One or more ingredients in the original hit (which you use to suspend the seed crystals in) can indeed be essential, and may be all you need to get nice crystals. In D'Arcy's original paper you will see that he needed Ca2+ to get nice crystals of one of his proteins. The same is true in Stoddards paper, which inspired D'Arcy, ref below. However in the majority of cases it is just the seed that you need, i.e. there was a nucleation problem in the new hits that appear with seed stock. You can see this very clearly because (in most cases) diluting the seed stock in the same solution gives a corresponding reduction in the number of crystals.
Ireton, Gregory C., and Barry L. Stoddard. "Microseed matrix screening to improve crystals of yeast cytosine deaminase." *Acta Crystallographica Section D: Biological Crystallography* 60, no. 3 (2004): 601-605. On 18 January 2014 00:18, Mahesh Lingaraju <[email protected]> wrote: > 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 < > [email protected]> 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 <[email protected]> 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 >> > >> > >> >> >> >> -- >> [email protected] 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 >> > > -- [email protected] 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
