After that I started to play with auto/manual refinement ('astigmatic' + 'regular'), with dozens of attempts, someone promising, but nothing really satisfying, perhaps due to my background limitations. I started with initial Res limit in the auto-refinement of 20A or later 16, 10, but I get only a worsening. When I employed 8,7,6 A as initial value, it started to produce something fine, but then playing with different smoothing factor I get completely different results.
3D refinement not improving
Mon, 10/21/2019 - 04:04#1
3D refinement not improving
I am a new cistem user, basically first experiences on data-processing. Currently I am trying to perform 3D refinement based on some ab-initio classes but I get stuck, so I would need guidance, perhaps also conceptual.
My dataset requires the VPP settings and unfortunately circa a third of the 2,4k micrographs had poor/absent CTF-fitting. About the 1,6K micrographs left (>310k particles) almost 50% are sligthly astigmatic, but maintaining good 1D plots, good CTF fitting. I was able to easily perform good particle picking and 2D classication runs. I can clearly see different orientations of my protein (known from literature) with micelles.
Q1) This loss of micrographs is due to the Volta PP settings?
Q2) Is there a way to fix it and recover micrographs lost? I tried slow CTF or other settings on a subset, but it employs too much and it doesn't give me anything good back (too close to focus?).
Despite there were apparently no changes between 2D runs between 'astigmatic' and 'regular' I later performed ab-initio run (80steps) using only the 50% with better CTF-fitting, no astigmatism (after several 2D classification runs > 50k particles) and among the 5 different classes, 3 were showing a very good profile and slightly different (but evident) conformations. I could see clearly the helices as tubes separated between each other and a CC of 0.77 when employing Chimera + PDB model from MDFF run.
Q3) I don't know if employing the 50% 'regular' for abinitio was a good thing or conceptually wrong, but since in my lab with relion they were experiencing some troubles with a reference model (suspect cause of flexibility) I wanted Cistem to calculate the initial 3D classes in some way.
Q4) Shouldn't be higher the initial CC or I am making an uncorrect consideration?
Q5) Estimating the resolution with Resmap of the 3 classes is reliable at this point?
Employing SF 0.5 I generally can see a gradual increasing of the FSC curve, while if I try 0.25 I can visually see the quality of the maps dropping down. No general change with SF 1. Some lecturers told me to use SF of 3.0 / 4.0 and employ a mask (done on Relion derived from the same density ) and apparently the density after that looks much better, with the helices assuming a jagged shape more than tubes. But at the same time I can clearly see that some parts are lost and not fitting the shape of the protein (in terms of CC I see a decreasing from 0.77 to 0.50) and in terms of FSC curve I don't see any increasing.
Q6) How many refinement runs are generally advisable? Generally we should start with autorefinement?
Q7) Which strategy I should follow? Perhaps it's a 'beginner' problem, but I would be glad if you could direct me in the right way. I can send you a small table with resumed all attempts, if necessary.
Many thanks in advance,