Vol.12 - Winter Issue 2011
One year with GeniX at Carlsberg Laboratory
An example of enzyme structural studies preliminary (anterior) to soaking experiments
Introduction
In September 2010 a GeniX microsource system has been installed at Carlsberg Laboratory replacing a Rigaku RUH3R rotating anode generator on a protein crystallography set-up. Research at Carlsberg Laboratory employs structural, kinetic and spectroscopic techniques to obtain a detailed molecular description of enzyme reactions. Primary research currently is focused on glycosyltransferases and glycosidases involved in the biosynthesis of oligosaccharides. This includes studying their use in enzymatic synthesis of oligosaccharides, the design and evaluation of glycosyltransferase inhibitors and assay development, including single cell enzymology. This research is part of a major new initiative on structure-function studies on barley starch synthases and detoxification enzymes.
Single crystal diffraction experiments at Carlsberg Laboratory provide static images of the enzymes with different kinds of substrates and inhibitors bound in the active site. This short note and the presented results illustrate the use of a laboratory crystallography set-up based on a low power high brilliance source in a 3D structure analysis of enzymes.
Experiment
The X-ray equipment at Carlsberg Laboratory is composed of a GeniX Cu HF System (50W, 50 µm Microsource with FOX 3D optics) coupled to a Rigaku R-axis IV++ Image plate detector (see figure 1).
One year after the installation of the GeniX, the X-ray equipment is very frequently used for screening crystal quality and determining suitable cryogenic conditions as well as obtaining space group and unit cell parameters of the crystal. However, occasionally the equipment is also used for collecting entire crystal diffraction data sets to obtain the 3-dimensional structure of the enzymes. An example is illustrated on figure 2 with preliminary structure studies of a human blood group glycosyltransferase mutant.
Results
An X-ray crystallography data set was collected to investigate the state of the enzyme binding site in crystals of the human blood group glycosyltransferase (conditions are summarized in table 1a). A highly redundant data set was recorded (see table 1b).
Often UDP is co-purified with the enzyme. Provided the binding site is empty, other ligand soaking experiments with other crystals from the same crystallization tray could be carry on. The data quality collected with the GeniX was suitable for obtaining a high quality electron density map of the enzymes structure. The result from the solved X-ray structure was that the enzyme molecules in the crystals contained UDP in the binding site. Therefore, is was concluded that in order to perform soaking experiments it is necessary to go through extra measures to remove the UDP from the binding site before crystallizing the enzyme. Furthermore, in addition to high data quality Dr René Jorgensen seems very satisfied with the low maintenance level of the GeniX . ''The start up and shutting down of the GeniX seems to be very stable and so far there has been no maintenance on the machine. With the old X-ray generator we had to change the filament every 1-2 months and we are very happy not to have to do this anymore.''
