Thank you all for your thoughts and suggestions in response to my query.
The rotamers, peptide omega angles, mol-probity data all are
perfectly fine. There are 3 outliers in Ramachandran plot ( may be
the ones causing the problem).
The protein has two engineered cysteines involved in disulfide bond
formation which restrict the conformation around the disulfide bond.
One of these cys residues is followed by pro which seems to be
constrained a bit. Nevertheless, the overall geometry is good.
There certainly are a couple of big positive density peaks which i
haven't been able to account for yet. The usual suspects from my
crystallization conditions do not seem to fit in those blobs.
I shall go back to 2.7A resolution and continue to try to resolve
these positive peaks. Thanks for your suggestions. Any other thoughts
on the matter would be greatly appreciated.
Thanks much,
Best,
Seema
On May 20, 2011, at 10:41 PM, Ethan Merritt wrote:
On Friday, 20 May 2011, you wrote:
Hi Ethan,
You are absolutely right. As a matter of fact, I had initially
processed the data to 2.7A and it looked pretty decent with R symm
less than 10%. The maps looked good too.
The problem arose during second round of refinement. The Rfree got
stuck at around 29-30 while the Rfactor kept decreasing to about
20-21.
That can simply mean there are many little things wrong with the
structure - rotamers, phi/psi, perhaps even a register shift.
I suggest that you use the 2.7A data, so that there is more local
information to drive the refinement, and take seriously any
hints that Molprobity provides about bad local geometry.
Another thought - are there any large positive peaks in the
difference density? Perhaps you are missing a solvent ion or
something of that sort. Even one missing sulfate could make
a noticeable difference in Rfree.
regards,
Ethan
The bond length and angle values are fine too.
I cut down the resolution to 3A hoping to improve the data quality by
removing some noise. But, it did not work. i also tried to put
restrains on the backbone B factors with limited success.
Any thoughts on how i can resolve this Rfree issue?
Thanks much,
Seema
On May 20, 2011, at 5:38 PM, Ethan Merritt wrote:
On Friday, May 20, 2011 02:28:26 pm Mittal, Seema wrote:
Hi All,
I am currently working on a 3A resolution dataset. The scaled file
shows the following statistics (scroll down to the end of this
email). It is P212121 space group with R merge of 8.8%.
Your data statistics look fine. In fact, it looks to me that your
crystal is
probably yielding good data to considerably better resolution
than 3A.
Why did you choose to cut it there?
My question is : Is there a way to selectively use only the data
with I/Sigma value of 2 and more for refinement?
That is a bad idea. By removing data you are throwing away
information.
Noisy data is still better than no data.
good luck with your [probably better than 3A] structure,
Ethan
And how do i achieve this using refmac? I am aware that this would
come at the cost of compromising data completeness. Any
suggestions/help would be greatly appreciated.
Thanks much,
Seema Mittal
Department of Biochemistry & Molecular Pharmacology
970L Lazare Research Building
University of Massachusetts Medical School
364 Plantation Street
Worcester, MA 01605
Shell I/Sigma in resolution
shells:
Lower Upper % of of reflections with I / Sigma less than
limit limit 0 1 2 3 5
10 20 >20 total
50.00 6.46 2.0 3.8 5.3 6.2 7.6 12.5 34.3
65.0 99.3
6.46 5.13 0.7 2.2 3.9 5.3 8.2 15.7 36.6
63.4 100.0
5.13 4.48 1.3 2.8 4.0 5.8 9.3 13.8 27.3
72.7 100.0
4.48 4.07 0.7 1.7 4.0 5.4 7.9 13.9 35.4
64.1 99.5
4.07 3.78 1.8 3.6 5.1 6.9 11.8 20.8 49.6
47.3 96.9
3.78 3.56 1.5 3.8 6.7 8.7 13.3 26.7 65.4
30.8 96.2
3.56 3.38 0.8 3.2 7.1 8.9 12.9 31.1 76.6
20.0 96.6
3.38 3.23 2.0 4.8 8.1 14.8 23.4 44.8 84.7
12.7 97.5
3.23 3.11 4.1 9.2 13.8 18.4 29.6 51.0 86.0
11.0 96.9
3.11 3.00 2.4 8.6 13.9 18.8 30.6 53.9 92.4
4.5 96.9
All hkl 1.7 4.3 7.1 9.8 15.3 28.0
58.1 39.9 98.0
Shell Lower Upper Average Average Norm. Linear Square
limit Angstrom I error stat. Chi**2 R-fac R-fac
50.00 6.46 511.7 20.0 8.8 1.098 0.065 0.073
6.46 5.13 284.6 10.1 6.3 1.047 0.062 0.064
5.13 4.48 500.9 17.0 8.8 1.007 0.062 0.069
4.48 4.07 446.1 17.4 9.2 1.032 0.069 0.070
4.07 3.78 307.1 14.5 8.4 1.065 0.089 0.092
3.78 3.56 243.4 13.8 7.9 1.033 0.108 0.112
3.56 3.38 182.3 12.0 8.3 1.083 0.132 0.134
3.38 3.23 136.5 10.4 7.7 1.048 0.155 0.151
3.23 3.11 107.4 9.2 7.3 1.096 0.184 0.163
3.11 3.00 91.0 8.7 7.3 1.044 0.215 0.201
All reflections 287.7 13.5 8.0 1.055 0.088 0.082
