Dear Dr. Van Raaji and Dr. Domínguez,

Thank you very much for your reply. The salt and pH of crystallisation recipe were same except PEG avg. mol wt. I agree, it may be due to different crystal packing but i am wondering whether disorderedness observed was intrinsic to the protein under PEG1500 or induced by different crystal packing. Also, is it possible that it may be the molten globule state of this protein? I read somewhere that its almost impossible to crystallise molten globule of any protein due to its transient nature. Is it possible that under the PEG1500 environment this collapsed state is more stable? Also looking at its active site, we are sure that it cant be in active form so i am not sure what may be its significance. Also if its a different conformation of active enzyme so is it possible to find out that whether its prior or later state of native form. The function of this enzyme is not related to polyols but hydrolyze cyclic amide substrate.

Kind regards

Shiv

On 6 August 2014 00:23, shivendra singh <shivendra22@gmail.com> wrote:

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