Dear Shiv, For me, the prime suspect is the pH. Did you check that besides the components the pH of both crystallizations was the same? Here I would measure the pH of the crystallization solution and not rely on the pH written on the bottles of the stock solutions. You could also measure the catalytic activity of the protein in the presence of PEG1500 and PEG3350 to see if that gives some hints what might be going on.
Good luck! Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von shivendra singh Gesendet: Dienstag, 5. August 2014 03:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] PEG dependent structural changes in crystal structure Dear All, I have been working on a protein which initially got crystallised in condition having PEG1500 as precipitant. The space group was P21 and got solved with reasonable Rfree. Analysis of its structure showed large deviation and very distinct active site architecture along with disorderedness in one of its long loop (no density) in comparison with the expected result, based on related homologous structures. The structure does not seem to be active with one of its active site residue moved apart from other catalytic amino acids. Also the substrate entry tunnel looks distorted. The purified enzyme used for crystallisation showed optimum activity in vitro. This led us to screen it again for some other crystallisation condition and got another crystal hit in condition having PEG3350 as precipitant. Rest of the components of crystallisation cocktail were same. The data belonged to P212121 space group. The regions which were disordered or distorted in earlier case were observed to be ordered and in their expected orientation and position. The enzyme is not reported to be in different structural or functional states as observed. I am wondering how the protein from the same batch showed two distinct structural organizations in conditions with varying PEGs. What may cause it to follow such transition. Whether it has some significant functional aspect or just a result of improper crystal packing. Thanks. Shiv
