Hello, If the homologous protein structure is available, or you have symmetry information of your crystal, then try engineering surface by putting in residues which has less entropy like replacing Lys to Arg, Met to Gln. We are able to fasten the crystal growth from 20 days to 3-4 days by creating phenylalanine Pi-stack. This is tricky but if you have an idea of crystal contact, then it should be straightforward without much trouble. Good Luck Appu
On 31 October 2014 11:43, Patrick Shaw Stewart <patr...@douglas.co.uk> wrote: > > Michael (and some others) > > You haven't mentioned - and I guess don't use regularly - the "random" MMS > approach, where crushed seed-crystals are added to random screens. This > really is a terrific method, and it will on average roughly double > productivity. It's the first thing I would think of in a case like > Vijaykumar's (as I told him this morning!). > > Galina Obmolova and her colleagues published a great paper earlier this > year about MMS. They were working with a set of 16 Fab fragments that > comprised all combinations of 4 different heavy chains and 4 different > light chains. Three structures were generated without MMS, but by various > very creative uses of microseeding they were able to get all 16/16 > structures. Ref below. > > Best wishes, > > Patrick > > __________________________ > > > Obmolova, G., Malia, T. J., Teplyakov, A., Sweet, R. W., & Gilliland, G. > L. (2014). Protein crystallization with microseed matrix screening: > application to human germline antibody Fabs. *Structural Biology and > Crystallization Communications*, *70*(8). > > > > > On 31 October 2014 16:07, R. M. Garavito <rmgarav...@gmail.com> wrote: > >> Although three months is a long time, it is no completely unheard of, and >> does not require the invocation of proteolysis. The longest time I have >> heard of is ~1 yr, so count yourself lucky. However to get good advice, as >> well as to use it, you need to ask yourself several questions: >> >> 1. What kind of crystals are they? Protein, salt, etc? If they are >> salt, don't pursue this condition. >> >> 2. How many crystals did you get? One or 2 in a drop or a microcrystal >> shower. And of what kind? Single, well shaped, rosettes, needle clusters, >> or something that looks crystalline. Screen more broadly around your >> initial hit. >> >> 3. How many times have your tried to repeat this? Once, twice, or >> more? Did you try setups in duplicate? If so, did you get reproducible >> results? Have you actually screened around these conditions, varying each >> component systematically (PEG, salt, pH, buffer, etc.)? >> >> 4. What method did you use? And in what kind of container? For one >> thing, we don't completely trust the integrity of our setups for longer >> than 2 months. All containers leak water slowly, so when crystals take >> longer than 2 months to grow (a) the real conditions are at much higher >> values than you naively think (i.e., the drop has dried out more than you >> expected) or (b) other components are crystallizing, for example a zinc >> salt. It depends what else is in your protein buffer, as well. >> >> To quicken protein crystallization (which is not always a good thing), >> increase your protein concentration (by 1.5-2x) and/or PEG concentration >> (such as screening up to 40% PEGmme 550). Sadly, crystallization is a >> combination of thermodynamic and kinetic factors: you can get crystals >> (sometimes a single crystal only) when just outside the truly optimal >> conditions, but this may be only a sporadic event. You got to keep >> screening. >> >> 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 <%28517%29%20355-9724> Lab: (517) >> 353-9125 <%28517%29%20353-9125>* >> *FAX: (517) 353-9334 <%28517%29%20353-9334> >> Email: rmgarav...@gmail.com <garav...@gmail.com>* >> ****************************************************************** >> >> >> >> >> On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri < >> vijaypkuma...@gmail.com> wrote: >> >> Dear all, >> >> I am trying to crystallize a 30 kD protein. Protein crystals are formed >> after 3 months. The crystals are formed in the following condition: >> 0.01M Zinc sulphate >> 0.1M MES monohydrate pH 6.5 >> 25% v/v PEG monomethyl ether 550 >> >> Please suggest me how to grow these crystals faster. >> >> Thanking you >> >> -- >> Vijaykumar Pillalamarri, >> UGC-JRF, >> C/O: Dr. Anthony Addlagatta, >> Senior Scientist, >> CSIR-IICT, Tarnaka, >> Hyderabad, India-500007 >> Mobile: +918886922975 >> >> >> > > > -- > 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 >
