Cyrus Bench has implemented DNA/RNA handling in order to allow loading and modeling with these highly biologically-relevant molecules. Rosetta is excellent at modeling with nucleotides present. It has been successful at modeling proteins with DNA and RNA for deriving affinity, specificity, and for exploring the effects of mutations. However, there are a few limitations that you should be aware of before working with DNA/RNA in CAD.
Warning: Our Structure Viewer has an issue that sometimes makes DNA/RNA look broken. To verify that an apparent break is not being modeled as a break, download your structure from CAD and open in another viewer such as pymol. If the DNA/RNA is not broken in this viewer, then you can be certain that the model is not modeled as broken. This is important because there are some non-canonical DNA/RNA types that will break and some that will just look broken.
Also, DNA and RNA are modeled as rigid-body structures. Rosetta would need additional score terms in order to model DNA flexibility properly. And RNA modeling is one of the most challenging things to do with Rosetta. So, we recommend partnering with an RNA specialist if you want RNA flexibility. For most modeling cases, rigid DNA/RNA is sufficient. Contact us if you would like further advice at support@cyrusbio.com.
LIMITATIONS
Our DNA/RNA modeling is new code. So this early version is not a comprehensive tool. The current version is mostly limited to canonical DNA/RNA.
There are many non-canonical DNA/RNAs that we will eventually be able to handle, but this early version is limited to A,U,T,G,C (for the most part). Your non-canonical residues will be initially deleted from the structure when you have a structure in Structure Loader. You can select the non-canonicals and undelete them before accepting changes and allowing everything to be loaded into CAD. However, you should verify that the residues are correct by downloading the file from CAD and manually inspecting the residues in another structure viewer. Our Structure Viewer has a known issue with DNA and RNA that makes some regions appear broken even though they are normal. Things that look broken in the Viewer will model normally.
So, some non-canonical DNAs will undelete and load into CAD normally. This should be verified because our Viewer is unreliable at viewing DNA at the point. This is an issue we are working hard to improve.
Non-ideal DNA/RNA structures will lose important features or not load at all
Many structures in the PDB or from other sources will have bond lengths or angles that are too far from the norm so will not be recognized properly. Commonly, the offending nucleotide will be treated as a separate small molecule than the DNA/RNA that it is connected to. Our Structure Loader will drop its covalent connection to up and downstream DNA/RNA and create a small molecule out of the remaining atoms. This would likely cause significant problems if you tried to model with this inaccurate variation, so you can either delete the small molecule or fix the input file. Feel free to email us the file so we can try to fix the molecule for you.
Example: PDB ID 6D0M: DNA Polymerase eta bound to DNA/RNA and Cytarabine
Above, you can see 6DOM in our Structure Loader. One nucleotide in chain T position 1 labeled DC has an X to the right. This indicates that the atoms are too far from ideal to be included in the structure so have been deleted. Below that are two nucleotides in chain P positions 1 and 2 labeled DA and DG. These had non-ideal atoms, but not so bad as to be deleted so they were modified in some way. To get more information, hover your mouse over the position on the structure or over its name to the right or above.
In the above example, DG at chain P position 2 had warning: altLocs: A, B. This means that during processing for this residue, it could place an atom in the residue at 2 locations, A and B. It discarded option B.
In the case of DA at chain P position 1, the warning reads the same, but you can see that it loses its covalent attachment to position 2 and is handled as a separate small molecule (below).
Again, DA at P1 gets the altLocs warning indicating non-Ideal atoms, but it has an additional problem that does not include a warning. You may note that the cartoon mode appears empty. This is a Viewer Error that occur for some cases. Above center, this structure has been loaded with all molecules accepted. Then it was opened in the default Structure Viewer which has more features than the Viewer available during Structure Loading. Here you can clearly see the molecule in sticks mode and it does not appear to be covalently attached. It only shows the adenine bound to pentose as if it was converted to a nucleoside. This is just a Viewer Error. If you download the file off CAD, you can see that the normal DNA is present. And more importantly, it models normally. We just need to fix the Viewer Error.
DNA/RNA will not work with DDG or Repack
Normally, you have the option to turn on or off Repack for any residue or small molecule present in your structure. This is because side-chains and small molecules have a set of alternative conformations that can be sampled during modeling. However, DNA and RNA are not handled this way. During modeling, the original conformation is held rigid. The protein and other molecules are allowed to move in relation to DNA/RNA, but DNA/RNA does not change.
Our DDG tool does not run with any non-protein molecule present. This tool may enable it in the future, but it needs to be benchmarked to see how predictive the tool will be with small molecules and/or with DNA/RNA. This is something we are working on, but cannot promise that DDG will be a good tool when small molecules are present. So, in the event that we find it effective, we will work on adding this feature in CAD.