Solving the structure of biological macromolecules has become an important phase in the understanding of biological mechanisms at the molecular level. This structural information can be used to manipulate proteins for industrial applications, such as catalysis, and is also often used as the first step towards the design of active molecules that will give birth to new medications (as an example, mention can be made of inhibitors of the HIV enzymes).
Among the different techniques available, X-ray crystallography is a widely used technique for providing a three-dimensional representation of macromolecules. An X-ray crystallography experiment generally consists in placing the macromolecule to be studied—usually a protein—in an intense X-ray beam, thus producing an image of the diffracted X-rays, the intensity thereof being then recorded by the means of a specific detector. From these data, the 3-D distribution of the electronic density of the studied macromolecule can be obtained, from which the 3-D structure of the studied molecule may be built.
Most currently used sample containers for macromolecule crystals include a sample that is mounted on a metallic sample holder closing a vial. Such containers each comprise a metallic base or cap, in which a pin is inserted. A loop, which is glued at the tip of this pin, supports the crystal sample, whose size is largely less than 1 mm and typically of about 10 μm to 300 μm. During transportation, the vial is thus closed by the cap of the metallic holder. This way, the sample is stored in the liquid nitrogen contained in the vial.
Due to the intensity of the X-ray beam, it is usually necessary to maintain the sample at a temperature close to that of liquid nitrogen. Thus, the crystal—typically an object of a dimension largely inferior to one millimeter—is to be placed a short time after its obtaining in a Dewar container full of to liquid nitrogen, and maintained under that condition during its transportation. Throughout data recording, the sample, placed on a goniometer, is constantly bathed in a flow of nitrogen gas at a temperature of about 100 K. During its transfer from the Dewar to the goniometer, the crystal must never be exposed to the ambient atmosphere, and must at all times be maintained at a temperature of no more than 130 K. This operation, when realized manually, is a very slow and delicate step of the process. Now, fully automated transfer systems exist, but each new system has to prove a very high degree of reliability before being accepted by the research community. Indeed, a sample sometimes has taken months to produce. The risk of spoiling it is generally considered to be too high to try anything else but what is already known as working with a reasonable rate of failure.
In order to answer the increasing demand in solving protein structure from the structural genomics projects as well as from pharmaceutical industry, the automation of the beamlines dedicated to protein crystallography has become a necessity. The sample mounting/dismounting on a goniometer is an important step in the automation processes and therefore important developments have been performed. The goniometer is the device which rotates the sample in order to record the diffraction pattern leading to the 3D structure of the protein. Several automated sample changers have been developed throughout the world, such as the system “CATS” (see Ohana J., Jacquamet L., Joly J., Bertoni A., Taunier P., Michel L., Charrault P., Pirocchi M., Carpentier P., Borel F., Kahn R., Ferrer J-L.: CATS: a Cryogenic Automated Transfer System installed on the beamline FIP at ESRF. J Appl Cryst 2004, 37:72-77), whose capabilities of the main core—a 6-axis robot from the Staubli Company—have been extended to another system named “G-Rob”. This last system also offers the possibility to record diffraction data, being a goniometer-robot that combines the two functions of the robotic sample changer and of the data recording without the transfer on the goniometer.
For “CATS” systems as well as for all other sample changers, the weakness in the mounting/dismounting processes is the sample transfer from the storage location (a liquid nitrogen Dewar) to the magnet allowing the sample to hold tight during the data collection. It is also noteworthy that this problem is even more general since it is also present in the manual transfer on the magnet of a laboratory X-ray source. The difficulty and weakness thus reside in the transfer of the sample from the Dewar to the sample holder. Since the sample cap is metallic, the mounting process is easy to make. The problem arises when the robot wants to retrieve the sample after X-ray exposures. The vial is equipped with a ring to hold the metallic cap. However, since the sample holder magnet is stronger than that of the vial magnetic ring, the robot can not retrieve the sample by pulling the vial, and the sample dismounting becomes an issue.
To solve this problem, different solutions have been carried out, all using pneumatic or electric external devices equipping the sample holder (see for instance G. Snell, C. Cork, R. Nordmeyer, E. Cornell, G. Meigs, D. Yegian, J. Jaklevic, J. Jin, R. Stevens, and T. Earnest, Automated Sample Mounting and Alignment System for Biological Crystallography at a Synchrotron Source. Structure, 2004, 12: 537-45).
The major drawbacks of all these solutions with such external devices are their non-reliability and complexity.