Re: [ccp4bb] turn granular to crystal
Dear Rui, I can only support Patrick's view on MMS. We've got some very good results with this technique in one tricky case in which the starting point wasn't much better than yours. In addition to the papers Patrick suggested the following might also be worth a look: The role of bias in crystallization conditions in automated microseeding. St John FJ, Feng B, Pozharski E. Acta Crystallogr D Biol Crystallogr. 2008 Dec;64(Pt 12):1222-7. Cheers Roberto On 25 Aug 2010, at 12:37, rui wrote: Hi, All, I'm trying to crystallize a soluble protein and got something like granular, they are rounded shaped and not so regular and also don't have sharp edges. See the attached pic. The current condition is PEG4000 and pH around 5. How can I improve this condition? Thanks a lot. --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk
Re: [ccp4bb] turn granular to crystal
Rui Assuming that they are protein, this is a perfect candidate for "MMS" microseeding into random screens You can do it with a robot or by hand See D'Arcy et al. Acta Cryst. (2007). D63, 550 - 554. 'An automated microseed matrix-screening method for protein crystallization' A very good recent article is Obmolova et al. Acta Cryst. (2010). D66, 927-933 'Promoting crystallization of antibody-antigen complexes via microseed matrix screening' (See also our website at http://www.douglas.co.uk/mms.htm) Yibin, what proteases do you use, what conc, how much do you add? Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, 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 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of rui Sent: 25 August 2010 13:37 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] turn granular to crystal Hi, All, I'm trying to crystallize a soluble protein and got something like granular, they are rounded shaped and not so regular and also don't have sharp edges. See the attached pic. The current condition is PEG4000 and pH around 5. How can I improve this condition? Thanks a lot.
Re: [ccp4bb] turn granular to crystal
Hi, You can easily test the crystals by adding a drop of "Ponceau S" staining solution. If you have protein crystals they will get stained. Best wishes, Tim yybbll wrote: > Hi, > A little danger this condition. I suggest you should firstly make sure > they are protein crystals. I know it is very difficult. I suggest you > can add proteases to the drops. If "crystals" dissolve, no problem, > they are protein crystals. > Best > Yibin > 2010-08-27 > > yybbll > > *发件人：* rui > *发送时间：* 2010-08-27 02:04:54 > *收件人：* yybbll > *抄送：* > *主题：* Re: [ccp4bb] turn granular to crystal > hi, > > no , I didn't use detergent. THe composition of the well buffer is > just PEG4000 and MgCl2 and NaAc. > > On Wed, Aug 25, 2010 at 9:52 AM, yybbll <mailto:yyb...@gmail.com>> wrote: > > Hi, Did you use detergent? It seems look like detergent crystals. > 2010-08-25 > > yybbll > -------- > *发件人：* rui > *发送时间：* 2010-08-25 20:37:18 > *收件人：* CCP4BB > *抄送：* > *主题：* [ccp4bb] turn granular to crystal > Hi, All, > > I'm trying to crystallize a soluble protein and got something like > granular, they are rounded shaped and not so regular and also > don't have sharp edges. See the attached pic. The current > condition is PEG4000 and pH around 5. How can I improve this > condition? Thanks a lot. > > > -- Tim Schulte Ruhr-Universita"t Bochum LS Biophysik AG Proteinkristallographie Universita"tsstr. 150 44780 Bochum Germany Tel. +49-234-32-25754
Re: [ccp4bb] turn granular to crystal
Hi, A little danger this condition. I suggest you should firstly make sure they are protein crystals. I know it is very difficult. I suggest you can add proteases to the drops. If "crystals" dissolve, no problem, they are protein crystals. Best Yibin 2010-08-27 yybbll 发件人： rui 发送时间： 2010-08-27 02:04:54 收件人： yybbll 抄送： 主题： Re: [ccp4bb] turn granular to crystal hi, no , I didn't use detergent. THe composition of the well buffer is just PEG4000 and MgCl2 and NaAc. On Wed, Aug 25, 2010 at 9:52 AM, yybbll wrote: Hi, Did you use detergent? It seems look like detergent crystals. 2010-08-25 yybbll 发件人： rui 发送时间： 2010-08-25 20:37:18 收件人： CCP4BB 抄送： 主题： [ccp4bb] turn granular to crystal Hi, All, I'm trying to crystallize a soluble protein and got something like granular, they are rounded shaped and not so regular and also don't have sharp edges. See the attached pic. The current condition is PEG4000 and pH around 5. How can I improve this condition? Thanks a lot.
Re: [ccp4bb] turn granular to crystal
I would try the following approach, some of which you may have done already: Broad pH screen, by 0.5 pH units to see if you are in the pH sweet spot. Finer PEG-4000 screen at the pH identified in the pH screen--the precipitate in the well would alert me to consider that either PEG-4000 is too high, or protein is insufficiently pure Try some of your best conditions at 4 deg C instead of RT, or some other intermediate temperature if you have the capability. (For me, the 4 deg C often makes a big difference; I've had minimal luck with intermediate temps.) Try an additive screen with 5-10% PEGS, polyols, and liquids (don't forget DMSO, DMF, MPD, as well as EG, glycerol or glucose, etc.), or 0.1-0.2 M salts. Whatever you have on hand, or more thorough march through the Hampton Research additives. If anything improves crystal form, try more (or less), or combine additives. At some point, try "reversing" your protein/precipitant ratios. You will typically have more luck in the higher protein/lower precipitant part of the crystallization phase diagram than the reverse. Screens are usually at the higher precipitant/lower protein part of the crystallization phase diagram. Cheers. On 8/25/2010 8:37 AM, rui wrote: Hi, All, I'm trying to crystallize a soluble protein and got something like granular, they are rounded shaped and not so regular and also don't have sharp edges. See the attached pic. The current condition is PEG4000 and pH around 5. How can I improve this condition? Thanks a lot. -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu
Re: [ccp4bb] turn granular to crystal
Hi Rui, I would try the additive screen. http://hamptonresearch.com/product_detail.aspx?cid=1&sid=36&pid=27 Best Regards, Georg rui schrieb: Hi, All, I'm trying to crystallize a soluble protein and got something like granular, they are rounded shaped and not so regular and also don't have sharp edges. See the attached pic. The current condition is PEG4000 and pH around 5. How can I improve this condition? Thanks a lot. -- Universität Tübingen Interfakultäres Institut für Biochemie Dr. Georg Zocher Hoppe-Seyler-Str. 4 72076 Tuebingen Germany Fon: +49(0)-7071-2973374 Mail: georg.zoc...@uni-tuebingen.de http://www.ifib.uni-tuebingen.de
Re: [ccp4bb] turn granular to crystal
Hi, Did you use detergent? It seems look like detergent crystals. 2010-08-25 yybbll 发件人： rui 发送时间： 2010-08-25 20:37:18 收件人： CCP4BB 抄送： 主题： [ccp4bb] turn granular to crystal Hi, All, I'm trying to crystallize a soluble protein and got something like granular, they are rounded shaped and not so regular and also don't have sharp edges. See the attached pic. The current condition is PEG4000 and pH around 5. How can I improve this condition? Thanks a lot.
