Reading a mmCIF from PyMOL in PyRosetta

The mmCIF (PDBx as in extended PDB) format is meant to replace PDB format. Soon the RCSB PDB will have to adopt 4-letter codes for novel chemical components, which will break the PDB format. PDBx format is space separate as opposed to the really annoying column position in the PDB format and in the PDBx format the metadata can be stored in a nearly sensible manner. However, PDBx is solely a deposition format, but it is not really used as analysis format regardless of what the PDB claims. I personally had to add support for it because a reviewer asked me to. This lack of adoption is often attributed to the "if it ain't broken don't fix it" principle. Although I personally would argue that it may due to how it's implemented: opening a PDBx from one program ought to work in another, but this is not often the case. An example of this is PyMOL files read in PyRosetta.

Covalents, patches and N-O-S bridges in PyRosetta

Crosslinked residues are common, but for sure make up for it by being simultaneously highly intriguing and highly technically problematic. Oddly, I seem to keep bumping into them. During my PhD a decade ago I saw a talk by the father of Kiwi structural biochemistry, Ted Baker, about a curious case where they found an isopeptide bonds hidden in their crystal density. In a postdoc I worked with isopeptide bonds —I blogged about isopeptide bonds in Rosetta four years ago. During the start of the pandemic I dis some covalent-docking of compounds with PyRosetta for the Covid Moonshoot project, which evolved into Fragmenstein. Most tools have a hard time with crosslinks. And last month the Twittersphere was abuzz with the news of lysine-hydroxylcysteine (N-O-S) bridges in protein.

PyMOL will strip LINK entries from PDBs on saving while NGL obeys only CONECT entries in PBDs. An exception is PyRosetta: it behaves very nicely with disulfides, isopeptide bonds ( cf. repo of PyRosetta code from Keeble et al.) and other crosslinks —mostly. As a result I thought I'd add a note on how to add them in PyRosetta.

Filling missing loops by cannibalising AlphaFold2

I could not resist this Photoshop.
But the process is not as dramatic
and the results not as bad as Temple of Doom...
If done right.

AlphaFold2 models have a complete sequence, but for innumerable reasons the crystal structure of the protein is better, but may have missing spans. As a result one may want, for illustrative purposes only, to rip out the required parts from the AlphaFold2 models (as fragments) and have them built into the target structure. Here is how to do it by threading.

Tweaking AlphaFold2 models with PyRosetta

In a previous post I explored the pitfalls of an AlphaFold2 model from EBI. Here I thought I'd share some PyRosetta methods that may be handy to use with AlphaFold2 models.

Per residue RMSD

Recently I calculated the local RMSD caused by each residue and I thought I'd share the methods I used using PyRosetta —it is nothing at all novel, but I could not find a suitable implementation. The task is simple given two poses, find out what residue's backbone is changing the most by scanning along comparing each a short peptide window from each.

Remodel in Pyrosetta

The Rosetta binary Remodel is a great tool as it allows interesting designs to be made. However, it is rather incompatible with Rosetta Scripts and Pyrosetta as it is heavily dependent on command line options for customisation and repeats some of the processes internally. Despite this, it can be cohersed rather effectively to work in Pyrosetta with some convenience and this is how.

XML to Pyrosetta: EvolutionaryDynamicsMover as an example

In the previous post I discuss the strategies to use a Pyrosetta class when the documentation lets you down. One topic discussed was the conversion of a Rosetta XML script to Pyrosetta. Here is an example, namely using the EvolutionaryDynamics mover as an example.

Pyrosetta scripting without a manual

I got recently asked how to figure out how to write a Pyrosetta script when there is no example. This is definitely the biggest weakness of Pyrosetta and Rosetta script, but it is not insurmountable. In fact, there is a wealth of information that is hidden that can be mined. Here is how and in the next post, I give an example.

Rosetta/Pyrosetta on a cluster or in the cloud

Due to licensing Rosetta and Pyrosetta cannot be installed via apt-get/pip but has to be downloaded from the Rosetta Commons website. This makes things harder if you are in a colabs notebook, ssh'ed into a machine or running off a remote jupyter notebook. Luckily it actually is straightforward.

Stay hydrated

Waters can be an integral part of a protein structure, in fact, it is common to find water crystallised tightly in an X-ray structure. These waters can change the calculated Gibbs free energy of a protein and give better experimental results. Explicit waters can be added in Rosetta/Pyrosetta thanks to the SPaDES algorithm described in Lai et al. 2017. Here is a guide to using it in Pyrosetta.

Switching ligand in a PDB with Fragmenstein

For the Covid Moonshot project, one question by Prof. Frank von Delft of Diamond XChem led to a series of events that culminated in Fragmenstein, a module to do fragment mergers when the followup is as faithful to the starting crystal hits as possible. Even if it's intended use is the hit-to-lead process, there is a nice use that make it rather handy for computational biochemistry in general: switching the ligand in a PDB to another in an energy minimised fashion that obeys the original ligand.

Filling missing loops —the proper way

Previously, I posted about how to join proteins and add missing loops the shoddy way. Now I'll address how to do it correctly, using Rosetta or Pyrosetta —I am sorry this has been so long overdue.
Since posting this, I realised one can do it even faster by hijacking the threading algorithm, which albeit not it's intended purpose works fine for fixing a structure without supervision —which the following discussed methods do.

Love thy neighbours, but select them with caution

In Rosetta NeighborhoodResidueSelector behaves differently than PyMOL's expand selector and it is good to be aware of it. Namely, in PyMOL the distance is from any atom, while in Rosetta it is from the center of mass atom, unless specified differently. In reality CloseContactResidueSelector works like PyMOL's expand selector.

How to set up an electron density scorefunction in Pyrosetta

Energy minimising structures in Rosetta/Pyrosetta is essential to avoid artifactual results. Say a mutation is introduced and in the protocol the neighbourhood is repacked: if the structure is not energy minimised properly the neighbourhood repacking step will spuriously reward the mutation a very negative ∆∆G. One worry is that the energy minimisation is not faithful to the crystal structure. This argument has two sides, on one the fudgey force fields in Rosetta do not truly model the chemical interactions while on the other crystal packing may be unnatural. Both points have merit. After all Rosetta does use implicit water, which do not behave like the stripped crystallographic waters and some residues may have non-standard protonations etc. But if one wants one can use a scorefunction that is weighted by the electron density map and here is how.

Working around segmentation faults of pyrosetta: threads & processes

Rosetta often does not die gracefully. Pyrosetta is the same. If the starting template is not great segmentation faults will result in the kernel issuing signal 11 to kill the process. The way around it is to spin it up as its own process via the multiprocessing module and not the threading module, because child threads use the same process.

Everything you wanted to know about isopeptide bonds in Rosetta, but were too afraid to ask

Rosetta is great at predicting (with some accuracy) the energies of variant proteins, however, to make the most out of it with proteins with internal isopeptide bonds a few considerations are needed.