Hi,
I recently collected Se-MAD data (resolution
--> p=2.4, e=2.8, hr=2.7) and currently attempting to find the heavy atom
sites, but have experienced several problems. I expect 20 Se sites
in my protein (~99 KDa).
Initially, I just used the peak dataset (2.4 angstroms) for SAD Phasing
procedures in CNS, AutoSol and Solve (via PHENIX), BnP, and
CCP4. With CNS, I found ~20 sites and they all showed up very nicely in
the maps. But when I did the density modification of both hands, neither
map seemed to be better than the other, which was very disappointing.
Next, I tried AutoSOL strategy in PHENIX, which found the sites also, but
listed varying degrees of occupancy for the sites, ranging from 0.7 to
0.05. Not being that familiar with PHENIX, I used the sites from AutoSOL
and made maps using CNS, but, map quality wasn't improved at all. I
have also tried using CCP4 and BnP to find the sites, hoping for better
results, but these programs failed to find all the sites, typically fewer than
6 or so, depending on the program. If they did list more sites, the
signal for the sites wasn't significant over background. My boss'
friend used SHELX to found all the sites with much improved
occupancies. Using these sites, the maps looked a bit better, but still,
no real discernable secondary structure, and still, alot of fragmented
density.
I am currently working on MAD phasing procedures, which have so
far, produced about the same results.
This is my first experimental phasing experiment, so I would like
some advice or guidance about either SAD or MAD phasing procedures in
general. For example, what programs do people typically use, and why
choose one program over another? Which is most reliable? I had
expected the experimental phases to give me a map with good enough quality to
at least build a rough model, but so far, this isn't the case. So what
steps can be taken now to improve the quality of the maps or phases to at
least get to a map with some discernable features?
Thanks in advance for your feedback and guidance.
Cheers,
Nicholas
