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 Lab: (517) 353-9125* > *FAX: (517) 353-9334 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
