I would also try Open-EPMR as well as Phaser. It
can sometimes find solutions that Phaser or Amore cannot, especially
for multiple chains and low resolution. EPMR is especially good at
handling high copy numbers of search models.
Cheers.
Pete Meyer wrote:
A few
things to try (or double-check):
1. If you ran phaser with SGALTERNATIVE ALL, make sure the mtz file you
gave to refmac has the same screw axes as the MR solution. If this is
off, it'll lead to higher R-factors.
2. As Fred mentioned, try with a single copy of the protein, and check
the FWT (sigma_a weighted 2Fo-Fc) maps. If the solution is reasonably
correct, you should be able to see some density for the other
components. Also, run some controls. Delete a portion of your search
model before MR, and insure that density for that region is present in
maps phased with the positioned model.
3. Use your seleniums. If your MR solution is correct, then you should
see peaks in model-phased anomalous difference maps. But confirm this
with your form_1 data. In my experience, these are not very sensitive
to model completeness (but that's with a different protein).
Since you've mentioned you're relatively new at this, you should
re-merge (scala or scalepack) as anomalous for anomalous difference
maps if you haven't done so already (normally I'd assume this was the
case for dealing with anomalous data and not mention it...sorry if I'm
telling you things you already know).
Good luck,
pm
Chao Quan wrote:
Dear CCP4 community:
I am a beginner to crystallography and therefore my apologies if this
question is too simple.
Basically we obtained several crystal forms of the same molecule, which
is a hetero-
trimer containing protein A(18kD), protein B(16kD) and a RNA
segment(40nt or about 15kD).
We have solved the structure of one crystal form(form_1); its
information is as follows:
space group = P 42 22;
unit cell = 126.514 126.514 76.766 90.00 90.00 90.00
resolution = 2.7A;
Rwork = 0.25, Rfree = 0.265;
solvent content = 60%;
Number of molecules per Asymmetric Unit = 1;
Data redundancy = 5;
Data Completeness = 94%;
I am now trying to solve the structure of form_2 crystal using
molecular replacement. So far the information I know about form_2 is as
follows:
space group = I 422 or I 41 22;
unit cell = 180.096 180.096 152.530 90.00 90.00 90.00
(unit cell is about 4 times the size of form_1)
resolution = 3.3A; (which is low)
Number of molecules per Asymmetric Unit = 2 or 3;
Data redundancy = 4;
Data Completeness = 92%;
There is no twinning(as shown by Sfcheck);
As shown in"Analyse Data for MR", the first peak is 100 and second is
68.92; I am not sure if this indicates translation in a Asymmetric
Unit;
The problem is, I can not get a good solution by MR using Phaser (both
I422 and I4122 are tried). When I searched for 3 molecules per
Asymmetric Unit, Phaser did not give solutions at all.
When I used 2/ASU instead, I was able to get some solutions, with
typical statistics as follows:
RFZ=14, TFZ=35.9, PAK=0, LLG=1036; However, these solutions had high R
values(like Rwork=0.59 and Rfree=0.58), which indicated that they are
not solutions at all. Still, I tried refinement using refmac5, but R
values did not go down even after 50 rounds; sometimes they even
increased after refinement.
Besides, the RMS values bond length, bond angle and chiral center were
all 0 as show by refmac5.
I tried limiting resolution range to 15-4A in Phaser, which did not
help either.
Now I am completely stuck. Could anyone give me some advice? I know
this situation is very strange, because I am using the SAME molecule
for MR but can not can a solution.
Thanks a lot,
P.S. 1) Both form_1 and form_2 crystals were grown using
Selenomethionine-containing samples. There are 3 Sel_Met in protein A
and 1 in protein B. 2) A 10-aa internal segment of protein B is missing
in the solved structure, which may indicate high flexibility.
Chao
--
Roger S. Rowlett
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
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