Thanks you for the suggestions, Patrick and Matthias. I was actually wondering if any of the components from the seeding solution actually were important but your explanations sound more logical.
I apologize for the large attachment. I did not realize that it was so big. Have a nice weekend ! Mahesh On Fri, Jan 17, 2014 at 6:18 PM, Patrick Shaw Stewart <patr...@douglas.co.uk > wrote: > > Mahesh, this is a very interesting and slightly controversial question. > > One approach is to mix together all of the crystals that you have in the > initial screen. The idea at the beginning of the project is to get as many > diverse hits as possible - you can worry about crystal size, space group > and quality later on. > > If you can collect data from crystals and determine the unit cell then you > can be more rational. You can construct a "library" of polymorphs having > different unit cells. This *may *allow you to push the space group in a > given condition to the unit cell or space group that you want - maybe you > find e.g. that your ligand can only diffuse into the active site with a > certain unit cell. > > There is a very interesting paper with several examples of how to use > polymorphs by the Stura group, see below. > > However, seeding can give rise to crystals with different but related > space groups. A very nice example is shown in the wikipedia article about > epitaxy - titanium oxide growing on iron oxide. Also Stura's paper on > epitaxial jumps is interesting and relevant. > > Make sure you dilute your seed stock in a systematic way as part of your > final optimization - one great advantage of microseeding is that oyu can > control the number of crystals per drop by diluting the seed stock. > > Also, make sure that you use fresh crystals to make the seed stock. For > some strange reason old crystals sometimes fail to act as seeds, even > though they can be crushed and still diffract. Maybe the unit cell > shrinks, or maybe the crystals become cross-linked. Make the seed stock as > soon as your crystals stop growing, then freeze them. Seed stocks can > almost always be frozen. > > Best wishes, Patrick > > > > *Library of polymorphs:* > Vera, Laura, Claudia Antoni, Laurent Devel, Bertrand Czarny, Evelyn > Cassar-Lajeunesse, Armando Rossello, Vincent Dive, and Enrico A. Stura. > "Screening Using Polymorphs for the Crystallization of Protein–Ligand > Complexes." Crystal Growth & Design 13, no. 5 (2013): 1878-1888. > > Stura, Enrico A., Jean-Baptiste Charbonnier, and Michael J. Taussig. > "Epitaxial jumps." Journal of crystal growth 196, no. 2 (1999): 250-260. > > > *Cross-seeding:* > Obmolova, Galina, Thomas J. Malia, Alexey Teplyakov, Raymond Sweet, and > Gary L. Gilliland. "Promoting crystallization of antibody-antigen complexes > via microseed matrix screening." Acta Crystallographica Section D: > Biological Crystallography 66, no. 8 (2010): 927-933. > > Abuhammad, Areej, Edward D. Lowe, Michael A. McDonough, P. D. Shaw > Stewart, Stefan A. Kolek, Edith Sim, and Elspeth F. Garman. "Structure of > arylamine N-acetyltransferase from Mycobacterium tuberculosis determined by > cross-seeding with the homologous protein from M. marinum: triumph over > adversity." Acta Crystallographica Section D: Biological Crystallography > 69, no. 8 (2013): 1433-1446. > > > *Seed stability etc* > Shaw Stewart, Patrick D., Stefan A. Kolek, Richard A. Briggs, Naomi E. > Chayen, and Peter FM Baldock. "Random microseeding: a theoretical and > practical exploration of seed stability and seeding techniques for > successful protein crystallization." Crystal Growth & Design 11, no. 8 > (2011): 3432-3441. > > > > *Original description of the "random" microseeding method*D'Arcy, Allan, > Frederic Villard, and May Marsh. "An automated microseed matrix-screening > method for protein crystallization." Acta Crystallographica Section D: > Biological Crystallography 63, no. 4 (2007): 550-554. > > > > > > > On 17 January 2014 22:24, Mahesh Lingaraju <mxl1...@psu.edu> wrote: > > > > Hi Folks > > > > The protein that I am working on gives several initial hits which are > needles. And at random, I picked the needles from a condition (0.2 M cacl2, > 0.1 M HEPES pH 7.5 & 28% PEG 400) and seeded into another screen where I > added 1µl protein + 1µl reservoir solution + 0.3 µl seed stock. I prepared > the seed stock fresh by adding 36 µl of the reservoir solution to > preexisting 2µl drop. > > One of the conditions from the seeded screen gave me a hit that looks > really promising ( see attached). I am sort of positive that these crystals > are protein as they are UV active. > > > > I tried to reproduce these but with needles from another condition which > actually look much nicer than the seeds i used to produce these crystals. > However, I failed to do so. > > > > While i understand that seed quality is important, I find it interesting > that the crystals do not reproduce with similar or better looking seeds. Is > it common that the seeds absolutely need to be from the same condition to > reproduce hits ? > > > > thanks for any suggestions > > > > Mahesh > > > > > > > > -- > patr...@douglas.co.uk Douglas Instruments Ltd. > Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK > Directors: Peter Baldock, Patrick Shaw Stewart > > http://www.douglas.co.uk > Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 > Regd. England 2177994, VAT Reg. GB 480 7371 36 >
