Michel Pirocchi

CATS: a Cryogenic Automated Transfer System installed on the beamline FIP at ESRF

CATS allows users to mount and dismount their crystal samples remotely on the diffractometer, without entering the experimental hutch. CATS has been integrated into the automated control of FIP, allowing users to choose the wavelengths, optimize the beam intensity, mount and screen their crystal sample automatically and finally record diffraction data on the best sample(s).

FIP: a highly automated beamline for multiwavelength anomalous diffraction experiments

FIP is a French Collaborating Research Group (CRG) beamline at the European Synchrotron Radiation Facility (ESRF) dedicated exclusively to crystallography of biological macromolecules, with a special emphasis on multiwavelength anomalous diffraction data collection in the 0.7-1.81 A wavelength range. The optics, consisting of long cylindrical grazing-angle mirrors associated with a cryocooled double-crystal monochromator, delivers an optimal beam in the corresponding energy range. The high level of automation, which includes automated crystal centring, automated data-collection management and data processing, makes the use of this beamline very easy. This is illustrated by the large number of challenging structures that have been solved since 1999.

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.

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.

Automated room-temperature ligand screening on beamline FIP at the ESRF

FIP, an automated beamline at the ESRF for protein crystallography, is designed to host a large variety of experiments such as anomalous, on-line absorption spectroscopy coupled, humidity controlled, and in situ diffraction experiments. Several of these techniques are made possible by a robot based system, named G-Rob. It is a fully integrated, multi-purpose automated and remotely controlled system that integrates several functions: multi-standard sample changer for frozen crystals; goniometer for frozen samples or capillaries [1]; crystallization trays handling for in situ (in the plate) screening and data collection [2]; beam monitoring. Another, but important, feature of beamline FIP is a web-based user interface, named WIFIP. With this interface, several users can share the control of the experiment, from sample handling to data reduction, through a web browser, on the beamline or from the lab or home. WIFIP includes the CrystalListing capability, that makes possible the listing of series of crystals from a crystallization plate, in order to collect dataset in a row in an automated way. It makes in situ experiments much easier and automated. X-ray crystallography is an established technique for ligand screening in fragment-based drugdesign projects, but the required manual handling steps – soaking crystals with ligand and the subsequent harvesting – are tedious and limit the throughput of the process. On FIP, an alternative approach is proposed: crystallization plates are pre-coated with potential binders from DMSO stock solutions [4], prior to protein crystallization, and X-ray diffraction is performed directly in situ. With these pre-coated plates, crystallographers can screen these libraries without any stock solution or crystal handling, and obtain the structure of complexes at room temperature. In the future, pre-coated plates will be made available, either with generic libraries or with custommade collections. The first one is prepared with a 3D 192-fragment library.

New developments for a full automation of the FIP beamline at the ESRF

24 European Crystallographic Meeting, ECM24, Marrakech, 2007 Page s116 Acta Cryst. (2007). A63, s116 SPACE [1] and beamline control software BSS [2]. For smooth communication with distant users via the Internet, we have newly developed the data management system DCha (Database for Crystallography with Home-lab Arrangements) which mediates between the users and SPring-8 beamlines. D-Cha provides the GUI for users to deposit the experimental conditions for samples and to browse / download the collected data on web browser. The mail-in system has been developed and operated for Structural Genomics Project at RIKEN Structural Genomics II (BL26B2) since September 2005. Then the system has been presented for public users at Structural Biology III (BL38B1) since December 2005 and at RIKEN Structural Genomics I (BL26B1) since December 2006. At BL26B1 and BL26B2, mail-in system has been operated on a daily basis. In addition, the commercial mail-in service has just started in July 2006, as the joint project among JASRI, RIKEN and analysis service companies. The mail-in data collection is our first step of remote beamline access at SPring-8. The next step is to achieve the fully remote control data collection based on the mail-in system.