*An essay by Charlie Strauss*

In the course of using Rosetta, often one wants to compare sets of structures. There are different use cases that require different strategies. Considerations include the following.

Are you comparing Experimental structures or model structures? Methods like DALI and CE that work well for comparing Experimental structure but perform very poorly on low resolution model structures, probably because certain protein characteristics they look for like hydrogen bonding and perfect SS types are absent in model structures. The best choice for model to experimental structure overlap is Mammoth (aka Mastodon).

Are you comparing models that all have the same sequence? Here there is no alignment ambiguity. For structures that are close in RMSD, then RMSD is a fairly good measure of similarity. When structures are poorly overlapped large values of RMSD fail to be useful as a rank ordering of similarity. A banded Distance Matrix Error works slightly better when the RMSD is large. When the alignment is known, an even more robust measure over large and small ranges is MaxSub, which looks for the largest well-overlapped subset of the protein. Rosetta has a built in mode that uses Mammoths version of the MaxSub comparison measure. This does not do the full mammoth procedure of attempting to discover the alignment, but simply the MaxSub portion of Mammoth.

Are you comparing Models at high resolution? RMSD is pretty darn good as a general measure.

Are you comparing models to structures of much different lengths? Mammoth's E-value does a primitive but effective normalization of the ratio of the size of the maximal overlapped region to the length of the proteins being compared. This number is fairly robust against protein size variations, but hey nothing is perfect.

Do you want to know how to compare RMSD values in comparisons taken in proteins of different lengths? For example, is overlapping a two 30 residue proteins to an rmsd of 4 angstroms, more or less statistically significant than overlapping two proteins of length 100 to 7 angstroms resolution? There are a variety of ways to try to normalize these. Skolnick has some useful suggestions. Mammoth has it's own built in. But an extremely easy to grasp, quick and dirty measure is the RMS100 (http://www.proteinscience.org/cgi/content/full/10/7/1470). This renormalizes any RSMD to the the value it would have if the proteins were of length 100 and at the same level of statistical significance. I recommend using this before trying anything fancy.

What does it mean to compare two structures. Nearly everyone mis-appreciates the subtlety of this issue. The starting place for thinking about this is that one wants to see how well one can overlap two different structures. Usually this boils down to finding the maximal number of superimposable residues at some level of accuracy. Superimposable usually means the RMSD is less than some value, although some methods like DALI use other measures that allow distortion. There is an obvious dilemma over which is better, a longer overlap at low resolution or shorter overlapped region at higher resolution. There's also dilemmas about whether long gaps should be allowed (which increases random overlaps) or if local accuracy (SS structure) should be weighted over global accuracy. There is the fact that when the alignment is unknown, the problem is NP hard and one may not be able to solve for the global optimum alignment for a given matching criteria. Finally, there is problem of how to compare measures for proteins of different lengths.

When the alignment is Known: besides RMSD and DME, the next more sophisticated and widely used method is MaxSub. GDT is another measure that finesses the trade-off between high-resolution/large-numbers-of residues, by using several different resolutions, computing the maximum number of residues overlapped at each, and then forming a weighted average these to a single number. When the alignment is unknown, some popular methods are DALI, CE, SAL, and Mammoth. These try to discover the optimal overlap.

A key insight that seems to have eluded nearly all methods, aside from Mammoth, is that finding the maximal superposition is not really the question you sought to answer. What you really wanted to know is if two proteins were structurally similar and there's more to that than maximal superposition. In particular you don't really want to know the number of overlapped residues since that varies according to the criteria: more is not better. What matters is the statistical significance of the result. It's very very frequently the case that one algorithm will find a better superposition than another on any given protein. SAL for example tends to find the most of any overall but it gets beat by others on some proteins. But what you really want is something whose statistical significance score has low variance. That is it is not useful to have a method that reports some rankable scalar quality score for an overlap if the correlation between that score and the true human generated comparison is poor or has high variance. When it has high variance it means you can't just consider the top-match as being the best but must go way deep in your list of ranked matches to have high confidence in the result. Tests have show that Mammoth is far more correlated with human rankings than other methods even though it often reports lower number of "overlappable" residues. It's critical to understand this, and this is why I am strongly biased towards the Mammoth measure. However if pure overlap measures are your only goal I recommend SAL. SAL is also especially good for comparative modeling. DALI is slightly better at comparing two experimental structures.

- Analyzing Results: Tips for analyzing results generated using Rosetta
- Rosetta on different scales: Guidelines for how to scale your Rosetta runs
- Solving a Biological Problem: Guide to approaching biological problems using Rosetta
- Resources for learning biophysics and computational modeling
- Getting Started: A page for people new to Rosetta
- Rosetta Servers: Web-based servers for Rosetta applications
- PyMOL: A tool for visualizing macromolecules