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Dear all I wanted to know the suggestion/opinion about anisotropy I am getting in my crystal diffraction. Things about crystal: one direction goes 2.8A and another is 6 A. cell parameters a=133, b=133, c=289, 90, 90, 120, SG=P3 Protein size: 45 KDa, membrane transporter protein, full size crystal grows at 20C in 10days, rod shaped. Things already tried: 1). additive screen from Hampton (has different 96 additives), 2).4C crystallization, 3). propylene glycol, 4). controlled dehydration, 5).memgold, memgold2, memsys and memstart screens as additive in my optimized condition, 6). incubation of crystals plates at 4 C before freezing for overnight, 7). crystallized with substrate, 8). pH screen, 9). Crosslinking using glutaraldehyde, 10). Seeding, 11). tried many different construct (all construct doesn't give crystal as well). 12). tried most of conventional cryo. Crystallization in LCP and with antibody are in consideration. Now, does some has experience that freezing can cause the anisotropy (I can try room temperature, I don't know crystal will survive or not). Your valuable suggestions are most welcome. Thanks Best Sitaram