Can anyone please comment on the distribution map? It seems that I don’t have enough information across the equatorial axis.
Does it indicate preferred orientation?
Can collecting data on tilted grid be a solution to this?
Your help is highly appreciated.
This looks fine - why do you think there is an issue here?
After focused refinement of a disordered portion of the complex, it shows ~4.5 Å but the map does not look like near to ~4 .5 Å
don’t know how to fix the issue !
I am using CS 2.15.
I have a data set of 490K (initial) particles of a complex comprising protein A(tetramer, 240 kDa), protein B(tetramer, 130 kDa) and a RNA(C, 63 kDa). After ab-initio reconstruction followed by homogenous refinement (distribution map shared) and heterogeneous refinement (132K particles), I have now 4 distinct classes (7-8 Å), three of which comprise one A(rigid part), one B(disordered), and one C (complex 450 kDa) and one of which is a hierarchical assembly (one A, two B, one C, 580 kDa).
Is “Force hard classification” recommended in heterogeneous refinement?
I carried out a 3DVA at this stage; each class display no movement of B except densities coming on and off.
Does it mean the map is not clean enough?
Interestingly, top half of each complex represents a fundamental unit required for reaction coordination which is one B, oneC and half of A and this intrigued me to combine the particle stack(homogenous refinement), subtract particles of top half of the refined map(>200 kDa), and local refinement (4.5 Å). However, map does not look like near to 4/5 Å.
It indicates my workflow is surely wrong. This also made me to think what if I had incomplete information across the top/bottom views (B is sitting on top of A)
Or does it mean I don’t have enough coverage for B compared to the whole complex?
Can I ask for a favour please to help me figuring out what wrong I have been doing and what would be an ideal workflow for my data set?
Many thanks in advance
I agree that the orientations look fine.
Since you are doubting the FSC resolution, it might be worth upgrading to v3 as some changes were made to the local refinement job type. You could also try just a normal homogeneous refinement or non-uniform refinement with your subtracted particles and a mask around the region of interest. In v2.15 those jobs are less sensitive to masking than local refinement can be in some cases.