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Hey Nick,
Maybe you should check the R_iso of your datasets (should be around 10)
to see if there is something wrong with one or more datasets. Since the
resolution dropped for the last two datasets you might have radiation
damages.
The next thing I would check is the anomalous correlation coefficient.
XPREP is fine for this. HKL2MAP is a good alternative.
If you want something automatic try the newest version of AUTOSHARP. It
does a lot of analysis for you and may give you some hints whats wrong.
regards
Christian
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
