I would also make sure that the spacegroup is correct. If you have instead P222, P2212, etc, you might find the solution at low resolution but the problem would become evident during advanced refinement steps, such as a stuck high Rfree or a noisy difference map. Good luck, Javier.
On Sat, May 21, 2011 at 4:44 PM, Seema Mittal <seema.mit...@umassmed.edu>wrote: > 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 >>>>> >>>>> >>>> >>>> >>> >>> >>> >> >> -- Javier M. Gonzalez, PhD. University of Maryland Baltimore Department of Pharmaceutical Sciences X-Ray Crystallography Shared Service (UMXSS) 20 Penn St., HSFII, Rm 514 Phone/Fax: 410-7061124/410-7060886 21201 Baltimore, MD http://www2.pharmacy.umaryland.edu/psc/xray/
