Thank you all. I think i have known the reason for the low occupancy. First, I co-crystallized the protein and peptide with a molar ratio 1:5. In this situation, the peptide likely would fully occupy the protein binding site. But the crystal is hard to freezen, so stepwise freezen method was used. At this step, I did not add peptide in the cryo-protectant and this step last for 24 hours. So, I think some peptdie in the crystal was lost at this step. Now, in the composite omit map, the protein-peptide binding site is clear to identify. But 6 residues out of the 14 visible residues can only be seen in the 2FoFc map but can not be seen in the composite omit map.
On 4/30/07, mesters <[EMAIL PROTECTED]> wrote:
Dear Jiamu Du, what were the exact concentrations (Molar values please) of protein and peptide in the co-crystallization experiment? This may help in estimating the (possible) occupancy of your peptide. Jeroen. JDwrote: > Does anyone know a program can perform the ocupancy refinement? > Or we always only refine B factor to reflect the occupancy? > > Thanks > > > On 4/30/07, *Eleanor Dodson* <[EMAIL PROTECTED] > <mailto:[EMAIL PROTECTED]>> wrote: > > Well - it is extremely likely that the peptide is partially > occupied and > the occupancy may well be < 0.5.. > > But at this resolution you are going to have great difficulty deciding > whether you should have > Occ=1.0 <B = 130> > > Occ = 0.5 <B = 100> > > Occ = 0.33 <B = ??? 80???> > > As your Rfactors show it makes very little difference to any scoring > system.. > > You can look at difference maps and try to see if one looks > flatter than > the other .. > > Even the overall Wilson plot B is not very well determined, so I > wouldnt > worry too much.. > > Eleanor > > Jiamu Du wrote: > > Dear All: > > According to your suggestion, I have set the peptide's occupency to > > 0.5. Two strategies were employed. > > 1. Direct using Refmac restrained refinement for 10 cycles. The B > > factor only drops to around 100. R/Rf did not change, either. > > 2. Direct CNS B-fator refinemen. The B factor drops to a moderate > > level 60-80, and the R/Rf each increases about 2%. > > 3. First using CNS B-fator refinemen nad next Refmac restrained > > refinement. The B factor drops to 60-80, and the R/Rf did not > change. > > > > I think next step TLS refinement should be carried out. > > > > > > On 4/30/07, *Philippe DUMAS* < [EMAIL PROTECTED] > <mailto:[EMAIL PROTECTED]> > > <mailto:[EMAIL PROTECTED] > <mailto:[EMAIL PROTECTED]>>> wrote: > > > > Jiamu > > > > According to the numbers you have mentioned I conclude that you > > peptide occupancy should be around 60-64 % > > I am interested to know what will be the value that you will > > obtain after refinement... > > > > > > Philippe Dumas > > IBMC-CNRS, UPR9002 > > 15, rue René Descartes 67084 Strasbourg cedex > > tel: +33 (0)3 88 41 70 02 > > [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]> > <mailto: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED] >> > > > > > > -----Message d'origine----- > > *De :* CCP4 bulletin board [mailto: > CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> > > <mailto: CCP4BB@JISCMAIL.AC.UK > <mailto:CCP4BB@JISCMAIL.AC.UK>>]*De la part de* Jiamu Du > > *Envoyé :* lundi 30 avril 2007 05:57 > > *À :* CCP4BB@JISCMAIL.AC.UK > <mailto:CCP4BB@JISCMAIL.AC.UK> <mailto:CCP4BB@JISCMAIL.AC.UK > <mailto:CCP4BB@JISCMAIL.AC.UK>> > > *Objet :* [ccp4bb] extra high B factor > > > > Dear All: > > I am refining a protein-peptide complex struture at 2.6 > > angstrom resolution. > > The data was obtain from a co-crystal and the wilson B > factor > > of the data is about 70. > > The affinity between protein and peptide is about 10E-7 to > > 10E-8 molar. > > Protein fragment of the structure has a common B facor > about 50. > > But surprisingly, the average B factor of the peptide is as > > high as 130, although the peptide can be clearly traced > from > > the the electron density map. All residues of the > peptide have > > such a high B factor. > > My question is how can I reduce the abnormal high B > factor to > > a common level or if this high B factor acceptable. > > And another question is if this high B fator will influence > > the final refiment level. > > > > Thanks. > > > > -- > > Jiamu Du > > State Key Laboratory of Molecular Biology > > Institute of Biochemistry and Cell Biology Shanghai > Institutes > > for Biological Sciences > > Chinese Academy of Sciences (CAS) > > > > > > > > > > -- > > Jiamu Du > > State Key Laboratory of Molecular Biology > > Institute of Biochemistry and Cell Biology Shanghai Institutes for > > Biological Sciences > > Chinese Academy of Sciences (CAS) > > > > > -- > Jiamu Du > State Key Laboratory of Molecular Biology > Institute of Biochemistry and Cell Biology Shanghai Institutes for > Biological Sciences > Chinese Academy of Sciences (CAS) -- Jeroen Raymundus Mesters, Ph.D. Institut fuer Biochemie, Universitaet zu Luebeck Zentrum fuer Medizinische Struktur und Zellbiologie Ratzeburger Allee 160, D-23538 Luebeck Tel: +49-451-5004070, Fax: +49-451-5004068 E-mail: [EMAIL PROTECTED] Http://www.biochem.uni-luebeck.de Http://www.iobcr.org Http://www.opticryst.org -- If you can look into the seeds of time and say which grain will grow and which will not - speak then to me (Macbeth) --
-- Jiamu Du State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences (CAS)
