Re: [ccp4bb] Crystals with Organic solvents
Hi Eswar Firstly, I would certainly try crystal seeding into random screens if you haven't already tried it. Refs below. Secondly, it's very convenient to grow the crystals under oil, and to soak the organic solvents into the drops, through the oil. This makes it much easier to harvest the crystals because the oil becomes saturated with the solvent and stops it from evaporating when you pick up the crystals. This approach can be used at the screening stage too. See Mortuza, et al. High-resolution structure of a retroviral capsid hexameric amino-terminal domain. Nature 431 (2004), pp 481-485. Also see http://www.douglas.co.uk/winner1.htm Good luck Patrick Allan D’Arcy, Frederic Villarda, May Marsh. 'An automated microseed matrix-screening method for protein crystallization'. Acta Crystallographica section D63 (2007) 550–554. On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444907007652 A. G. Villaseñor, A. Wong, A. Shao, A. Garg, A. Kuglstatter and S. F. Harris. 'Acoustic matrix microseeding: improving protein crystal growth with minimal chemical bias.' Acta Crystallographica Section D66 (2010) 568-576. On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444910005512 Galina Obmolova,* Thomas J. Malia, Alexey Teplyakov, Raymond Sweet and Gary L. Gilliland. 'Promoting crystallization of antibody–antigen complexes via microseed matrix screening.' Acta Crystallographica Section D66 (2010) 927–933. Open-access at http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen and Peter F.M. Baldock. 'Getting the most out of the random microseed matrix-screening method in protein crystallization'. Cryst. Growth Des., 2011, 11 (8), pp 3432–3441. On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442 See also http://www.douglas.co.uk/mms.htm On Fri, Aug 26, 2011 at 11:55 AM, eswar reddy eswar.uo...@gmail.com wrote: Dear All I was working on a Human protein and expression and solubility is good in E.coli and purification is One step (His-Tag), and i need to cleave the Histag before screens, if not the protein will precipitated and Aggregated, but after trying for 1.2 years i have crystals and they are with Organic solvents, (10 conditions), these crystals are inter grown like broccoli shaped and i tried seeding, but it is not successful, and even i tried with additive screen but the result is the same is there is any idea to increase the size and shape of my protein crystals. Any suggestions will be helpful for me Thanks in Advance -- 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
[ccp4bb] Crystals with Organic solvents
Dear All I was working on a Human protein and expression and solubility is good in E.coli and purification is One step (His-Tag), and i need to cleave the Histag before screens, if not the protein will precipitated and Aggregated, but after trying for 1.2 years i have crystals and they are with Organic solvents, (10 conditions), these crystals are inter grown like *broccoli *shaped and i tried seeding, but it is not successful, and even i tried with additive screen but the result is the same is there is any idea to increase the size and shape of my protein crystals. Any suggestions will be helpful for me Thanks in Advance
Re: [ccp4bb] Crystals with Organic solvents
Depending on how big your broccoli is I would simply crush them and mount as big of a piece as possible and see how well it diffracts. I assume you have tried already: - glycerol - changing protein:reservoir ratios - temperature - adding oil to either / and the reservoir, your drop -crushed up some broccoli and recreened your original sparse matrix screen while seeding with chunks of broccoli seeds - when you say the His-tag needs to be cleaved otherwise it will precipitate, that mitt also just be either leaching Ni from your column or simply the imidazole - You don't say how you purify your protein, I hope you are not just setting up after the Ni capture step and at least have some other method following e.g. anion or SEC Just a few incomplete suggestions, Jürgen On Aug 26, 2011, at 6:56 AM, eswar reddy wrote: Dear All I was working on a Human protein and expression and solubility is good in E.coli and purification is One step (His-Tag), and i need to cleave the Histag before screens, if not the protein will precipitated and Aggregated, but after trying for 1.2 years i have crystals and they are with Organic solvents, (10 conditions), these crystals are inter grown like broccoli shaped and i tried seeding, but it is not successful, and even i tried with additive screen but the result is the same is there is any idea to increase the size and shape of my protein crystals. Any suggestions will be helpful for me Thanks in Advance .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] Crystals with Organic solvents
I would definitely try gelfiltration (how do you get rid of the cleaved tag anyway, sample buffer exchange?) but especially ion exchange. A homogeneous sample on SDS-PAGE and/or gelfiltration is often not (at all) homogeneous in ion exchange. Beyond that i would make some point mutations on surface residues and focus on those (ie try the surface entropy method). Good luck, Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of eswar reddy [eswar.uo...@gmail.com] Sent: Friday, August 26, 2011 6:56 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystals with Organic solvents Dear All I was working on a Human protein and expression and solubility is good in E.coli and purification is One step (His-Tag), and i need to cleave the Histag before screens, if not the protein will precipitated and Aggregated, but after trying for 1.2 years i have crystals and they are with Organic solvents, (10 conditions), these crystals are inter grown like broccoli shaped and i tried seeding, but it is not successful, and even i tried with additive screen but the result is the same is there is any idea to increase the size and shape of my protein crystals. Any suggestions will be helpful for me Thanks in Advance
