEL sources offer the potential for time resolved studies exquisitely sensitive to rapid structural biological processes while at the same time potentially extending the information beyond that provided by current solution scattering methods. Cryogenic approaches offer the potential to increase signal and/or improve sample demands opening up the technique to a wider array of studies. Finally, use of the atomic scattering properties reveals biological information in the case of nucleic acids and has the potential to provide molecular rulers able to resolve dynamic structural changes as they occur. This chapter provides a glimpse of what is possible and a starting point for further exploration.

This chapter describes preparation before collecting solution scattering data. SAXS requires a sample concentration similar to that typically used for crystallization, and a volume on the order of tens of μ‎l. SANS can require an order of magnitude more than this. The sample should be well characterized, and free from impurities. Standard laboratory techniques are suitable for characterization. Buffer choice and accurate matching is a component of experimental success and after sample preparation is the next most critical step. The sample should be monodisperse, stable, and well characterized.

In this chapter we note that solution scattering data can be divided into four regions. At zero scattering angle, the scattering provides information on molecular weight of the particle in solution. Beyond that, the scattering is influenced by the radius of gyration. As the scattering angle increases, the scattering is influenced by the particle shape, and finally by the interface with the particle and the solution. There are a number of important invariants that can be calculated directly from the data including molecular mass, radius of gyration, Porod invariant, particle volume, maximum particle dimension, particle surface area, correlation length, and volume of correlation. The meaning of these is described in turn along with their mathematical derivations.

This chapter describes the basic background for small angle scattering starting with a simple description of the experiment and geometric theory of scattering. It describes the nature of the data produced and the information contained within that data and how relationship between real and reciprocal space in the context of solution scattering. The concept of spherical averaging is described along with its implications and effects on the available structural information from experiment. The chapter describes important fundamental concepts in solution scattering such as the pair distribution function, contrast, and resolution and information content. The chapter is presented so as to promote an intuitive understanding of the theoretical foundations of solution scattering, rather than a rigorous treatment of them.

The technique of small angle solution scattering has been revolutionized in the last two decades. Exponential increases in computing power, parallel algorithm development, and the development of synchrotron, free-electron X-ray sources, and neutron sources, have combined to allow new classes of studies for biological specimens. These include time-resolved experiments in which functional motions of proteins are monitored on a picosecond timescale, and the first steps towards determining actual electron density fluctuations within particles. In addition, more traditional experiments involving the determination of size and shape, and contrast matching that isolate substructures such as nucleic acid, have become much more straightforward to carry out, and simultaneously require much less material. These new capabilities have sparked an upsurge of interest in solution scattering on the part of investigators in related disciplines. Thus, this book seeks to guide structural biologists to understand the basics of small angle solution scattering in both the X-ray and neutron case, to appreciate its strengths, and to be cognizant of its limitations. It is also directed at those who have a general interest in its potential. The book focuses on three areas: theory, practical aspects and applications, and the potential of developing areas. It is an introduction and guide to the field but not a comprehensive treatment of all the potential applications.

This chapter discusses recovering shape or structural information from SAXS data. Key to any such process is the ability to generate a calculated intensity from a model, and to compare this curve with the experimental one. Models for the particle scattering density can be approximated as pure homogenenous geometric shapes. More complex particle surfaces can be represented by spherical harmonics or by a set of close-packed beads. Sometimes structural information is known for components of a particle. Rigid body modeling attempts to rotate and translate structures relative to one another, such that the resulting scattering profile calculated from the model agrees with the experimental SAXS data. More advanced hybrid modelling procedures aim to incorporate as much structural information as is available, including modelling protein dynamics. Solutions may not always contain a homogeneous set of particles. A common case is the presence of two or more conformations of a single particle or a mixture of oligomeric species. The method of singular value decomposition can extract scattering for conformationally distinct species.

Direct electron density determination from SAXS data opens up new opportunities. The ability to model density at high resolution and the implicit direct estimation of solvent terms such as the hydration shell may enable high-resolution wide angle scattering data to be used to calculate density when combined with additional structural information. Other diffraction methods that do not measure three-dimensional intensities, such as fiber diffraction, may also be able to take advantage of iterative structure factor retrieval. While the ability to reconstruct electron density ab initio is a major breakthrough in the field of solution scattering, the potential of the technique has yet to be fully uncovered. Additional structural information from techniques such as crystallography, NMR, and electron microscopy and density modification procedures can now be integrated to perform advanced modeling of the electron density function at high resolution, pushing the boundaries of solution scattering further than ever before.

Extracting information from scattering data is very sensitive to the quality of the data. In this chapter data quality characterization is described, including initial data processing procedures to alert the user to potential data quality issues. Accurate buffer subtraction is crucial for correct modeling and analysis of SAS data, and mechanisms for identifying buffer subtraction errors are discussed. Examining SAS parameters such as a function of concentration or exposure is very useful for identifying concentration dependent artifacts or radiation damage that, if unnoticed, can be very detrimental to further analysis, including misinterpreting the results and drawing erroneous conclusions. SAS is often used for analyzing flexible molecules in solution that may be difficult to study with other structural techniques. Qualitative and quantitative assessments of flexibility are described.

Thic chapter describes some of the processes that are often carried out within specific data processing software associated with an instrument but are invisible to the user. It is useful to be aware of them. These include dealing with detector artifacts and limitations, and the integration of the signal from a two-dimensional image to produce a one-dimensional scattering profile, among other steps.

This chapter provides an introduction to small angle solution scattering with particular reference to the complementary technique of X-ray crystallography and the relationship between the two. It describes at its most basic level the theoretical underpinnings of solution scattering starting from a single molecule and how this information is sampled in crystals versus in solution. A brief introduction is given to some of the primary types of structural information that can be obtained from experiments. The chapter concludes discussing some of the most common applications of the technique in structural biology, and where the future is likely headed.

Protein structural vibrations impact biology by steering the structure to functional intermediate states; enhancing tunneling events; and optimizing energy transfer. Strong water absorption and a broad continuous vibrational density of states have prevented optical identification of these vibrations. Recently spectroscopic signatures that change with functional state were measured using anisotropic terahertz microscopy. The technique however has complex sample positioning requirements and long measurement times, limiting access for the biomolecular community. Here we demonstrate that a simplified system increases spectroscopic structure to dynamically fingerprint biomacromolecules with a factor of 6 reduction in data acquisition time. Using this technique, polarization varying anisotropy terahertz microscopy, we show sensitivity to inhibitor binding and unique vibrational spectra for several proteins and an RNA G-quadruplex. The technique’s sensitivity to anisotropic absorbance and birefringence provides rapid assessment of macromolecular dynamics that impact biology.

The Machine Recognition of Crystallization Outcomes (MARCO) initiative has assembled roughly half a million annotated images of macromolecular crystallization experiments from various sources and setups. Here, state-of-the-art machine learning algorithms are trained and tested on different parts of this data set. We find that more than 94% of the test images can be correctly labeled, irrespective of their experimental origin. Because crystal recognition is key to high-density screening and the systematic analysis of crystallization experiments, this approach opens the door to both industrial and fundamental research applications., 11 pages, 4 figures, minor text and figure updates

Oligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology-based methods, protein-protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet-V1 superfamily, Aha1 from Colwellia psychrerythraea. This family displays a broad range of crystallographic interfaces none of which can be reconciled with the NMR and SAXS data collected for Aha1. Unlike conventional methods relying on a dense network of experimental restraints, the sparse data are used to limit conformational search during optimization of a physically realistic energy function. This work highlights a new approach for studying minor conformational changes due to structural plasticity within a single dimeric interface in solution.

Nearly all protein functions require structural change, such as enzymes clamping onto substrates, and ion channels opening and closing. These motions are a target for possible new therapies; however, the control mechanisms are under debate. Calculations have indicated protein vibrations enable structural change. However, previous measurements found these vibrations only weakly depend on the functional state. By using the novel technique of anisotropic terahertz microscopy, we find that there is a dramatic change to the vibrational directionality with inhibitor binding to lysozyme, whereas the vibrational energy distribution, as measured by neutron inelastic scattering, is only slightly altered. The anisotropic terahertz measurements provide unique access to the directionality of the intramolecular vibrations, and immediately resolve the inconsistency between calculations and previous measurements, which were only sensitive to the energy distribution. The biological importance of the vibrational directions versus the energy distribution is revealed by our calculations comparing wild-type lysozyme with a higher catalytic rate double deletion mutant. The vibrational energy distribution is identical, but the more efficient mutant shows an obvious reorientation of motions. These results show that it is essential to characterize the directionality of motion to understand and control protein dynamics to optimize or inhibit function.

As technology advances, the crystal volume that can be used to collect useful X-ray diffraction data decreases. The technologies available to detect and study growing crystals beyond the optical resolution limit and methods to successfully place the crystal into the X-ray beam are discussed., Structural biology has contributed tremendous knowledge to the understanding of life on the molecular scale. The Protein Data Bank, a depository of this structural knowledge, currently contains over 100 000 protein structures, with the majority stemming from X-ray crystallography. As the name might suggest, crystallography requires crystals. As detectors become more sensitive and X-ray sources more intense, the notion of a crystal is gradually changing from one large enough to embellish expensive jewellery to objects that have external dimensions of the order of the wavelength of visible light. Identifying these crystals is a prerequisite to their study. This paper discusses developments in identifying these crystals during crystallization screening and distinguishing them from other potential outcomes. The practical aspects of ensuring that once a crystal is identified it can then be positioned in the X-ray beam for data collection are also addressed.

X-ray crystallography typically requires the mounting of crystals, which can make the sample difficult to manipulate when it is small and the microscope objective is close to the crystallization plate. By simply moving the objective to the bottom of a clear crystallization plate (inverting the normal view), crystals were able to be manipulated and harvested from wells having a 0.9 mm diameter and 5.0 mm depth. The mounting system enabled the structural solution of the 187 amino acid N-terminal domain ofSaccharomyces cerevisiaeglutaminyl-tRNA synthetase from crystals that appeared during high-throughput screening but proved recalcitrant to scale-up and optimization. While not a general mounting solution, the simple expedient of removing the objective lens from the area where manipulation and harvesting occur greatly facilitates the manual, or even automated, process.

Ketol-isomerases catalyze the reversible isomerization between aldoses and ketoses. D-Xylose isomerase carries out the first reaction in the catabolism of D-xylose, but is also able to convert D-glucose to D-fructose. The first step of the reaction is an enzyme-catalyzed ring opening of the cyclic substrate. The active-site amino-acid acid/base pair involved in ring opening has long been investigated and several models have been proposed. Here, the structure of the xylose isomerase E186Q mutant with cyclic glucose bound at the active site, refined against joint X-ray and neutron diffraction data, is reported. Detailed analysis of the hydrogen-bond networks at the active site of the enzyme suggests that His54, which is doubly protonated, is poised to protonate the glucose O5 position, while Lys289, which is neutral, promotes deprotonation of the glucose O1H hydroxyl groupviaan activated water molecule. The structure also reveals an extended hydrogen-bonding network that connects the conserved residues Lys289 and Lys183 through three structurally conserved water molecules and residue 186, which is a glutamic acid to glutamine mutation.