The knowledge of an accurate molecular structure is necessary for rational drug design and the development of effective therapeutic agents and drugs. The importance of X-ray crystallography to determine these structures can be highlighted by the 28 Nobel Prizes that have been obtained by scientists for discoveries in this field. In 2013 we celebrated 100 years of crystallography and 2014 has been declared as the International Year of Crystallography (IYCr) by UNESCO.
In our most recent labCrystal journal, we take a look at some of the new research coming out of X-ray crystallography labs around the world, from protein folds named after chocolate bars to crystals in oil.
Producing the perfect diffracting crystals frequently requires multiple experiments, tweaking one variable at a time, in order to optimise the crystallisation conditions. Following an initial crystallisation hit, the conditions are usually refined further by setting up a new plate and varying the concentrations of the components along the x- and y-axes in the reservoir. Devising unique approaches to this can often lead to uniquely successful results.
In one example from UCLA, Michael Collazo and colleagues optimised proteinase K crystals in a hanging drop format using an unconventional method (Figure 1). A gradient of reagents was set up over a reservoir of the original condition using mosquito® Crystal’s multi-aspirate function. Unlike a traditional optimisation process, in which components are mixed in the source reservoir together (Figure 1A), here they are equilibrating back towards the original hit condition which makes up the alternative reservoir (Figure 1B).
Figure 1. Optimising proteinase K crystal formation using an unconventional hanging drop method. (A) the traditional method of crystallisation, (B) the unconventional ‘alternative reservoir’ method of forming crystals, (C) a proteinase K crystal formed from the unconventional method of crystal optimisation (400 μm)
Being able to build this level of versatility into experimental design, and deviate from convention, will allow you make steps towards an optimal crystallisation condition.
Toblerones and herbicides
New research coming out of Australia’s Collaborative Crystallisation Centre (C3), conducted by the CSIRO structural biology group, has demonstrated the first X-ray structure of cyanuric acid hydrolase (AtzD), an enzyme involved in the detoxification of the pesticide atrazine. Making use of seeded crystallisation experiments – sped up via mosquito® LCP – the research revealed an interesting ring-opening chemistry and a novel fold in the protein structure, termed the ‘Toblerone fold’, that up until now had not been observed.
Figure 2. A ribbon representation of a tetramer of cyanuric acid hydrolase (AtzD), an enzyme involved in the detoxification of atrazine.
Thanks to the high resolution structure, the team were also able to successfully identify the binding pocket residues (Figure 2) involved in substrate specificity.
Microbatch, or ‘under oil’ protein crystallisation has been around since the 90s and allows for very stable experimental conditions due to the almost complete lack of vapour diffusion. This means the reagent concentrations do not change considerably throughout the experiment since the oil used is relatively water impermeable. However, alternative research using the dynamic vapour diffusion methods suggests that that the gradual change in conditions occurring via diffusion may in fact be essential to crystal formation.
A potential compromise comes in the form of water permeable oils or mixes serving as a barrier between the reservoir and crystallisation drop in a method called ‘vapour diffusion rate controlled under oil’. Allan D’Arcy later modified the microbatch under oil approach using a 1:1 silicon oil and paraffin oil mix, dubbed ‘Al’s Oil’, which allows nucleation to happen at any time when the protein concentration falls into the nucleation phase of the process.
Interestingly, the crystallisation under oil process has been especially successful with membrane proteins. This is thought to be due to the favourable kinetics this process provides, helping to identify otherwise impossible to obtain proteins (e.g. chlorophyll binding protein 43 of the Photosystem II complex from spinach (Figure 3)).
Figure 3. The first transmembrane protein crystallised using the microbatch technique (chlorophyll binding protein 43).
Finding the right optimisation process or technique can be a challenging and complex task. However, with the aid of great technological insight and advances, like TTP Labtech’s dragonfly® and mosquito, discovering the next novel protein structure is that much easier.