There are many many kinases with different conformations of the C
terminal and N terminal domains.
I would first refine the Cterminal as far as you can go - correct the
sequence etc etc.
Then align a selection of kinases on your C-terminal coordinates (
MSDfold at www.ebi.ac.uk/msd will return you a set )
Then look at them in the density and see if one looks better than the
others..
Maybe you can try MOLREP with phase information to position the N
terminal domain.
Or ffear using the difference map coefficients as input.
But kinases are notoriously difficult.
Eleanor
PS - Soetinmes Arp-Warp can do miracles - never for me, but for others..
Das, Debanu wrote:
Hi Eric,
If your Phaser results show a high Z-score (> 8) AND high LLG AND your
solution packs without clashes AND refines (even though starting
R/Rfree is high) AND reproduces density for the model portion AND
produces some Fo-Fc density for the missing portion, most probably
your solution is correct.
Starting R/Rfree can be high, especially given that you have only ~50%
of the molecule and which may have structural differences from the
target.
What you could try out is to manually position the second
domain/portion based on the partial density and any other information
you may have, draw a mask around this complete molecule and use this
mask for further density modification to see if you can improve the
density in the missing region. You can try several different molecular
masks with different positions of the missing portion.
Unfortunately, if that does not help, you will have to resort to
experimental phasing. The phasing power from the partial model may not
be good enough to get complete phase information for the whole
molecule. This should be easier in your case assuming the MR solution
is right. Even if you get weak/poor derivatives, you may be able to
find heavy atoms sites using the partial MR phases and then combine
MR+experimental phases.
Regards,
Debanu.
*From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf
Of *Eric Liu
*Sent:* Tuesday, July 31, 2007 2:24 PM
*To:* [email protected]
*Subject:* [ccp4bb] how to bring back the missing density for half of
the structure
Hi All,
I would like to get some help from here for a data set I recently
worked on. I have been working on a new kinase data set which does not
have a close homolog. The data was collected to 2.1A resolution in
space group P212121 however the difference between a and b is only
0.5A. If I index the data as P4, Rmerge is increased from 13% to 39%.
I used the most close homologs which have about 37% sequence identity
as search model for molecular replacement and it seemed I have got the
solution by using Phaser with only the c-terminal part of the search
model and also a long loop removed. After changed the different
residues back to the target protein, the structure was refined to
Rfree/R 46% and 43% to 2.1 A resolution. The existing c-terminal
structure has well defined density except 25ish residue at the very
c-terminal end doesn't have well connected density. Current
model contains about 50% of overall target residues. I can see some
extented difference density for several residues going to the
N-terminal part and also extented density for the C-terminal loop for
several residues. I also see tones of not well-conncted difference
density in the N-terminal region. There was no sever clashes between
molecules after mount all symmetry related molecules. My question is
the following:
1. Have I got the correct solution for the molecular replacement?
2. How can I bring back the missing density for the N-terminal
residues and the loop region?
I would really appreciate any inputs or suggestions.
Eric
