As I have talked about in previous blog posts (here and here, if interested!), the majority of my research so far has focussed on improving our ability to generate loop decoys, with a particular focus on the H3 loop of antibodies. The loop modelling software that I have been developing, Sphinx, is a hybrid of two other methods – FREAD, a knowledge-based method, and our own ab initio method. By using this hybrid approach we are able to produce a decoy set that is enriched with near-native structures. However, while the ability to produce accurate loop conformations is a major advantage, it is by no means the full story – how do we know which of our candidate loop models to choose?
In order to choose which model is the best, a method is required that scores each decoy, thereby producing a ranked list with the conformation predicted to be best at the top. There are two main approaches to this problem – physics-based force fields and statistical potentials.
Force fields are functions used to calculate the potential energy of a structure. They normally include terms for bonded interactions, such as bond lengths, bond angles and dihedral angles; and non-bonded interactions, such as electrostatics and van der Waal’s forces. In principle, they can be very accurate, however they have certain drawbacks. Since some terms have a very steep dependency on interatomic distance (in particular the non-bonded terms), very slight conformational differences can have a huge effect on the score. A loop conformation that is very close to the native could therefore be ranked poorly. In addition, solvation terms have to be used – this is especially important in loop modelling applications since loop regions are generally found on the surface of proteins, where they are exposed to solvent molecules.
The alternatives to physics-based force fields are statistical potentials. In this case, a score is achieved by comparing the model structure (i.e. its interatomic distances and contacts) to experimentally-derived structures. As a very simple example, if the distance between the backbone N and Cα of a residue in a loop model is 2Å, but this distance has not been observed in known structures, we can assume that a distance of 2Å is energetically unfavourable, and so we can tell that this model is unlikely to be close to the native structure. Advantages of statistical potentials over force fields are their relative ‘smoothness’ (i.e. small variations in structure do not affect the score as much), and the fact that all interactions do not need to be completely understood – if examples of these interactions have been observed before, they will automatically be taken into account.
I have tested several statistical potentials (including calRW, DFIRE, DOPE and SoapLoop) by using them to rank the loop decoys generated by our hybrid method, Sphinx. Unfortunately, none of them were consistently able to choose the best decoy out of the set. The average RMSD (across 70 general loop targets) of the top-ranked decoy ranged between 2.7Å and 4.74Å for the different methods – the average RMSD of the actual best decoy was much lower at 1.32Å. Other researchers have also found loop ranking challenging – for example, in the latest Antibody Modelling Assessment (AMA-II), ranking was seen as an area for significant improvement. In fact, model selection is seen as such an issue that protein structure prediction competitions like AMA-II and CASP allow the participants to submit more than one model. Loop model selection is therefore an unsolved problem, which must be investigated further to enable reliable predictions to be made.