Micro-crystal electron diffraction (MicroED) has shown great potential for structure determination of macromolecular crystals too small for X-ray diffraction. However, specimen preparation remains a major bottleneck. Here, we report a simple method for preparing MicroED specimens, named Preassis, in which excess liquid is removed through an EM grid with the assistance of pressure. We show the ice thicknesses can be controlled by tuning the pressure in combination with EM grids with appropriate carbon hole sizes. Importantly, Preassis can handle a wide range of protein crystals grown in various buffer conditions including those with high viscosity, as well as samples with low crystal concentrations. Preassis is a simple and universal method for MicroED specimen preparation, and will significantly broaden the applications of MicroED.

Traditionally small-molecule crystallographers have not usually observed or recognized significant radiation damage to their samples during diffraction experiments. However, the increased flux densities provided by third-generation synchrotrons have resulted in increasing numbers of observations of this phenomenon. The diversity of types of small-molecule systems means it is not yet possible to propose a general mechanism for their radiation-induced sample decay, however characterization of the effects will permit attempts to understand and mitigate it. Here, systematic experiments are reported on the effects that sample temperature and beam attenuation have on radiation damage progression, allowing qualitative and quantitative assessment of their impact on crystals of a small-molecule test sample. To allow inter-comparison of different measurements, radiation-damage metrics (diffraction-intensity decline, resolution fall-off, scaling B-factor increase) are plotted against the absorbed dose. For ease-of-dose calculations, the software developed for protein crystallography, RADDOSE-3D, has been modified for use in small-molecule crystallography. It is intended that these initial experiments will assist in establishing protocols for small-molecule crystallographers to optimize the diffraction signal from their samples prior to the onset of the deleterious effects of radiation damage.

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 100K and the progression of radiation damage was tracked over increasing dose (0.13-3.88MGy). 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.

Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UK, National Crystallography Service, School of Chemistry, Faculty of Engineering and Physical Sciences, University of Southampton, Southampton SO17 1BJ, UK, and Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. *Correspondence e-mail: elspeth.garman@bioch.ox.ac.uk, s.j.coles@soton.ac.uk

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 of 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 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 metal shift 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.

The author wishes to make the following corrections to this paper [1]:[...]

Radiation damage still remains a major limitation and challenge in macromolecular X-ray crystallography. Some of the high-intensity radiation used for diffraction data collection experiments is absorbed by the crystals, generating free radicals. These give rise to radiation damage even at cryotemperatures (~100 K), which can lead to incorrect biological conclusions being drawn from the resulting structure, or even prevent structure solution entirely. Investigation of mitigation strategies and the effects caused by radiation damage has been extensive over the past fifteen years. Here, recent understanding of the physical and chemical phenomena of radiation damage is described, along with the global effects inflicted on the collected data and the specific effects observed in the solved structure. Furthermore, this review aims to summarise the progress made in radiation damage studies in macromolecular crystallography from the experimentalist’s point of view and to give an introduction to the current literature.

Radiation chemistry causes structural perturbations with significant impact to the accurate structural understanding of mechanism, especially in metalloproteins.Xylose isomerase (XI) is an example of this issue: it catalyzes the reversible interconversion of the aldoses D-xylose, D-ribose, and D-glucose to the corresponding ketoses.XI requires a divalent metal ion such as Mn2+, Mg2+ or Co2+ as a cofactor [1].It is an important industrial enzyme; XI is used extensively in the food industry, for example in producing high-fructose corn syrup, and it also holds enormous potential for application in converting biomass into fuels and chemicals.Thus understanding its mechanism and thereby potentially improving its suitability for industrial applications is even more important.The reaction mechanism has been studied in increasingly detailed studies, many discussing the mobility of the catalytic metal ion.We show that this mobility can be driven by the Xrays used to reveal the structure, and that it may not be mechanistically critical.X-rays generate free radicals when interacting with macromolecule crystals.These radicals propagate changes that result in both global effects and specific structural changes, even when cryogenic temperatures (around 100 K) are used to minimize the effects.Global radiation damage is observed as a loss of diffraction intensity with the weakest higher resolution reflections fading first, an increase in unit-cell volume, higher Wilson B-factors, and often overall higher mosaicity of the crystal [2].These effects can preclude structure solution.On the other hand, specific damage, contributing to the global effects, is seen from the electron density maps after refinement, and manifests as the breakage of disulfide bonds, decarboxylation of acidic side chains and photoreduction of metal atoms [3] potentially leading to incorrect biological deductions being made from the resultant structural model.The effects of radiation damage on the metalloprotein, XI, have been investigated.By collecting consecutive synchrotron Xray diffraction datasets at 100 K on a substrate free XI crystal, radiation induced changes in the structure were tracked as a function of dose.The metal and the surrounding amino acid environment experience a buildup of free radicals, making it more susceptible to damage than the rest of the protein.The radiation damage induced structural perturbations are similar to structural alterations that have been attributed to the mechanism, i.e. the metal ion used as a cofactor occupied several different positions.Thus the X-ray induced metal movement observed with XI appears to be an artifact of the method used to derive the structure, and neutron based structural studies have since illuminated the mechanism.All metals will be sensitive to radiation effects, as they have a high X-ray absorption cross-section.This has profound implications for the high resolution study of metalloproteins in general and is especially pertinent to the study of enzymes, since metalloproteins make up over half their population.Understanding and identifying radiation induced structural perturbation is critical to an accurate structural interpretation of mechanism.

The Gfo/Idh/MocA protein family contains a number of different proteins, which almost exclusively consist of NAD(P)-dependent oxidoreductases that have a diverse set of substrates, typically pyranoses. In this study, to clarify common structural features that would contribute to their function, the available crystal structures of the members of this family have been analyzed. Despite a very low sequence identity, the central features of the three-dimensional structures of the proteins are surprisingly similar. The members of the protein family have a two-domain structure consisting of a N-terminal nucleotide-binding domain and a C-terminal α/β-domain. The C-terminal domain contributes to the substrate binding and catalysis, and contains a βα-motif with a central α-helix carrying common essential amino acid residues. The β-sheet of the α/β-domain contributes to the oligomerization in most of the proteins in the family.

Aldose-aldose oxidoreductase (Cc AAOR) is a recently characterized enzyme from the bacterial strain Caulobacter crescentus CB15 belonging to the glucose-fructose oxidoreductase/inositol dehydrogenase/rhizopine catabolism protein (Gfo/Idh/MocA) family. Cc AAOR catalyses the oxidation and reduction of a panel of aldose monosaccharides using a tightly bound NADP(H) cofactor that is regenerated in the catalytic cycle. Furthermore, Cc AAOR can also oxidize 1,4-linked oligosaccharides. In the present study, we present novel crystal structures of the dimeric Cc AAOR in complex with the cofactor and glycerol, D-xylose, D-glucose, maltotriose and D-sorbitol determined to resolutions of 2.0, 1.8, 1.7, 1.9 and 1.8 Å (1 Å=0.1 nm), respectively. These complex structures allowed for a detailed analysis of the ligand-binding interactions. The structures showed that the C1 carbon of a substrate, which is either reduced or oxidized, is close to the reactive C4 carbon of the nicotinamide ring of NADP(H). In addition, the O1 hydroxy group of the substrate, which is either protonated or deprotonated, is unexpectedly close to both Lys(104) and Tyr(189), which may both act as a proton donor or acceptor. This led us to hypothesize that this intriguing feature could be beneficial for Cc AAOR to catalyse the reduction of a linear form of a monosaccharide substrate and the oxidation of a pyranose form of the same substrate in a reaction cycle, during which the bound cofactor is regenerated.

Agrobacterium tumefaciens (At) strain C58 contains an oxidative enzyme pathway that can function on both d-glucuronic and d-galacturonic acid. The corresponding gene coding for At keto-deoxy-d-galactarate (KDG) dehydratase is located in the same gene cluster as those coding for uronate dehydrogenase (At Udh) and galactarolactone cycloisomerase (At Gci) which we have previously characterized. Here, we present the kinetic characterization and crystal structure of At KDG dehydratase, which catalyzes the next step, the decarboxylating hydrolyase reaction of KDG to produce α-ketoglutaric semialdehyde (α-KGSA) and carbon dioxide. The crystal structures of At KDG dehydratase and its complexes with pyruvate and 2-oxoadipic acid, two substrate analogues, were determined to 1.7 Å, 1.5 Å, and 2.1 Å resolution, respectively. Furthermore, mass spectrometry was used to confirm reaction end-products. The results lead us to propose a structure-based mechanism for At KDG dehydratase, suggesting that while the enzyme belongs to the Class I aldolase protein family, it does not follow a typical retro-aldol condensation mechanism.

In vitro toxicological studies together with atomistic molecular dynamics simulations show that occupational co-exposure with C60 fullerene may strengthen the health effects of organic industrial chemicals. The chemicals studied are acetophenone, benzaldehyde, benzyl alcohol, m-cresol, and toluene which can be used with fullerene as reagents or solvents in industrial processes. Potential co-exposure scenarios include a fullerene dust and organic chemical vapor, or a fullerene solution aerosolized in workplace air. Unfiltered and filtered mixtures of C60 and organic chemicals represent different co-exposure scenarios in in vitro studies where acute cytotoxicity and immunotoxicity of C60 and organic chemicals are tested together and alone by using human THP-1-derived macrophages. Statistically significant co-effects are observed for an unfiltered mixture of benzaldehyde and C60 that is more cytotoxic than benzaldehyde alone, and for a filtered mixture of m-cresol and C60 that is slightly less cytotoxic than m-cresol. Hydrophobicity of chemicals correlates with co-effects when secretion of pro-inflammatory cytokines IL-1β and TNF-α is considered. Complementary atomistic molecular dynamics simulations reveal that C60 co-aggregates with all chemicals in aqueous environment. Stable aggregates have a fullerene-rich core and a chemical-rich surface layer, and while essentially all C60 molecules aggregate together, a portion of organic molecules remains in water.

D-galacturonic acid is the main component of pectin. It could be used to produce affordable renewable fuels, chemicals and materials through biotechnical conversion. Keto-deoxy-D-galactarate (KDG) dehydratase is an enzyme in the oxidative pathway of D-galacturonic acid in Agrobacterium tumefaciens (At). It converts 3-deoxy-2-keto-L-threo-hexarate to α-ketoglutaric semialdehyde. At KDG dehydratase was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 169.1, b = 117.8, c = 74.3 Å, β = 112.4° and an asymmetric unit of four monomers. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The three-dimensional structure of At KDG dehydratase will provide valuable information on the function of the enzyme and will allow it to be engineered for biorefinery-based applications.