Equipment to Support Protein Crystallisation in the York Structural Biology Laboratory

The determination of the structure of biological macromolecules using X-ray crystallography is providing information about how biological systems work at the level of individual molecules. This information has transformed our understanding of some of the fundamental processes of life. One example is the crystal structures of the protein haemoglobin in the presence and absence of oxygen which explain how blood cells are able to transport oxygen from the lungs to the tissues. A second example would be how the activity of metabolic enzymes such as glycogen phosphorylase are regulated so that cells either burn or store food depending on the nutritional state of the organism. In addition, crystal structures of key proteins can give important information about what happens at the molecular level in disease or infection and can guide the development of new drugs. A striking and topical example is the determination of the structure of the enzyme neuraminidase from the influenza virus. The structure was used to direct the development of the drugs relenza and subsequently tamiflu. Moreover, structures of neuraminidase and other influenza proteins allow us to understand why variants of flu (such as avian influenza or the Spanish flu of 1918) are so virulent, perhaps providing guidance on developing even better drugs. There are a number of steps in protein crystallography before a structure can be determined. The process starts with the production of large quantities of the protein of interest. The next, key step is to produce crystals of the protein. This can be a long and difficult process, finding the right solution conditions under which crystals will form. Crystals are necessary as when you shine X-rays on a crystal, you obtain a diffraction pattern from which, with a lot of effort, you can extract an image of what the structure of the molecule looks like. Therefore, the success of X-ray analysis is underpinned and determined by successful provision of crystals. In recent years, there have been continual improvements in both the design of the solution conditions and the robotics equipment available for setting up large numbers of crystallisation trials. A particularly important development has been the use of very small, nano-litre sized drops. These reduce the amount of protein that needs to be used, increases the number of crystallisation trials that can be conducted and in some cases, the small drops have increased the success of forming crystals. The Structural Biology Laboratory at York (YSBL) is one of the largest laboratories in Europe dedicated to the determination and analysis of protein structure. Scientists in YSBL have made major contributions by not only determining the structures of many important proteins, but also in developing the experimental and computational methods that are required for X-ray crystal structure determination. One element of this methods development has been devising new crystallisation screen solutions and also working with the manufacturers in developing improved robotics equipment. This application is for an upgrade to the robotics equipment that supports crystallisation trials in YSBL. This will allow scientists at York to benefit from some of the advances in equipment, to determine the structures of more proteins, more rapidly, but also to continue to work with manufacturers in making further improvements.

Crystallisation is a key step in the determination of protein structure by X-ray crystallography. Over the past 4 years, we have worked to adapt and improve various robotics systems to increase the quantity and quality of crystallisation trials. This application is to replace old, prototype models used in this work with the latest improved versions to facilitate structural biology research at York. Specifically, we request funds to (i) replace an old model of the crystallisation robot (Mosquito), (ii) replace an old screen making robot (Tecan) - both with state-of-the art new models, and (iii) to purchase a screen transfer robot (Hydra) to distribute screen solutions into the crystallisation plates, currently a manual process. The Mosquito and Tecan robots were purchased as advanced prototypes 4 years ago and they transformed protein crystallisation in the York Structural Biology Laboratory (YSBL) into a modern high throughput (HT) approach. We have collaborated with the manufacturers to optimise these instruments and new generations of equipment are now the first choice for many structural laboratories worldwide. However, the YSBL models have become obsolete and inefficient. The systems are indispensable for the everyday HT crystallisation process in York and there is an urgent need for their replacement. A key bottleneck is the manual distribution of screen solutions into the crystallisation plates. The proposed Hydra robot will facilitate screen-to-plate liquid transfer with efficiency that would match the other 2 robots. Together, these three instruments will create hardware-software consistent and complementary crystallisation suite. The manufacturers' appreciation of the very productive collaborations with the YSBL resulted in remarkably substantial (66%, 50%,20%) discounts on the list prices. It has to be stressed that the new instruments are also vital for research programmes for the development of new crystallisation methods and image recognition.