Re: [ccp4bb] cryo for high salt crystal
Hi Anna, Actually I just tried lithium sulfate: I made a cryo with 1M KCl, 1.3M Lithium sulfate to replace the ammonia sulfate totally at pH7. it is not freezing clear. I guess I should try to increase the amount of LiSO4. But from all the warm responses here, I found sometimes people don't necessary keep the same concentration of component in the cryo as in the well solution + cryoprotectant. I usually make the artificial mother liquor as Roger or Michael mentioned early on. So my question is: will that hurt the crystal if the concentration of the component of cryo change? Want to thanks to all for your warm suggestions. I haven't finish all the reading suggested here. but will. Thanks,Manqing Date: Thu, 12 Jul 2012 13:40:39 -0700 From: anna.s.gardb...@gmail.com Subject: Re: [ccp4bb] cryo for high salt crystal To: CCP4BB@JISCMAIL.AC.UK Hello, Min.Lithium sulfate is a cryoprotectant at 2.0 M and sometimes even less (much lower than the concentrations needed for ammonium sulfate), so I would try replacing your ammonium sulfate with lithium sulfate, creating a cryoprotectant with 0.5-1.0 M KCl, 1.4-2.0 M LiSO4, at pH 7. You might need to transfer your crystals through a couple of intermediate drops. Regarding the reservoir precipitation - is there any chance you could control the humidity of the area in which you're working? Even filling a couple of adjacent reservoirs with water might help buy you a few extra crucial seconds. Also, working in a cold room to harvest your crystals will help reduce the evaporation rate. Good luck! Best,Anna On Tue, Jul 10, 2012 at 9:28 AM, m zhang wrote: regaentDear All, I am sure this question was discussed before. But I am wondering if anyone got the same experience as I do. I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil, or ammonium sulfate itself: The problem is that all the cryo plus original reagents in the reservoir precipitate the salts out. And more serious problem is because of high salt in the condition, while I am trying to loop the crystal, both the drop and cryoprotectant drop form salt crystals (not sure it is KCl or ammonia sulfate) significantly and very quickly, that cause my crystal dissolved. My crystal doesn't seem to survive paraton-N oil. Does anyone here have similiar case? any suggestion will be appreciated. Thanks,Min
Re: [ccp4bb] cryo for high salt crystal
Hi Jim, 25% is w/v. Thanks for the information. Will check the webinar. Thanks,Min From: jim.pflugr...@rigaku.com To: mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] cryo for high salt crystal Date: Tue, 10 Jul 2012 17:39:56 + Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that w/v or v/w?), try using 100% saturated in reservoir, 75% saturated in reservoir, or 50% saturated in reservoir. You will have to TEST these. See also this webinar on cryocrystallography which shows how to make these solutions: http://www.rigaku.com/node/1388 You could also try high salt solutions with similar technique. Good luck! Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m zhang [mzhang...@hotmail.com] Sent: Tuesday, July 10, 2012 11:28 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cryo for high salt crystal regaentDear All, I am sure this question was discussed before. But I am wondering if anyone got the same experience as I do. I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil, or ammonium sulfate itself: The problem is that all the cryo plus original reagents in the reservoir precipitate the salts out. And more serious problem is because of high salt in the condition, while I am trying to loop the crystal, both the drop and cryoprotectant drop form salt crystals (not sure it is KCl or ammonia sulfate) significantly and very quickly, that cause my crystal dissolved. My crystal doesn't seem to survive paraton-N oil. Does anyone here have similiar case? any suggestion will be appreciated. Thanks, Min
[ccp4bb] cryo for high salt crystal
regaentDear All, I am sure this question was discussed before. But I am wondering if anyone got the same experience as I do. I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil, or ammonium sulfate itself: The problem is that all the cryo plus original reagents in the reservoir precipitate the salts out. And more serious problem is because of high salt in the condition, while I am trying to loop the crystal, both the drop and cryoprotectant drop form salt crystals (not sure it is KCl or ammonia sulfate) significantly and very quickly, that cause my crystal dissolved. My crystal doesn't seem to survive paraton-N oil. Does anyone here have similiar case? any suggestion will be appreciated. Thanks,Min
Re: [ccp4bb] AW: [ccp4bb] crystallization of complex and ...
To all and Alex, -The Kd is around 500nM from Biacore. -One of my protein tends to precipitate and is only stable below 3mg/m. If I concentrated it to higher concentration, it will precipitate a lot after 1hr. And it was previously crystallized at low concentration. But when I inject on gel filtration, I do try to load large amount both proteins. Then this leads to another question: How high should the concentration of complex be in co-crystallization or gel filtration? I set mine at 6mg/ml for the complex and I get some precipitate. What concentration of complex would people start with to co-crystallize? -Yes, I use the same buffer system for both proteins and complex. Thank you all for your suggestions. Min Date: Fri, 16 Sep 2011 09:13:46 +0200 From: alexander.paut...@boehringer-ingelheim.com Subject: [ccp4bb] AW: [ccp4bb] crystallization of complex and ... To: CCP4BB@JISCMAIL.AC.UK Dear Min,regarding #1 some things come to my mind:- What is the Kd that you got from the Biacore? And did you make sure that the sample is concentrated enough (both on gel filtration and in crystallization) to have a “sufficient” amount in the complex.- did you use the same buffer systems? Your Kd might be different in different buffers.BestAlex Dr. Alexander Pautsch Boehringer Ingelheim Pharma GmbH & Co. KG Dept. Lead Identific. and Optim. Sup. Ge Tel.: +49 (7351) 54-4683 Fax: +49 (7351) 54-97924 mailto:alexander.paut...@boehringer-ingelheim.com Boehringer Ingelheim Pharma GmbH & Co. KG, Sitz: Ingelheim am Rhein; Registergericht Mainz: HR A 22206; Komplementär Boehringer Ingelheim Deutschland GmbH; Geschäftsführung: Dr. Engelbert Günster (Vorsitzender), Ralf Gorniak, Mark Hagmann, Michael Klein, Dr. Martin Wanning; Vorsitzender des Aufsichtsrates: Prof. Dr. Dr. Andreas Barner; Sitz: Ingelheim am Rhein; Registergericht Mainz: HR B 23260 Diese E-Mail ist vertraulich zu behandeln. Sie kann besonderem rechtlichen Schutz unterliegen. Wenn Sie nicht der richtige Adressat sind, senden Sie bitte diese E-Mail an den Absender zurück, löschen die eingegangene E-Mail und geben den Inhalt der E-Mail nicht weiter. Jegliche unbefugte Bearbeitung, Nutzung, Vervielfältigung oder Verbreitung ist verboten. / This e-mail is confidential and may also be legally privileged. If you are not the intended recipient please reply to sender, delete the e-mail and do not disclose its contents to any person. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von m zhang Gesendet: Freitag, 16. September 2011 04:20 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] crystallization of complex and ... Dear all, I have two questions: First, I was trying to crystallize a complex of two proteins. Both proteins has been crystallized before. The two proteins bind to each other based on Biacore study, but they didn't form a single peak on gel filtration. When I mixed them at 1:1 ratio, the crystals I got contain only one of the two proteins. I was suggested to increase the ratio, for example 1.5:1, to increase the probability of co-crystallization which I will try. But I do want to hear if there are other possible ways to try. What would you try if you were in my situation? Second is about reusing of Ni-NTA resin. According to Qiagen's instruction, after using fresh Ni-NTA resin, one only needs to wash the used Ni resin first with 0.5M NaOH, then with your own buffer. After that the resin is ready to be reused until it needs being recharged. But my question is: Once immidazole competes with His-tagged protein and binds to Ni-resin, how can immidazole be rinsed off with the same buffer(usually pH is above 7) one uses to purify the protein? Thank you for any suggestion or comment. Min
[ccp4bb] crystallization of complex and ...
Dear all, I have two questions: First, I was trying to crystallize a complex of two proteins. Both proteins has been crystallized before. The two proteins bind to each other based on Biacore study, but they didn't form a single peak on gel filtration. When I mixed them at 1:1 ratio, the crystals I got contain only one of the two proteins. I was suggested to increase the ratio, for example 1.5:1, to increase the probability of co-crystallization which I will try. But I do want to hear if there are other possible ways to try. What would you try if you were in my situation? Second is about reusing of Ni-NTA resin. According to Qiagen's instruction, after using fresh Ni-NTA resin, one only needs to wash the used Ni resin first with 0.5M NaOH, then with your own buffer. After that the resin is ready to be reused until it needs being recharged. But my question is: Once immidazole competes with His-tagged protein and binds to Ni-resin, how can immidazole be rinsed off with the same buffer(usually pH is above 7) one uses to purify the protein? Thank you for any suggestion or comment. Min
Re: [ccp4bb] stoichiometry of complex and incubator
Dear all, Thank you all very much for your kind suggestions. Now we have some preliminary data of SPR. But we might try other new ideas suggested here. Best,Min Date: Thu, 4 Aug 2011 13:19:41 +0200 From: krishna@gmail.com Subject: Re: [ccp4bb] stoichiometry of complex and incubator To: CCP4BB@JISCMAIL.AC.UK Dear Min, You can use microscale thermophoresis for this kind of tricky cases when you have limited amount of protein and ligands. There is enough literature on this topic. have a look in the following reference: Proc Natl Acad Sci U S A. 2006 Dec 26;103(52):19678-82 Hope this helps. Regards, Krishna Krishna Chinthalapudi Graduate Student Hannover Medical School Germany On Thu, Aug 4, 2011 at 4:32 AM, m zhang wrote: Dear all, I have two unrelated questions. Any suggestion on any of them will be greatly appreciated. First, we have two proteins that bind each other without a doubt. But since we have very limited amount of proteins and it takes a long time to reproduce them, we are very hesitating to try ITC and AUC to find out the stoichiometry of these complex. So I am wondering if there is any other way we can try to find the ratio without consuming much proteins? Second, we are thinking about getting a new incubator for crystallization. Could anyone here recommend a model or a brand? Thank you, Min
[ccp4bb] stoichiometry of complex and incubator
Dear all, I have two unrelated questions. Any suggestion on any of them will be greatly appreciated. First, we have two proteins that bind each other without a doubt. But since we have very limited amount of proteins and it takes a long time to reproduce them, we are very hesitating to try ITC and AUC to find out the stoichiometry of these complex. So I am wondering if there is any other way we can try to find the ratio without consuming much proteins? Second, we are thinking about getting a new incubator for crystallization. Could anyone here recommend a model or a brand? Thank you, Min
[ccp4bb] setting up additive screen
Dear all, I am trying to optimize my crystal with additives. Since the yield of my protein purification is very limited, I am wondering what is the most efficient way to set up drops with additive to save my protein and not wasting additives? I am setting up 1 to 1 drops with 0.2 ul additives. But I feel 0.2ul is not very actuate, even if I use a p2. Would you share your ways to set up drops with additives? If I want to screen some additives, what additives would you suggest to try first, especially those from the 96 additive conditions from Hampton? By the way, just wondering, what kind of p2 pipettor work better? Any input is greatly appreciated! Thank you, Min
Re: [ccp4bb] low expression and aggregation of protein
Thanks, Lin. I am sorry for being short of information. I was actually not sure what kind of information is needed. Well, the cells were grown at 27 degree in Insect-Xpress. I tried to grow cells both in shaker and stationery flask. Anything else I can try? Best, Min Date: Sat, 22 Jan 2011 21:57:02 +0100 Subject: Re: [ccp4bb] low expression and aggregation of protein From: yyb...@gmail.com To: mzhang...@hotmail.com CC: CCP4BB@jiscmail.ac.uk Actually, it is very difficult to say something just based on your simple information. You should give more informations about your work, for example, temperature, cuture, etc. Best, Lin On Fri, Jan 21, 2011 at 8:12 PM, m zhang wrote: Dear All, Recently I am trying to express a glycoprotein in Drosophila S2 cells. However, the expression yield is every low and most of the protein expressed aggregate. I was suggested making new constructs. But I am wondering if anyone here can give me some suggestions to improve it beyond that since I am new to glycoprotein? All your input will be greatly appreciated! Regards, Min
[ccp4bb] low expression and aggregation of protein
Dear All, Recently I am trying to express a glycoprotein in Drosophila S2 cells. However, the expression yield is every low and most of the protein expressed aggregate. I was suggested making new constructs. But I am wondering if anyone here can give me some suggestions to improve it beyond that since I am new to glycoprotein? All your input will be greatly appreciated! Regards, Min
[ccp4bb] model-mask
Hi All, I was trying to make a model-mask file for ncs-averaging in DM. I noticed that in CNS, the mask file has to be O-compressed. Does that mean if I read in O the mask file and write it out, the output file is already in O-compressed format? Related to the first question, the second one is: if I want to use just heavy atom sites to produce a mask file, will that work if there is NCS between them? My understanding is that the mask file just tell the DM program what is to be averages and where is the density to be averaged, am I right? Any suggestion are very welcome! Thanks, Zhang _ Can you find the hidden words? Take a break and play Seekadoo! http://club.live.com/seekadoo.aspx?icid=seek_wlmailtextlink
[ccp4bb] questions about CNS
Hi everyone, I have some basic questions about CNS. First, I am wondering if there is anywhere I can set my initial B-factor during my early refinement. When I generate my initial model in generate.inp, I can set the B-factore. However, after I did the rigid.inp and anneal.inp, the B-factor just go up to more than 100 by itself. I look through the input files, and couldn't find where to set the B-factor. Do I have to set the B-factor by hand each time? Or will the crazy B-factor affect my refinement? Second, I am refining a new protein/DNA complex. I started with the DNA alone. After rigid body refinement I got ~1% decrease of Rf. But when I tried anneal after that, the Rf just went up much higher than where I started? If you work on a protein/DNA complex, what is your stategy? Will the anneal help refine the DNA alone? How much decrease of Rf usually you can get from refining just DNA? Third, I was wondering if I want to extend my resolution, do I have to make sure my cv file was made with the highest resolution? Thank you for your suggestion! Cortney _ Don't get caught with egg on your face. Play Chicktionary! http://club.live.com/chicktionary.aspx?icid=chick_wlmailtextlink
