Combining 'dry' co-crystallization and in situ diffraction to facilitate ligand screening by X-ray crystallography.

X-ray crystallography is an established technique for ligand screening in fragment-based drug-design projects, but the required manual handling steps - soaking crystals with ligand and the subsequent harvesting - are tedious and limit the throughput of the process. Here, an alternative approach is reported: crystallization plates are pre-coated with potential binders prior to protein crystallization and X-ray diffraction is performed directly 'in situ' (or in-plate). Its performance is demonstrated on distinct and relevant therapeutic targets currently being studied for ligand screening by X-ray crystallography using either a bending-magnet beamline or a rotating-anode generator. The possibility of using DMSO stock solutions of the ligands to be coated opens up a route to screening most chemical libraries.

In-plate protein crystallization, in situ ligand soaking and X-ray diffraction.

X-ray crystallography is now a recognized technique for ligand screening, especially for fragment-based drug design. However, protein crystal handling is still tedious and limits further automation. An alternative method for the solution of crystal structures of proteins in complex with small ligands is proposed. Crystallization drops are directly exposed to an X-ray beam after cocrystallization or soaking with the desired ligands. The use of dedicated plates in connection with an optimal parametrization of the G-rob robot allows efficient data collection. Three proteins currently under study in our laboratory for ligand screening by X-ray crystallography were used as validation test cases. The protein crystals belonged to different space groups, including a challenging monoclinic case. The resulting diffraction data can lead to clear ligand recognition, including indication of alternating conformations. These results demonstrate a possible method for automation of ligand screening by X-ray crystallography.

X-ray structure reveals a new class and provides insight into evolution of alkaline phosphatases.

The alkaline phosphatase (AP) is a bi-metalloenzyme of potential applications in biotechnology and bioremediation, in which phosphate monoesters are nonspecifically hydrolysed under alkaline conditions to yield inorganic phosphate. The hydrolysis occurs through an enzyme intermediate in which the catalytic residue is phosphorylated. The reaction, which also requires a third metal ion, is proposed to proceed through a mechanism of in-line displacement involving a trigonal bipyramidal transition state. Stabilizing the transition state by bidentate hydrogen bonding has been suggested to be the reason for conservation of an arginine residue in the active site. We report here the first crystal structure of alkaline phosphatase purified from the bacterium Sphingomonas. sp. Strain BSAR-1 (SPAP). The crystal structure reveals many differences from other APs: 1) the catalytic residue is a threonine instead of serine, 2) there is no third metal ion binding pocket, and 3) the arginine residue forming bidentate hydrogen bonding is deleted in SPAP. A lysine and an aspargine residue, recruited together for the first time into the active site, bind the substrate phosphoryl group in a manner not observed before in any other AP. These and other structural features suggest that SPAP represents a new class of APs. Because of its direct contact with the substrate phosphoryl group, the lysine residue is proposed to play a significant role in catalysis. The structure is consistent with a mechanism of in-line displacement via a trigonal bipyramidal transition state. The structure provides important insights into evolutionary relationships between members of AP superfamily.

Automated analysis of vapor diffusion crystallization drops with an X-ray beam.

Crystallogenesis, usually based on the vapor diffusion method, is currently considered one of the most difficult steps in macromolecular X-ray crystallography. Due to the increasing number of crystallization assays performed by protein crystallographers, several automated analysis methods are under development. Most of these methods are based on microscope images and shape recognition. We propose an alternative method of identifying protein crystals: by directly exposing the crystallization drops to an X-ray beam. The resulting diffraction provides far more information than classical microscope images. Not only is the presence of diffracting crystals revealed, but also a first estimation of the space group, cell parameters, and mosaicity is obtained. In certain cases, it is also possible to collect enough data to verify the presence of a specific substrate or a heavy atom. All these steps are performed without the sometimes tedious necessity of removing crystals from their crystallization drop.

A new highly integrated sample environment for protein crystallography.

Protein crystallography is becoming a popular technique that is routinely used to access structural information. At one end of the process, sample preparation is now facilitated by commercially available crystallization kits. At the other end, structure determination has been made easier by automated software. Data collection, the step in between, is now usually performed on synchrotron sources. However, it is still restricted to experienced users and requires significant help from beamline staff. Part of this difficulty arises from the sophisticated experimental setup, which comprises a goniometer, a magnetic head, a device for changing the sample and monitoring accessories. It was proposed that this setup could be simplified by replacing these elements by a robotic arm that can perform all of the required tasks. In the present paper, it is demonstrated that this new setup can be used on a synchrotron beamline to mount and centre the sample and to collect diffraction data. This new system completely changes the design of the experimental setup by merging functions that were previously considered to be distinct. Moreover, automation of sample handling need not be considered as a specific development and is now included in a unique multipurpose device.