Dear All: Thank you for help. Now I will make a summary of this topic.
First, the actual situation is that: 1. Protein and peptide have a affinity about 10E-7 to 10E-8 molar. 2. The 2.6 angstrom data were got from a co-crystal and had a wilson B about 70, in the experiment protein/peptide have a molar ratio 1:5. 3. Stepwise cryo-protection was employed in the frozen step and last for 24 hours. 4. In the refinement, the protein has a common B factor about 50. But the peptide has a extra high B factor about 130. For this situation, many friends give me suggetion. The summary is below. 1. Four friends suggested the occupancy of the peptide should be set to 0.5. For this solution, after B factor refinement in CNS and continuing restrained refinement in Refmac, B factor of the peptide fall to a acceptable level 70. 2. Juergen Bosch suggested TLS parameters should be considered. For this solution, after TLS refinement, the B fator of the peptide have little change. It seems not due to this. Another related topic is how to refine occupancy. 1. Three friends suggested that in CNS. Qindividual.inp can do this. 2. Peter Zwart suggested PHENIX can refine the occupancy of ligand in the latest version. 3. Two friends suggested Shelx. But, some firends mentioned that, occupancy refinement can only be done at atomic resolution (say, 1.2 A). Then I think the reason for this situation is probably for the reason below. 1. I did not add peptide in the cryo-protectant. 2. Stepwise frozen last for 24 hours. This two reasons result in the lost of peptide in crystal. The low occupancy of the peptide leads to the extra high B fator when I set the occupancy to 1.0. Thank you ALL. -- Jiamu Du State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences (CAS)
