Re: [ccp4bb] Reproducing crystals.
Jun Yong and Jobichen (I've mentioned this before, but ) - both of your projects jump out as very good targets for microseeding with random screens. This method often gives extra hits and better crystals because it is more likely that crystals will grow in the metastable zone. It often reduces the *need *for optimization. Read Allan D'Arcy's excellent paper, plus Obmolova et al for a spectacular example. Our web site has some recent tips that may help you too. Good luck Patrick D'Arcy, A., Villard, F., Marsh, M. An automated microseed matrix screening method for protein crystallization, 2007, Acta Crystallographica, D63, 550-554. G. Obmolova, T. J. Malia, A. Teplyakov, R. Sweet and G. L. Gilliland. Promoting crystallization of antibody-antigen complexes via microseed matrix screening Acta Cryst. (2010). D66, 927-933 http://www.douglas.co.uk/mms.htm * * On Tue, Apr 12, 2011 at 1:07 PM, Jun Yong Ha j...@princeton.edu wrote: Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance. -- 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
Re: [ccp4bb] Reproducing crystals.
Hey Jun, If it's an old batch check and see if you have microorganisms living in the stock or proliferating in the drop - see the paper by Bai et al: doi:10.1107/S1744309107002904 In this paper they demonstrated how they could not reproduce a crystal hit from an old screen up until they realized a fungi that grew in the drop has been secreting protease that chewed up certain part of their protein - once they have utilized in-situ proteolysis they managed to reproduce there crystals with their home-made ingredients and, of course, solve the structure. In line with this, and if it is possible, pickup one of those crystals and run it on a gel as they did so to make sure it is in the correct size and not a truncated version. Good luck, Chen --- Chen Guttman The Zarivach laboratory for Macromolecular Crystallography Building 39, Room 009B Ben-Gurion University of the Negev POBox 653 Zip Code 84105 Beer-Sheva Israel http://lifeserv.bgu.ac.il/wb/zarivach Tel. +972-8-6479519 Fax. +972-8-6472970 On Tue, Apr 12, 2011 at 13:56, Jun Yong Ha j...@princeton.edu wrote: Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance.
Re: [ccp4bb] Reproducing crystals.
I once had a similar problem. The crystals only reproduced when grown in condition containing PEG 8000 from SIGMA but not from FLUKA. This difference may not affect crystals from other proteins but some proteins are more sensitive to slight changes than others. Since you found out that the stock is not available, it will be worthwhile to try making stock containing PEG 4000 and maybe also try PEG 4000 from other suppliers. -Raj
[ccp4bb] Reproducing crystals.
Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance.
Re: [ccp4bb] Reproducing crystals.
Maybe your old solution evaporated then you end up in your old tube with a more concentrated solution in PEG and NaCl so try to screen with new conditions with higher PEG and/or NaCl concentration Mick 2011/4/12 Jun Yong Ha j...@princeton.edu Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance. -- -- Mickael Blaise PhD Department of molecular biology Centre for structural biology Aarhus university Gustav wieds vej 10 8000 Aarhus-Denmark
Re: [ccp4bb] Reproducing crystals.
or PEG 4000 got old. ask around in the department or university for old PEG 4000 bottles. good luck! Berta On Apr 12, 2011, at 3:28 PM, mickael blaise wrote: Maybe your old solution evaporated then you end up in your old tube with a more concentrated solution in PEG and NaCl so try to screen with new conditions with higher PEG and/or NaCl concentration Mick 2011/4/12 Jun Yong Ha j...@princeton.edu Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance. -- -- Mickael Blaise PhD Department of molecular biology Centre for structural biology Aarhus university Gustav wieds vej 10 8000 Aarhus-Denmark
Re: [ccp4bb] Reproducing crystals.
Hi, Some anecdotes here for your reference: One paper I read says that the authors were having trouble reproducing a crystal from an initial screen. After some debugging, they realized that it was because that they used a same pipette tip when making screens. Adding a little solution from the condition prior to the hit condition solved the mystery. Another paper I read said that they failed reproducing crystals until realizing that the only success they had was that they forgot to add well solution to the hanging drop(a very small protein domain though). Of course there comes the famous example that the pioneer crystallographer used a metal container to transfer meat from the butcher's for making his protein. Then it turned out that the magic bullet for his success in crystallization was the galvanized Zinc(or nickel?). I guess there must be tons of such stories if we read through Acta D. :) PEG is known to decompose in solution, I think something like lactate might be generated. If your solution is strongly buffered by hepes (100mM at pH7.5 for example), the pH may not changed much even if you have a few mM of lactate. -- From: Jun Yong Ha j...@princeton.edu Sent: Tuesday, April 12, 2011 7:56 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Reproducing crystals. Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance.
Re: [ccp4bb] Reproducing crystals.
PS, it might be a good time to start an additive screen. -- From: Jun Yong Ha j...@princeton.edu Sent: Tuesday, April 12, 2011 7:56 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Reproducing crystals. Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance.
Re: [ccp4bb] Reproducing crystals.
Frances Jurnak published a paper in 1986 on PEG impurities and purification. As I recall, it turns out that different manufacturers put different additives in PEGs as preservatives. These are generally anti-oxidants. PEGs do get oxidized. I suggest you heat up your new PEG solutions to say 80 deg C and cool them down, then use them. Let us know what happens. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jun Yong Ha Sent: Tuesday, April 12, 2011 6:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Reproducing crystals. Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance.
Re: [ccp4bb] Reproducing crystals.
You might also try to control the degree of oxidation using the microwave, and setting up trials after different numbers of cycles of heating. Kendall On Apr 12, 2011, at 12:41 PM, Jim Pflugrath wrote: Frances Jurnak published a paper in 1986 on PEG impurities and purification. As I recall, it turns out that different manufacturers put different additives in PEGs as preservatives. These are generally anti-oxidants. PEGs do get oxidized. I suggest you heat up your new PEG solutions to say 80 deg C and cool them down, then use them. Let us know what happens. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jun Yong Ha Sent: Tuesday, April 12, 2011 6:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Reproducing crystals. Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance.
