There is often one more round of #ISOLDE + final refinement to do, if only to check visually that the "final" refinement didn't mess anything up. Of note, #servalcat produces a sharpened map and a difference map, both extremely helpful to spot and fix problems. I have found very subtle modeling errors that I had totally missed until I examined the model against these maps. So, whenever I use servalcat, I always do at least a second round of visual inspection and manual fitting in ISOLDE.
16/19

This step is important for two reasons. The MD force field will produce a distribution of bond lengths and angles that doesn't quite match the distribution expected by the validation suite run by the #PDB deposition server, so omitting this last refinement will cause many things to be flagged as outliers. The other reason is that #ISOLDE doesn't (yet) refine atomic b-factors, so omitting this last refinement would leave nonsensical b-factors in the model.
15/19

Once I am happy with the current state of the atomic model, the next step depends on the global resolution of the map:
1. For low resolution (~5 to 2.5 Å), I run the model through #phenix.real_space_refine using the parameter file generated by #ISOLDE. This turns off most things, but refines the coordinates with reference-model restraints (so they won't move far from where I left them with my manual fitting) and the atomic b-factors.
13/19

After this exhaustive first pass inspecting each residue, I try to clear the biggest problems flagged by #ISOLDE's validation tools (there are always some). Clashes are rare because atoms that are too close strongly repel each other under the MD force field. Ramachandran and rotamer outliers are rare in #AF2 models, but can arise when the simulation distorts the model. These problems often point to regions where the map is ambiguous, and where the model needs more attention.
12/19

2. A post-processed map from #deepEMhancer used only as a visual guide (so not allowed to pull on the atoms in the iMDFF procedure in #ISOLDE) is often helpful in regions of lower resolution. I have seen cases where I couldn't find a stable conformation for a long side chain, until I placed it where the deepEMhancer map suggested: suddenly it was nicely stable in the combined pull of the raw map and MD force field.
11/19

Then comes the fun part in #ISOLDE: tug the atoms and they respond in a physically realistic way, how cool is that?! This step can also be very tedious if the model is large. Using the `isolde step next` command, I walk along each chain from its first to last residue (in a symmetric map, only for the ASU), fixing problems. This includes placing water molecules, ions and ligands, where there is density supporting their presence. This is super fun to do, and teaches a lot about chemistry.
9/19

At this point, I use #ISOLDE to do manual fitting. If the map has a low resolution, I apply secondary structure restraints to the model. I start with a global simulation to let the model settle, which I let run until the dots in the interactive Ramachandran plot have reached an equilibrium. With #AF2 models, there shouldn't be Ramachandran outliers in the first place, but this is a good overview of how many residues are moving. I want them to settle in the nearest density before I continue.
8/19

2/2
Aber eigentlich passiert alles „nur“ im Inneren der Figuren.
Schon mit Beginn der Ouvertüre - dem magischen Tristan-Akkord - ist die große Frage in den Raum gestellt. Ob sie beantwortet wird? Man wird es sehen - oder besser gesagt erleiden…