Re: [ccp4bb] Help needed finding hit condition
Hello everyone. I remember the screen was again from Jenabioscience and this had happened with one of my protein. The screen was very old and the condition was peg3350, tris pH 8, lithium sulfate and NaCl as the salt. Hit was obtained which was never reproducible. Luckily I solved the structure from the hit itself which diffracted to 2.2A resolution. But it is still a mystery for us but this is all crystallography is. Strange things happen. Faisal Postdoc PHRI, NJ, USA On Jul 31, 2017 5:19 PM, "Janet Newman" wrote: > Hi Jonathan, > > > Hopefully you know about the trick of making any precious condition last > longer - use the 'magic' solution only in the drop itself, and use the best > approximation you can make in the reservoir of the experiment (if you are > doing vapour diffusion) > > > Regards, Janet > > > Janet Newman > Principal Scientist / Director, Collaborative Crystallisation Centre (C3) > CSIRO Material Science and Engineering > 343 Royal Parade > Parkville. VIC. 3052 > Australia > Tel +613 9662 7326 <+61%203%209662%207326> > Email janet.new...@csiro.au > -- > *From:* CCP4 bulletin board on behalf of Jonathan > Bailey > *Sent:* 31 July 2017 22:34 > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Help needed finding hit condition > > > Dear CCP4bb community > > > I apologies for the slightly off topic post. > > > We have recently had success crystallizing a membrane protein (diffraction > > 3 Å at a synchrotron source) using the *in meso* method, the hit > condition was from the Jena Bioscience screen Pi-minimal condition number > #57. > > > Hit condition – 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium > bromide > > > The screen is old and expired 12/20/2013 (lot # JBS00013133), we have > tried to reproduce the crystals using homemade optimization screens around > the hit condition but have not had any success. We have tried reproducing > the hit using a new (not expired) Pi-minimal screen but had no success. We > are only able to reproduce the crystals using the expired screen and we do > not have much of it left. > > > > We went back and tested the pH of the condition that had given crystals, > the expected pH was 7.9 but we found it to be 6 – 6.5 using a pH indicator > strip. We believe the drop in pH is caused by oxidative degradation of the > PEG1000 resulting in the formation of carboxylic acid species. > > > We have contacted Jena Bioscience to try and get some of the old screen > stock but unfortunately they do not have any. > > > My question is does anyone out there happen to have any expired screen > stocks of this Pi-minimal condition (#57), ideally from the same lot (lot # > JB200013133), that they would be willing to send us. > > > > Does anyone have any advice as to how to reproduce the condition? We’ve > considered bubbling oxygen through and heating the sample to accelerate the > oxidation process. > > > > King Regards > > > Jonathan Bailey (PhD student) > > > Professor Martin Caffrey Lab MS&FB group Trinity College Dublin >
Re: [ccp4bb] Help needed finding hit condition
?Hi Jonathan, Hopefully you know about the trick of making any precious condition last longer - use the 'magic' solution only in the drop itself, and use the best approximation you can make in the reservoir of the experiment (if you are doing vapour diffusion) Regards, Janet Janet Newman Principal Scientist / Director, Collaborative Crystallisation Centre (C3) CSIRO Material Science and Engineering 343 Royal Parade Parkville. VIC. 3052 Australia Tel +613 9662 7326 Email janet.new...@csiro.au From: CCP4 bulletin board on behalf of Jonathan Bailey Sent: 31 July 2017 22:34 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Help needed finding hit condition Dear CCP4bb community I apologies for the slightly off topic post. We have recently had success crystallizing a membrane protein (diffraction > 3 Å at a synchrotron source) using the in meso method, the hit condition was from the Jena Bioscience screen Pi-minimal condition number #57. Hit condition - 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium bromide The screen is old and expired 12/20/2013 (lot # JBS00013133), we have tried to reproduce the crystals using homemade optimization screens around the hit condition but have not had any success. We have tried reproducing the hit using a new (not expired) Pi-minimal screen but had no success. We are only able to reproduce the crystals using the expired screen and we do not have much of it left. We went back and tested the pH of the condition that had given crystals, the expected pH was 7.9 but we found it to be 6 - 6.5 using a pH indicator strip. We believe the drop in pH is caused by oxidative degradation of the PEG1000 resulting in the formation of carboxylic acid species. We have contacted Jena Bioscience to try and get some of the old screen stock but unfortunately they do not have any. My question is does anyone out there happen to have any expired screen stocks of this Pi-minimal condition (#57), ideally from the same lot (lot # JB200013133), that they would be willing to send us. Does anyone have any advice as to how to reproduce the condition? We've considered bubbling oxygen through and heating the sample to accelerate the oxidation process. King Regards Jonathan Bailey (PhD student) Professor Martin Caffrey Lab MS&FB group Trinity College Dublin
Re: [ccp4bb] Help needed finding hit condition
Hi Jonathan, Old buffer may have slow evaporation with some side reactions. The concentration of each component may increase a little bit. In this case, I would consider the condition as the origin for further optimization. Each component may need to be considered separately. For 150mM Tris pH 8.000, The pH and concentration may have a slight change (increase ?). I will try the concentration of 150mM, 155 mM and 160mM. For pH, pH 8.1 and pH 8.2... For KBr, the concentration may be 80, 85, 90mM or higher. For 47.1% w/v PEG1K (Pretty high concentration), As the result for ring-opening epoxide reaction, it is not very stable anyway. The reaction always continued. Basic condition made the case even worse. In this case, the Mn (MW) of PEG1K may not be that average anymore. It is very possible that the polymer chain is elongated. Of course, the concentration of PEG is increased too. In the meanwhile, the presence of KBr may cause further chemical modification on the PEG chains. You may try PEG 95--1050 (Sigma P3515), PEG 1305-1595 (Sigma 202136), PEG3K or PEG 3350, etc. Best, Kevin P.S. If you take a look from the top of your old solution in the tube, what's the color? Slight Yellow? You can use a tube with dd water a reference. On Mon, Jul 31, 2017 at 5:34 AM, Jonathan Bailey wrote: > Dear CCP4bb community > > > I apologies for the slightly off topic post. > > > We have recently had success crystallizing a membrane protein (diffraction > > 3 Å at a synchrotron source) using the *in meso* method, the hit > condition was from the Jena Bioscience screen Pi-minimal condition number > #57. > > > Hit condition – 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium > bromide > > > The screen is old and expired 12/20/2013 (lot # JBS00013133), we have > tried to reproduce the crystals using homemade optimization screens around > the hit condition but have not had any success. We have tried reproducing > the hit using a new (not expired) Pi-minimal screen but had no success. We > are only able to reproduce the crystals using the expired screen and we do > not have much of it left. > > > > We went back and tested the pH of the condition that had given crystals, > the expected pH was 7.9 but we found it to be 6 – 6.5 using a pH indicator > strip. We believe the drop in pH is caused by oxidative degradation of the > PEG1000 resulting in the formation of carboxylic acid species. > > > We have contacted Jena Bioscience to try and get some of the old screen > stock but unfortunately they do not have any. > > > My question is does anyone out there happen to have any expired screen > stocks of this Pi-minimal condition (#57), ideally from the same lot (lot # > JB200013133), that they would be willing to send us. > > > > Does anyone have any advice as to how to reproduce the condition? We’ve > considered bubbling oxygen through and heating the sample to accelerate the > oxidation process. > > > > King Regards > > > Jonathan Bailey (PhD student) > > > Professor Martin Caffrey Lab MS&FB group Trinity College Dublin > -- Kevin Jin Sharing knowledge each other is always very joyful.. Website: http://www.jinkai.org/
Re: [ccp4bb] Help needed finding hit condition
Jonathan, While your claim of oxidative degradation of PEG1000 may be true -- I gather you mean that the conversion of the ends of the PEG polymers to aldehydes or peroxides, then to carboxylates -- you should check out Fran Jurnak’s old paper (Journal of Crystal Growth 76, 577, 1986). The synthesis of PEG often contains some of phosphoric acid due to the way they terminated the chain elongation. It is variable from batch to batch and from supplier to supplier; Merck (Germany) is a fairly good source, but Baker/Union Carbide isn't. Unless Jena took the time and effort to purify the PEG (see Bill Ray's article in the same issue, p. 562), you have another factor that can drop the pH. You might ask where Jena buys their PEG stock. Also, the way many companies make the screens are not always clear. Some just mix stocks, meaning the pH of the Tris stock (perhaps at 1 M) was pH 8, but when diluted down with the other components to make the 150 mM concentration for the screen mixture, the pH can be significantly different. The dilution of Tris would drop the pH. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com > On Jul 31, 2017, at 8:34 AM, Jonathan Bailey wrote: > > Dear CCP4bb community > > > > I apologies for the slightly off topic post. > > > > We have recently had success crystallizing a membrane protein (diffraction > > 3 Å at a synchrotron source) using the in meso method, the hit condition was > from the Jena Bioscience screen Pi-minimal condition number #57. > > > > Hit condition – 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium > bromide > > > > > The screen is old and expired 12/20/2013 (lot # JBS00013133), we have tried > to reproduce the crystals using homemade optimization screens around the hit > condition but have not had any success. We have tried reproducing the hit > using a new (not expired) Pi-minimal screen but had no success. We are only > able to reproduce the crystals using the expired screen and we do not have > much of it left. > > > We went back and tested the pH of the condition that had given crystals, the > expected pH was 7.9 but we found it to be 6 – 6.5 using a pH indicator strip. > We believe the drop in pH is caused by oxidative degradation of the PEG1000 > resulting in the formation of carboxylic acid species. > > > > We have contacted Jena Bioscience to try and get some of the old screen stock > but unfortunately they do not have any. > > > > My question is does anyone out there happen to have any expired screen stocks > of this Pi-minimal condition (#57), ideally from the same lot (lot # > JB200013133), that they would be willing to send us. > > > Does anyone have any advice as to how to reproduce the condition? We’ve > considered bubbling oxygen through and heating the sample to accelerate the > oxidation process. > > > King Regards > > > > Jonathan Bailey (PhD student) > > > > Professor Martin Caffrey Lab MS&FB group Trinity College Dublin >
Re: [ccp4bb] Help needed finding hit condition
Hello, Such pH drifts are rather common with crystallisation screens. Have you tried to produce other precipitant solutions with ca. 47% PEG 1000, 80 mM Potassium bromide but having a pH of (say) 6.2 ? A pH rangle close to that where your crystals were obtained. This would mean changing buffering agent to… MES perhaps ? Cheers, Fred. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jonathan Bailey Sent: Monday, July 31, 2017 2:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Help needed finding hit condition Dear CCP4bb community I apologies for the slightly off topic post. We have recently had success crystallizing a membrane protein (diffraction > 3 Å at a synchrotron source) using the in meso method, the hit condition was from the Jena Bioscience screen Pi-minimal condition number #57. Hit condition – 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium bromide The screen is old and expired 12/20/2013 (lot # JBS00013133), we have tried to reproduce the crystals using homemade optimization screens around the hit condition but have not had any success. We have tried reproducing the hit using a new (not expired) Pi-minimal screen but had no success. We are only able to reproduce the crystals using the expired screen and we do not have much of it left. We went back and tested the pH of the condition that had given crystals, the expected pH was 7.9 but we found it to be 6 – 6.5 using a pH indicator strip. We believe the drop in pH is caused by oxidative degradation of the PEG1000 resulting in the formation of carboxylic acid species. We have contacted Jena Bioscience to try and get some of the old screen stock but unfortunately they do not have any. My question is does anyone out there happen to have any expired screen stocks of this Pi-minimal condition (#57), ideally from the same lot (lot # JB200013133), that they would be willing to send us. Does anyone have any advice as to how to reproduce the condition? We’ve considered bubbling oxygen through and heating the sample to accelerate the oxidation process. King Regards Jonathan Bailey (PhD student) Professor Martin Caffrey Lab MS&FB group Trinity College Dublin - Upozornění: Není-li v této zprávě výslovně uvedeno jinak, má tato E-mailová zpráva nebo její přílohy pouze informativní charakter. Tato zpráva ani její přílohy v žádném ohledu Biotechnologický ústav AV ČR, v. v. i. k ničemu nezavazují. Text této zprávy nebo jejích příloh není návrhem na uzavření smlouvy, ani přijetím případného návrhu na uzavření smlouvy, ani jiným právním jednáním směřujícím k uzavření jakékoliv smlouvy a nezakládá předsmluvní odpovědnost Biotechnologického ústavu AV ČR, v. v. i. Disclaimer: If not expressly stated otherwise, this e-mail message (including any attached files) is intended purely for informational purposes and does not represent a binding agreement on the part of Institute of Biotechnology CAS. The text of this message and its attachments cannot be considered as a proposal to conclude a contract, nor the acceptance of a proposal to conclude a contract, nor any other legal act leading to concluding any contract; nor does it create any pre-contractual liability on the part of Institute of Biotechnology CAS