Shiv, PEG1500 and PEG3350 are not the same because of the nature of their synthesis and manufacture. They are a polydisperse and semi-purified products of an ethylene oxide condensation. So while they are described as H-(CH2-CH2-O)<n>-OH, the value of <n> and its ranges are quite different. Some of the PEG molecules in both with be the same size, but PEG1500 will have a lot more small PEGs than PEG3350; it is the small PEGs that might bind more tightly to proteins.
On top of this, there is the manufacturing issue. All PEG is made in bulk and then fractionated; different manufacturers have slightly different recipes and purification schemes (which are trade secrets, so good luck in finding more information from any company). Take a look at an old paper by Fran Jurnak (J. Cryst, Growth, 76, 577-582, 1986) for the trials and tribulations of working with PEG from different manufacturers of PEG (as well as how the purify it if you really get worried). She got at least 4 different space groups, and the key was the differing amount of phosphoric acid used in the neutralization step in PEG production. EACH batch of PEG from the same manufacturer can be different! So Herman's comment about pH should be considered. But as Herman said check the enzyme activity of your protein in each and get different batches of PEG to test. Is it a particular size of PEG or a PEG contaminant? Remember that stock solutions you may buy from Hampton or other X-ray specialty companies can be equally suspect because of where they get the bulk product to begin with. Even with purified PEGs (i.e., cleaned up of metal contaminants, aldehyde, and peroxided) age. So think about how old your stocks are. One last comment, just about your terminology. I would avoid the use of the phrase "improper crystal packing." There is nothing really improper about it, just unfortunate from your point of view. The crystal packing is just the natural consequence of physical interactions in solution that you have prepared. You just have to be lucky sometimes. Good luck, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com **************************************************************** On Aug 4, 2014, at 9:20 PM, shivendra singh <shivendr...@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
