Sonali,
How did your MR search(es) fail?
1. Too many clashes? (allow more clashes or remove likely floppy bits
of the protein, e.g. N- and/or C-termini)
2. Could not place all molecules in the asymmetric unit? (Consider
searching for fewer molecules in asymmetric unit. A partial solution
may give you a better idea of the number of molecules in the ASU by
examining symmetry packing in Coot or Pymol. The Matthews Calculator
does not always give a definitive solution, especially for >2
molecules per unit cell. Consider examining a partial solution as
suggested above.)
3. Failure to converge? Phaser will almost always come up with
something, even if it is not right. Examine symmetry packing in Coot
or Pymol to see if it makes sense. Maybe you still have the wrong
space group.
4. Twinning? I've forgotten to do this on occasion, and twinning can
lead you on a merry chase that won't refine properly.
With 46% identity, the likelihood of finding a solution with Phaser
should normally be very high. An Rfree of >50% is normally indicative of
a random fit (e.g., non-solution). If you can get an Rfree of <50% with
the out-of-the-box MR solution, you may have a valid solution. I solved
one structure where the good MR fit had an Rfree of 46-48%, and the
"random" solutions were 52-55%.
You may have considered these issues already, but if not, hope this helps,
Cheers,
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 6/20/2012 2:13 PM, sonali dhindwal wrote:
Dear All,
I am working on a protein for last so many years and for which i have
got crystal now in a tray which i kept 1 years ago. It diffracts well
and resolution is 2.2A, which is good.
I indexed in HKL2000, mosflm and automar and it shows P21 space group
in all data reduction packages. But when I tried using molrep or
phaser then I do not get any solution. The sequence of my protein is
having 46% identity with other available crystal structure.
Also when I tried to get matthews coffecient, it calculates its
molecular mass less ( about 35 kDa) than which should be (original
54kDa) with solvent content 47%.
I have also run the silver staining gel of the protein which contained
crystal that shows about 45 kD protein band which is 10 less than the
original. Also I tried to run gel on crystal but it did not give
anything as it was a small crystal.
I have tried all combinations of the search model and tried to break
available pdb many ways to make different search models but have not
got any good solution. Molrep gives contrast even 10 or more but no
good electron density map yet. Free R and figure of merit becomes 52%
and 42% respectively in Refmac with all the solutions.
I will highly appreciate all the suggestions for this kind of problem.
Thanks and regards
--
Sonali
