Hi Haitao, I need to ask you a few questions first:
1. Did you mean you could not solve your structure by molecular replacement? Did you compare your crystal's unit cell with the PDB file? Are they significantly different? If the assymetric unit has more than one monomer, have you tried doing a molecular replacement search with one monomer only? 2. Can you give us the PDB number so that we can take a look at the protein? The reason for that is, I suspect that your protein was not degraded from either end, but only got clipped some where on the surface - so the structure is basically unperturbed. If the PDB structure turns out to be single-domain, then you should do your molecular replacement search with the whole protein. If it is a two-domain structure, and one of them is ~20kD, then try use that 20kD domain to do the search again. R-fac~=0.5 is probably saying that your current solution is totally wrong. As I remember, R-factor for a totally radom acentric (for example, protein) structure is 0.59. Also, Even if you follow the published crystallization conditions, your protein may still crystallize in a totally different way. But unless the protein itself has changed its shape (which normally does not happen), you should be able to do a molecular replacement. If molecular replacement does not work at all, then maybe it is time to send your sample to mass spec to see what it really is. But I highly suspect what you need to do now is nothing but to optimize your molecular replacement parameters. Zhijie Li Graduate student, Univeristy of Toronto ----- Original Message ----- From: Haitao ZHANG To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, July 07, 2008 10:27 PM Subject: [ccp4bb] Truncated protein structure Dear all, I repeated a protein crystallization which is reported in PDB with the almost same condition, and got a 2.6A data. But the problem is that I can not determine the right phases with neither CCP4 nor CNS. Then I found the protein in my crystal had been degraded from ~30kD to ~20kD by SDS-PAGE, but did not know from which terminus it was truncated. So I used truncated PDB templates which N-, C- or both terminal were cut to fit the length of my shorter protein, and tried many different templates. But always high R-factor (~0.5). With COOT I found the backbone did not fill the electron density map perfectly. The only difference of my crystallization condition is 277K(mine) vs 295K(reported) in temprature. Any suggestions will be appreciated. Haitao ZHANG, Ph.D Student, Shanghai Institute of Materia Medica, Chinese Academy of Sciences
