Structural studies
Fermentation
One of the major activities in the Unit is the structural analysis of proteins, especially of membrane proteins and membrane bound complexes. Some of these proteins are obtained by recombinant over-expression in various hosts; others, especially membrane protein complexes, are isolated from mitochondria and bacteria where they are expressed at their natural abundance levels. The Unit's large scale fermentation facility has a 70 litre fermentor for culturing micro-organisms including several fungal species, Escherichia coli, Lactococcus lactis, and Thermus thermophilus.
Protein purification
Before proteins can be crystallized, they have to be purified. Therefore, we have developed rapid and efficient methods to purify the proteins of interest based on affinity chromatography, ion exchange chromatography and size exclusion chromatography.
Protein crystallization
The Unit has a high throughput Innovadyne robot for setting up crystallisation trials in 96 well MRC crystallisation plates using sitting drops with a volume of 100 nl. Solutions for systematic screening are generated with a Tecan robot. Crystallisation trials are evaluated with high magnification stereo microscopes.
X-ray crystallography
The Unit is equipped with a Rigaku FR-E+, one of the brightest home X-ray sources available. It produces two slightly different X-ray beams. One beam passes through high flux optics which facilitates the screening of small or weakly diffracting crystals. The other beam passes via high resolution optics which can be used for screening crystals or data collection. Each beam is coupled to a MAR 345dtb detector.
Dehydration of protein crystals can lead to dramatic improvement in their diffraction properties and in the mosaicity of crystal lattices. A Maatel HC-1b device allows protein crystals to be dehydrated in a controlled, accurate and reproducible manner. It has been used with great benefit with crystals of F1-ATPase.
In addition, we collect X-ray diffraction data at the European Synchrotron Facility in Grenoble, France, at the Swiss Light Source and increasingly at DIAMOND, near Harwell, UK.
Structural modelling
If we are to understand the functions and mechanisms of proteins, we must know their atomic structures. To help in the process of modelling structures of proteins, the Unit has two state-of-the-art desktop stereographic 3D displays and a bespoke 93" stereographic 3D projection system for the visualisation of protein structures. They are especially useful in the manual refinement of atomic models from X-ray crystallography and the docking of ligands to protein molecules.