Guillaume Pompidor

P13, the EMBL macromolecular crystallography beamline at the low-emittance PETRA III ring for high- and low-energy phasing with variable beam focusing

The P13 macromolecular crystallography beamline, based on the low-emittance source PETRA III, enables X-ray diffraction experiments on macromolecular crystals over a wide wavelength range (0.7–3.1 Å). The beam has a variable focus size and a small divergence enabling data collection on micrometre-sized crystals.

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

The new version MS2 of the in situ on-axis micro-spectrophotometer at the macromolecular crystallography beamline X10SA of the Swiss Light Source supports the concurrent acquisition of Raman, resonance Raman, fluorescence and UV/Vis absorption spectra along with diffraction data.

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 (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) 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.

Challenges and solutions for the analysis of in situ, in crystallo micro-spectrophotometric data

The particular challenge of the analysis of optical absorption and Raman spectroscopic data measured from protein crystals and how the SLS-APE software toolbox supports scientists in dealing with such data is described.

Human Cellular Retinaldehyde-Binding Protein Has Secondary Thermal 9- cis -Retinal Isomerase Activity

Cellular retinaldehyde-binding protein (CRALBP) chaperones 11-cis-retinal to convert opsin receptor molecules into photosensitive retinoid pigments of the eye. We report a thermal secondary isomerase activity of CRALBP when bound to 9-cis-retinal. UV/vis and (1)H NMR spectroscopy were used to characterize the product as 9,13-dicis-retinal. The X-ray structure of the CRALBP mutant R234W:9-cis-retinal complex at 1.9 Å resolution revealed a niche in the binding pocket for 9-cis-aldehyde different from that reported for 11-cis-retinal. Combined computational, kinetic, and structural data lead us to propose an isomerization mechanism catalyzed by a network of buried waters. Our findings highlight a specific role of water molecules in both CRALBP-assisted specificity toward 9-cis-retinal and its thermal isomerase activity yielding 9,13-dicis-retinal. Kinetic data from two point mutants of CRALBP support an essential role of Glu202 as the initial proton donor in this isomerization reaction.

Experimental phasing at low energy at EMBL beamline P13

The P13 Macromolecular Crystallography beamline, operated by the EMBL at Petra III, enables diffraction data collection over a wide energy range, from 4 keV to 17.5 keV (0.7 – 3.1 Å)[1]. In order to fully exploit the beamline capability at low energy (1012 ph.s-1 at 5 keV), the PILATUS 6M-F detector, with a 450 μm Si sensor thickness and with a custom calibration at low energy, is mounted on a 2 θ-angle stage to increase the maximum resolution achievable (2.4 Å at 4keV). We will present and describe the typical experimental setup for long-wavelength data collection. For low-energy experiments, typically below 5.5 keV, the air absorption can be drastically reduced by the use of a helium path. The gas is circulating in a homemade cone, which can be fixed on the detector upon user request. In this configuration, at the minimal crystal to detector distance (135mm), only the first 30 mm of the path of the diffracted beams is in air. The beamline can be configured for low-energy data collection in about 10 minutes and remains fully compatible with the use of the MARVIN sample changer for high-throughput screening, standard cryogenic cooling, standard SPINE pins for sample mounting, and the mini-κ of the MD2 diffractometer allowing the reorientation of the crystal for anomalous strategic data collection. To illustrate the beamline capabilities, several examples of structure determination using intrinsic anomalous scatterers, i-e sulfur SAD or calcium (MAD at the Ca K edge at 4.05 keV) will be presented. In particular, we will demonstrate the efficiency of the He-path in terms of improvement of diffraction data quality. To extend the experimental opportunities, a small laboratory dedicated to the preparation of derivative is available to users close to the beamlines. With more than 150 compounds available, the heavy atom library contains many elements (Cd, Pd, U...) exhibiting strong anomalous signal at low energy, which are excellent candidates for SAD and MAD experiment at long wavelength. The EMBL Integrated Facility for Structural Biology in Hamburg provides services for high-throughput crystallization. Crystals can be harvested with a Crystal Direct Harvester system.

Serial synchrotron crystallography at EMBL PETRA III beamline P14

Serial synchrotron crystallography (SSX) combines single-shot X-ray images of randomly oriented crystals, recorded at a synchrotron source, into a single dataset. The method requires a high-brilliance synchrotron source with a beam size similar to the sample size, an appropriate sample delivery method, a detector with sufficient frame rate and a data processing pipeline. SSX can act as a pre-screening method for samples intended for XFEL experiments or a stand-alone experiment to determine the protein structure when crystal growth to larger size cannot be achieved, e.g. in vivo grown crystals. Proof of principle experiments [1] have shown the feasibility of the method.

Monoclinic HEWL derivative structure at 0.75 Å resolution

Tris-dipicolinate lanthanide complex, Na3.[Ln(DPA)3], where DPA stands for pyridine-2,6 dicarboxylate, has been proven to be especially interesting to produce derivative crystals with high phasing power [1]. The lanthanide complex can act as a cross-linking agent, which binds at the interface between protein molecules, leading to a supramolecular interaction at the crystal scale [2]. In the case of hen egg-white lysozyme (HEWL), derivative crystals obtained by co-crystallization with Yb(DPA)33− belong to the space group C2, in similar crystallization conditions that normally lead to the tetragonal form P43212. Data were collected on a derivative crystal up to a resolution of 0.75 Å at the EMBL beamline P13 at PETRA III at DESY (Hamburg, Germany). Taking advantage of the high the resolution and the strong anomalous signal of the Yb3+ (f′′ = 5.2 e− at 17 keV), the structure was solved by both SAD and ab initio methods. Data collection, experimentally phased electron density maps and the structure, especially with respect to the vicinity of the lanthanide binding sites, will be discussed.