NU refine resolution-5.5 A, map doesn't have those features

[Nan_12]

Hi,

I am working on a flexible protein (~128 kDa). I did Topaz picking and performed one round of 2D classification to remove obvious junk. I used the remaining particles to do Het Refinement with 3 models of paralog structures and 2 junk volumes (obtained after killing ab initio). The particles obtained after Het Refinement were used in an NU Refinement job, with a good volume from the previous Het Refinement as its input model. This gave a 5.5 Å map, but the map lacks those features.

I am doing the processing at bin 4 due to space constraints (pixel size at bin 4 is 2.6 Å/pixel). In parallel, I did multiple rounds of Het Refinement, but the map quality did not improve. Also, the cFAR value is always less than 0.5.

The central two core domains are stable, whereas the extended helical domains are quite flexible. Each of them has one domain attached via a linker.

I am a newbie to processing, so any advice on how to improve the map quality would be greatly appreciated.

[jcoleman]

Rather than using references from homologous proteins, why not try to do ab initio to get an initial reference? It’s usually much better to generate your initial reference this way as maps generated from pdbs will induce bias and there may be meaningful differences between your protein and these other related proteins.