Dear Sonali -
It seems very likely that your original protein (which did not
crystallize) was proteolytically degraded over the one year storage, and
you now have a fragment of the original protein which is capable of
crystallizing. You should analyze all of the homologous structures in
the PDB to see if there are break points, like domain boundaries, which
could produce a fragment of the appropriate size upon cleavage. Then
use those fragments as your search models. Don't forget to consider
more distantly related proteins, or structures that match only a
subdomain of your protein.
However, it sounds as if you've already done that. Furthermore, if I
read your email correctly, the original crystal is now gone, so there is
no chance to collect additional data. (If this is not the case, you
might attempt to get enough data for sulfur SAD phasing.)
So what I suggest is that you try to reproduce the crystallization, not
by waiting a year, but by limited proteolysis of the full-length
protein. You should be able to get a stable fragment corresponding to
the thing that's in your tray, and analyze it by mass spec and/or
N-terminal sequencing. (You might be able to N-terminal sequence from
your existing crystallization drop as well.) Once you know what the
fragment is, you should try cloning and expressing just that fragment,
then setting up more trays, perhaps with selenomethionine labeling.
Good luck!
- Matt
On 6/20/12 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
--
Matthew Franklin, Ph. D.
Senior Scientist
New York Structural Biology Center
89 Convent Avenue, New York, NY 10027
(212) 939-0660 ext. 9374
