Re: [ccp4bb] zinc with HEPES/seeding
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.ukDouglas 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) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] zinc with HEPES/seeding
mitegen loops might help, particularly micromesh... JPK On Fri, May 11, 2012 at 12:35 PM, 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.ukDouglas 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) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] zinc with HEPES/seeding
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.ukDouglas 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) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- patr...@douglas.co.ukDouglas Instruments Ltd.
