Challenges and solutions for the analysis of<i>in situ</i>,<i>in crystallo</i>micro-spectrophotometric data

Combining macromolecular crystallography within crystallomicro-spectrophotometry yields valuable complementary information on the sample, including the redox states of metal cofactors, the identification of bound ligands and the onset and strength of undesired photochemistry, also known as radiation damage. However, the analysis and processing of the resulting data differs significantly from the approaches used for solution spectrophotometric data. The varying size and shape of the sample, together with the suboptimal sample environment, the lack of proper reference signals and the general influence of the X-ray beam on the sample have to be considered and carefully corrected for. In the present article, how to characterize and treat these sample-dependent artefacts in a reproducible manner is discussed and theSLS-APEin situ,in crystallooptical spectroscopy data-analysis toolbox is demonstrated.</jats:p

Fingerprinting redox and ligand states in haemprotein crystal structures using resonance Raman spectroscopy

It is crucial to assign the correct redox and ligand states to crystal structures of proteins with an active redox centre to gain valid functional information and prevent the misinterpretation of structures. Single-crystal spectroscopies, particularly when appliedin situat macromolecular crystallography beamlines, allow spectroscopic investigations of redox and ligand states and the identification of reaction intermediates in protein crystals during the collection of structural data. Single-crystal resonance Raman spectroscopy was carried out in combination with macromolecular crystallography on Swiss Light Source beamline X10SA using cytochromec′ fromAlcaligenes xylosoxidans. This allowed the fingerprinting and validation of different redox and ligand states, identification of vibrational modes and identification of intermediates together with monitoring of radiation-induced changes. This combined approach provides a powerful tool to obtain complementary data and correctly assign the true oxidation and ligand state(s) in redox-protein crystals.</jats:p

A novel β-xylosidase structure from Geobacillus thermoglucosidasius:The first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds

Geobacillus thermoglucosidasius is a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding a G. thermoglucosidasius β-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed in Escherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6Å) structure of the enzyme-substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of the G. thermoglucosidasius β-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity

Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M^(pro)), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M^(pro). In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2

Le tris-dipicolinate de lanthanide (un complexe luminescent pour la résolution de structures de macromolécules biologiques par les méthodes sad et mad )

La résolution de structure de macromolécules biologiques par diffraction des rayons X nécessite la détermination des phases associées aux facteurs de structure. Les méthodes MAD et SAD sont devenues des méthodes de choix pour la détermination de nova de structures de protéines par cristallographie. Dans cette optique, les lanthanides sont particulièrement intéressants car leur facteur de diffusion anomale est très élevé, f' :::: 28 e' à leur seuil d'absorption Lm, et reste significatif avec le rayonnement CuKa d'un générateur de laboratoire (f' = ] ],0 e' pour Eu3+ à 1 = ],54]8 A). Le complexe tris-dipicolinate de lanthanide a permis d'obtenir des cristaux dérivés de 6 protéines différentes et de résoudre leur structure par les méthodes SAD ou MAD. Dans le cas de 2 protéines, dont les structures ont pu être résolues au laboratoire, les cristaux dérivés obtenus par co-cristallisation appartiennent à de nouvelles formes cristallines résultant des interactions fortes entre le complexe et la protéine. Le complexe se lie aux protéines par des liaisons hydrogène entre ses groupements carboxylate et les acides aminés donneurs de liaison hydrogène. Par ailleurs, les propriétés de luminescence des complexes d'europium et de terbium permettent de tester rapidement la fixation du complexe dans les cristaux dérivés. La luminescence des complexes d'europium et de terbium par excitation à un et deux photons a été caractérisée dans les cristaux dérivés. La forte interaction entre le tris-dipicolinate de lanthanide et certains acides aminés peut être mise à profit pour la co cristallisation et l'obtention de cristaux dérivés de protéines permettant la détermination de nova de structures.Solving the structure ofbiological macromolecules using X-ray diffraction requires the determination of phases of structure factors. The SAD and MAD methods making use of anomalous scattering are now widely used for de nova determination ofprotein structures by crystallography. From this point ofview, lanthanide elements are particularly interesting because their anomalous scattering factors are very high at their Lm absorption edge with f' :::: 28 e' and remain significant using CuKa radiation trom a in house generator (f' = ] ].0 e' for Eu3+ at Â. = ] .54] 8 A). Tris-dipicolinate lanthanide complexes were used to obtain derivative crystals of 6 different proteins and to solve their structures using SAD or MAD methods. ]n the case of2 proteins, for which the structures were solved using CuKa radiation, derivative crystals belong to new crystal forms due to strong interactions between lanthanide complex and protein molecules. The complex binds proteins through hydrogen bonds between its carboxylate groups and hydrogen bond donor amino acids. Unique luminescence properties of europium and terbium complexes allow quick checking of the lanthanide complex binding in derivative crystals. One and two-photon induced luminescence of europium and terbium complexes were characterized in derivative crystals of lysozyme.Strong supramolecular interaction between tris-dipicolinate lanthanide complex and hydrogen-bond donor residues could be used for co-crystallization and for obtaining protein derivative crystals for de nova structure determination.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

A new on-axis micro-spectrophotometer for combining Raman, fluorescence and UV/Vis absorption spectroscopy with macromolecular crystallography at the Swiss Light Source

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.ISSN:0909-0495ISSN:1600-577