Thank you all for your replies. I tried seeding also. But it did not help me.
I ran SDS-PAGE. The fresh protein and crystal are showing bands at same molecular weight. So there might not be protein degradation. On 3 November 2014 22:24, Appu kumar <[email protected]> wrote: > 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 <[email protected]> > 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 <[email protected]> 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: [email protected] <[email protected]>* >>> ****************************************************************** >>> >>> >>> >>> >>> On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri < >>> [email protected]> 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 >>> >>> >>> >> >> >> -- >> [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 >> > > -- Vijaykumar Pillalamarri, UGC-JRF, C/O: Dr. Anthony Addlagatta, Senior Scientist, CSIR-IICT, Tarnaka, Hyderabad, India-500007 Mobile: +918886922975
