It sounds as though microseeding worked very well, but the crystals are still growing far too quickly.
Try diluting the protein and/or the reservoir solution to half the concs you are using or lower. To have enough protein you need larger drops, say 500 + 500 + 200 by hand if you can't do it with the Phoenix (wrong robot of course ;) Dispense the seed with a Hamilton syringe, rinsing the needle in the reservoirs before adding to the drops. You could also try using large volumes, say 4 ul total, with dilute ingredients, and make a small hole in the tape with a pin to let the wells slowly dry out. On 11 May 2012 18:35, Rajesh Kumar <ccp4...@hotmail.com> wrote: > > Dear Patrick, > > You along with others had made some suggestions last time. May be its a > good time to update. > > With classical screening, I got a crystal like appearances/shower with > HEPES 7.5 and LiSo4 1.5M. Trying to vary the pH of Hepes or using Tris and > with different conc of Lithium I could only get very very thin needles > which shower and difficult to pick even with 0.05 loop. changing conc of > protein, salt, adding oil on well, changing drop ratio, adding 5% PEGs, > glycerol, ethylene glycol, didnt reduce shower. > > I prepared the seeds from 7.6 ph and 1.25 M Liso4 conditions and MMS > screened with all the 4 screens (Qiagen procomplex, classic, peg, JCSG) > 100nl+100nL+50nL seeds using Phoenix. > > Got several nice hits which very good size individual crystals in > conditions with 30% glycerol and 14% Isopropanol, 20% PEG8K, 30 PEG400. > When I optimized them I got beautiful crystals and tried at ALS 5.0.3 but > no spots. I tried picking crystals from 30 min to 4 hrs to 4 days after > plates were set and there was no luck. Crystals started to appear from 20 > min onwards and keep growing in next couple of hours. I thought of trying > dehydration but they were already in dehydrating conditions them selves. I > wanted to ask if anyone ever failed with MMS but thought not > waste others time on this. Cross seeding to full length protein and Se met > protein also gave beautiful crystal but again no diffraction. I have not > checked SeMet as they were bit small. > > I am still open to ideas if you have any thing on seeding. I did try in > microbatch and hanging drop as well (1.5 ul protein+ 1.4ul reservor+ 0.4 ul > seeds, same as sitting drop which gave me very good looking crystals) but I > didn't get any thing. I tried streaking with reduced LiSO4 to 1.1 M but > it didn't give me anything. > > Currently, I am making entropy mutations, new constructs > of different lengths to solve the above problem. > > I still want to improve this condition with needles, because I collected a > 4.5A data on one of the needle (just one). I need phases. I have sent some > Iodide soaks to synchotron (yet to collect data) but manipulating these > crystal drops with more than 100 tiny needles with a tough membrane on it > has been frustrating as I end up loosing several drops to just to fish out > 1-2 needles. I am ready to try if there any trick left. > > Thanks for lots and lots of help. > > Regards, > Rajesh > ------------------------------ > Date: Fri, 11 May 2012 18:09:54 +0100 > Subject: Re: [ccp4bb] zinc with HEPES > From: patr...@douglas.co.uk > To: ccp4...@hotmail.com > > Rajesh > > How did you do the MMS? By hand or with a robot, and what screens did you > use? > > and why did you change to HEPES out of interest? > > Patrick > > > On 11 May 2012 18:05, Rajesh Kumar <ccp4...@hotmail.com> wrote: > > The rationale was to see if Zn could make differences in crystal > morphology. This is because the protein has CxxC and CxxH similar to a zinc > finger motif. > All my efforts, additive screening, MMS, streaking, micro batch, hanging > drop, changing drop ratio, drop shape, did not help me to either increase > thickness or change the shape of very very thin needle crystals. > Yes, I will try very less, 50uM. > Thanks for helping me to understand. > Rajesh > > > Date: Fri, 11 May 2012 12:53:53 -0400 > > Subject: Re: [ccp4bb] zinc with HEPES > > From: liehy...@gmail.com > > To: ccp4...@hotmail.com > > > > Rajesh, > > 10mM zinc seems a bit too high. I normally used it at <50uM conc. > > ray > > > > On Fri, May 11, 2012 at 12:26 PM, Rajesh Kumar <ccp4...@hotmail.com> > wrote: > > > Dear All, > > > > > > This question sounds simple but I dont know the answer. > > > I was preparing a 24 well crystal screen. When I try to use 10 mM > ZnSO4 > > > with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn > > > acetate the effect is same. > > > I dont know why this Zn in not compatible with HEPES. > > > Could you please tell me why is this? > > > I appreciate your help. > > > > > > Thanks > > > Rajesh > > > > > -- > 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 > > -- 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
