auxotroph, and then grow this strain in the presence of selenomethionine (a selenium-
containing analogue of methionine). Selenomethionine is therefore incorporated
into the protein in the place of methionine, and the final purified and crystallised
protein has the selenium heavy metal conveniently included in its structure.- Diffraction data and phase information having been collected, these data are
processed by computer to construct an electron density map. The known sequence of
the protein is then fitted into the electron density map using computer graphics, to
produce a three-dimensional model of the protein (Fig. 8.5.). In the past there had been
concern that the three-dimensional structure determined from the rigid molecules
found in a crystal may differ from the true, more flexible, structure found in free
solution. These concerns have been effectively resolved by, for example, diffusing
substrate into an enzyme crystal and showing that the substrate is converted into
product by the crystalline enzyme (there is sufficient mother liquor within the crystal
to maintain the substrate in solution). In a more recent development, it is now
becoming possible to determine the solution structure of protein using NMR. At
present the method is capable of determining the structure of a protein up to about
20 000 kDa but will no doubt be developed to study larger proteins. Although the
time-consuming step of producing a crystal is obviated, the methodology and data
analysis involved are at present no less time-consuming and complex than that for
X-ray crystallography.
Fig. 8.5(Relaxed-eye stereo pair): A Ca-trace of herpes simplex virus type 1 thymidine kinase from a
crystallographic study of a complex of the enzyme with one of its substrates, deoxythymidine. The enzyme is an
a–bprotein, having a five-stranded parallelb-sheet surrounded by 14a-helices. The active site, occupied
by deoxythymidine, is a volume surrounded by four of the helices, the C-terminal edge of theb-sheet and a short
‘flap’ segment; a sulphate ion occupies the site of theb-phosphate of the absent co-substrate ATP. (Short missing
regions of chain indicate where electron density calculated from the X-ray data could not be interpreted.)
(Picture provided by John N. Champness, Matthew S. Bennett and Mark R. Sanderson of King’s College London.)339 8.4 Protein structure determination