Re: [ccp4bb] Plate crystals
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Jahan, is your diffraction useless, or do you call 4A resolution useless? 4A is not too bad and can be a start for structure determination while you are waiting for better crystals. You give very little information about what you actually have done, and if you search the bb-archive for how to get better crystals you can probably make a book from the number of hits. If you got a large number of crystals while seeding, try harder - the idea of micro seeding is to adjust the conditions below nucleation concentration and seed there. Dilute your cat whisker further until you have only very few seeds left (you may want to have a look at Fig. 11.2.(a) on p. 63 of http://shelx.uni-ac.gwdg.de/~tg/thesis.pdf to get an idea of the effect. If all else fails, you can also try restricted proteolysis, although I would put this in routine optimization methods nowadays. Good luck, Tim On 10/16/2012 12:01 AM, Jahan Alikhajeh wrote: Dear Friends, I am trying to crystalize a 70 kDa nasty protein but I got plate shape crystals with high mosaicity and useless diffraction (up to 4A). I tried to improve/optimize crystallization but either I got the same or nothing. I tried seeding but I had so many crystals without any improvement. Does anyone have better idea than routine optimization method in the lab? Thanks in advance. Jahan - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQfQ68UxlJ7aRr7hoRAqsOAKCI5hIkO2VkI4EGEuQQfggWQASFqQCg2BHo 6QaulOnNvPRocZ9r4BlQTVg= =FM/p -END PGP SIGNATURE-
[ccp4bb] Plate crystals
Jahan It sounds as though the protein crystallizes well, so microseeding (done the right way) is very likely to solve the problem. Make a strong seed stock with as much crystalline material as possible from several wells, including different hits if possible. Just mix them all together, but keep PEG conditions separate from salt conditions (or you will get two layers). Make a set of serial dilutions from neat up to 1 in 100,000 and freeze them at -80. You need to seed into *random screens*, so that you can pick up new conditions. Then you should optimize two or three new conditions by using the diluted seed stock. For example, if you estimate that there are 1000 crystals in the drop, you use the 1:1000 dilution. For info and references see http://www.douglas.co.uk/mms.htm On 15 October 2012 23:01, Jahan Alikhajeh ja...@graduate.org wrote: Dear Friends, I am trying to crystalize a 70 kDa nasty protein but I got plate shape crystals with high mosaicity and useless diffraction (up to 4A). I tried to improve/optimize crystallization but either I got the same or nothing. I tried seeding but I had so many crystals without any improvement. Does anyone have better idea than routine optimization method in the lab? Thanks in advance. Jahan -- 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. 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] Plate crystals
apart from optimising the crystallisation conditions, it might be worth optimising the protein preparation or do some limited proteolysis - or even express short N-terminal and/or C-terminal deletions. Mark Quoting Patrick Shaw Stewart: Jahan It sounds as though the protein crystallizes well, so microseeding (done the right way) is very likely to solve the problem. Make a strong seed stock with as much crystalline material as possible from several wells, including different hits if possible. Just mix them all together, but keep PEG conditions separate from salt conditions (or you will get two layers). Make a set of serial dilutions from neat up to 1 in 100,000 and freeze them at -80. You need to seed into *random screens*, so that you can pick up new conditions. Then you should optimize two or three new conditions by using the diluted seed stock. For example, if you estimate that there are 1000 crystals in the drop, you use the 1:1000 dilution. For info and references see http://www.douglas.co.uk/mms.htm On 15 October 2012 23:01, Jahan Alikhajeh ja...@graduate.org wrote: Dear Friends, I am trying to crystalize a 70 kDa nasty protein but I got plate shape crystals with high mosaicity and useless diffraction (up to 4A). I tried to improve/optimize crystallization but either I got the same or nothing. I tried seeding but I had so many crystals without any improvement. Does anyone have better idea than routine optimization method in the lab? Thanks in advance. Jahan -- 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. 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 Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] Plate crystals
You might get better help if you post links (not attachments) to diffraction images from several angles and an image of the crystal that gave the image, and maybe several images of typical crystals. Also, you could post dimensions of the crystal, and if your lab is set up for it, a picture of the crystal in the loop. 4 Å diffraction for a plate crystal could be promising, but plate crystals require proper handling and optimization. You should also try to work with your 4 Å data and see if you can get a structure with MR (or even MIR), if that is at all possible. You might find that you can trim some extra sequence from one or both ends that hinder good crystallization. James On Oct 15, 2012, at 4:01 PM, Jahan Alikhajeh wrote: Dear Friends, I am trying to crystalize a 70 kDa nasty protein but I got plate shape crystals with high mosaicity and useless diffraction (up to 4A). I tried to improve/optimize crystallization but either I got the same or nothing. I tried seeding but I had so many crystals without any improvement. Does anyone have better idea than routine optimization method in the lab? Thanks in advance. Jahan
[ccp4bb] Plate crystals
Dear Friends, I am trying to crystalize a 70 kDa nasty protein but I got plate shape crystals with high mosaicity and useless diffraction (up to 4A). I tried to improve/optimize crystallization but either I got the same or nothing. I tried seeding but I had so many crystals without any improvement. Does anyone have better idea than routine optimization method in the lab? Thanks in advance. Jahan
Re: [ccp4bb] Plate crystals
During optimization, have you tried Hampton's additive screen? Gopal From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jahan Alikhajeh [ja...@graduate.org] Sent: Monday, October 15, 2012 6:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Plate crystals Dear Friends, I am trying to crystalize a 70 kDa nasty protein but I got plate shape crystals with high mosaicity and useless diffraction (up to 4A). I tried to improve/optimize crystallization but either I got the same or nothing. I tried seeding but I had so many crystals without any improvement. Does anyone have better idea than routine optimization method in the lab? Thanks in advance. Jahan Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Plate crystals
Hi Jahan, Since I can't tell what you tried in terms of improving/optimizing crystallization, here are some methods that my some colleagues and I have had good luck with, in addition to what Gopal has suggested. (1) Sitting drop technique under oil (2) Varying ratios of protein to precipitant (3) Seeding with several serial dilutions of the seeded material Since you write that you have high mosaicity, perhaps it is also worth double-checking your crystal cryoprotectant and flashcooling conditions. Like Elspeth Garman likes to remind everyone, cryoprotectant and crycooling conditions that mitigate ice formation do not automatically ensure the best diffraction conditions, which often need further optimization. Good luck! Raji On Mon, Oct 15, 2012 at 9:30 PM, Parthasarathy, Gopal parth...@merck.comwrote: During optimization, have you tried Hampton's additive screen? Gopal From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jahan Alikhajeh [ja...@graduate.org] Sent: Monday, October 15, 2012 6:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Plate crystals Dear Friends, I am trying to crystalize a 70 kDa nasty protein but I got plate shape crystals with high mosaicity and useless diffraction (up to 4A). I tried to improve/optimize crystallization but either I got the same or nothing. I tried seeding but I had so many crystals without any improvement. Does anyone have better idea than routine optimization method in the lab? Thanks in advance. Jahan Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Plate crystals
Hi Jahan, You could also try dehydrating the crystals by moving them over well solutions of ammonium sulfate, sodium chloride, or high molecular weight PEG, starting with low concentrations, and slowly increasing over time. Good luck, Sara On Mon, Oct 15, 2012 at 10:06 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Jahan, Since I can't tell what you tried in terms of improving/optimizing crystallization, here are some methods that my some colleagues and I have had good luck with, in addition to what Gopal has suggested. (1) Sitting drop technique under oil (2) Varying ratios of protein to precipitant (3) Seeding with several serial dilutions of the seeded material Since you write that you have high mosaicity, perhaps it is also worth double-checking your crystal cryoprotectant and flashcooling conditions. Like Elspeth Garman likes to remind everyone, cryoprotectant and crycooling conditions that mitigate ice formation do not automatically ensure the best diffraction conditions, which often need further optimization. Good luck! Raji On Mon, Oct 15, 2012 at 9:30 PM, Parthasarathy, Gopal parth...@merck.comwrote: During optimization, have you tried Hampton's additive screen? Gopal From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jahan Alikhajeh [ja...@graduate.org] Sent: Monday, October 15, 2012 6:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Plate crystals Dear Friends, I am trying to crystalize a 70 kDa nasty protein but I got plate shape crystals with high mosaicity and useless diffraction (up to 4A). I tried to improve/optimize crystallization but either I got the same or nothing. I tried seeding but I had so many crystals without any improvement. Does anyone have better idea than routine optimization method in the lab? Thanks in advance. Jahan Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
