Before putting in the ligand, there is a clear density for three extra atoms at more than 3 sigma in the Fo-Fc map but for other atoms the density appears around those 3 peaks in the 2Fo-Fc map only and not in the Fo-Fc.
Thanks for your reply. Amit On Wed, Mar 19, 2014 at 8:57 PM, Sampson, Jared <jared.samp...@nyumc.org>wrote: > Hi Amit - > > I don't know of a universally accepted minimum sigma level, but I tend not > to attempt to fit anything that doesn't have clearly interpretable density > at 1.0 sigma (in a 2Fo-Fc map). A map level of 0.5-0.6 sigma is very low, > and to me, it's very likely that you're seeing "what you want to see" and > fitting your ligand into noise. Is there any positive Fo-Fc density > (greater than about 2-3 sigma) at that location if you remove the ligand? > Do you see an appreciable decrease in Rfree when you add the ligand? That > would be an indicator to me that you have placed it correctly. > > Especially if identification of the ligand binding site is the primary > purpose of this structure, I think you need stronger evidence of its > presence. > > You may also want to try co-crystallization, which may reduce the effect a > full soak has on the diffraction of the crystal. > > Cheers, > Jared > > -- > Jared Sampson > Xiangpeng Kong Lab > NYU Langone Medical Center > 550 First Avenue > New York, NY 10016 > 212-263-7898 > http://kong.med.nyu.edu/ > > > On Mar 19, 2014, at 10:39 AM, Amit Kumar <amit7urmcc...@gmail.com> wrote: > > > > > Hello, > > > > My protein is 26 kDa and the resolution of the data is 1.90 angs. My > ligand is 174 Daltons. and it was soaked into the crystal. Ligand is > colored and the crystal after soaking takes up intense color. However if we > soak more than optimum, the color deepens in intensity but the crystal > diffracts no more. So perhaps the ligand's occupancy can not be the 1.00. > > > > After model building I see ligand density, starting to appear at 0.7 > sigma and clear at 0.5-0.6 sigma, close to the protein residue where it > "should" bind. Occupancy is ~0.6 after the refinement and B factors for the > atoms of the ligand range from 30-80. > > > > Questions I have > > (1) What is the acceptable sigma level for very small ligands for peer > review/publication? > > (2) I did refinement by Refmac and by Phenix refine, separately. The map > quality for the ligand is better after the refmac refinement than after the > Phenix refinement. Why is such a difference and which one should I trust? I > used "mostly" default parameters for both (Phenix and Refmac) before the > refinement. > > > > Thanks for your time. > > Amit > > > > > ------------------------------------------------------------ > This email message, including any attachments, is for the sole use of the > intended recipient(s) and may contain information that is proprietary, > confidential, and exempt from disclosure under applicable law. Any > unauthorized review, use, disclosure, or distribution is prohibited. If you > have received this email in error please notify the sender by return email > and delete the original message. Please note, the recipient should check > this email and any attachments for the presence of viruses. The > organization accepts no liability for any damage caused by any virus > transmitted by this email. > ================================= > >
