Re: [ccp4bb] off topic: problematic protein
I would like to thank all of you who promptly replied to my posting with so many ideas and suggestions (18 answers so far). I will post a summary soon. best wishes to all Savvas On Apr 19, 2011, at 12:48 PM, Savvas Savvides wrote: Dear colleagues We are working on a large bacterial protein (featuring a large number of repeats) that appears to copurify with a lot of other proteins after Ni-affinity chromatography and gel-filtration. We have tried adjusting the ionic strength of these runs and have gone to as high as 5M NaCl but only saw marginal improvements. It appears that the protein likes to stick to a lot of stuff, and in fact the number of repeats in a given construct appears to correlate with the extent of contaminants in our purification steps. We have admittedly never seen anything like this among the so many different, and often challenging, proteins, we have worked on in our group over the last few years. We are now thinking of trying detergents in the buffers (at non-micellar concentrations), in conjunction with playing a bit with the pH to see if such an approach provides a 'stripping' effect. Interestingly, the protein has a calculated pI of 3.5 ! As the options for handling this protein are indeed quite numerous, we would be grateful for any additional input and possible tips/tricks. I will prompty post a summary of the thread. Best regards Savvas et al. Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html
Re: [ccp4bb] Rcullis and phasing power
Hi Kenneth, I think the FOM reported by BP3 is a good indication of the quality of a solution: the FOM is an estimate of the mean cosine of the phase error. BP3 does not work with calculated anomalous differences with SAD data, but uses the observed and calculated F+ and F-: thus, an R-cullis or phasing power has less meaning with BP3's algorithm. Best wishes, Raj On Wed, 20 Apr 2011, Kenneth A. Satyshur wrote: sirs: I am running BP3 in crank on SeMet data SAD and getting a solution, but we need a number like Rcullis which is nowhere to be found, and comes from Mlphare. What is best to report? thanks kas -- Kenneth A. Satyshur, M.S.,Ph.D. Associate Scientist University of Wisconsin Madison, Wisconsin 53706 608-215-5207
Re: [ccp4bb] Crystal Optimization: Summary
Dear All, Thank you for the replies to my post. I received more than 14 response. The summary is given below. The suggestions were - add another purification step. * Ni-column * remove His-tag * Ni-column * gel filtration if you have done that: add another step to test for better crystals - additive screens - room temperature data set to check if freezing hampers the crystal - seeding - crystallisation at different temperatures - removal of the His-tag (or leave it on if it is cleaved) Dehydration. Reductive methylation Add PEG400 (or set your PEG concentration ~5% higher) into the cryosolution. Cryoprotection with DMSO. Grew the crystals in the presence of 5% glycerol. Crosslink the crystals with glutaraldehyde by vapor diffusion. Adding 20% peg200 as cryoprotectant. Using the Hampton Research Crystal Screen HT as additive screen (adding 5% into the mother liquor). Slowly increase the PEG concentration to 28 - 30% Add 1-2% Glycerol or MPD or Ethylene Glycol or other cryo-agent to your protein buffer you may find similar or even identical crystallization condition in which your crystal may tolerate higher concentration of cryo-agent. To include any salts in the cryo (at least half of the concentration) that may be already in the protein solution.
[ccp4bb] Jobs in Australia
Just a note to alert interested users of some (post-doctoral) research associate opportunities in Australia: http://employment.uow.edu.au/cgi-bin/job_details.cgi?id=23878
[ccp4bb] Puzzle with MALS
Dear All, I am having a consistency issue processing my SEC-MALS data. The sample is a 80KD protein which may form a dimer. The experiment I run a year ago shows a major peak at 11.4 ml (GE S200 column), which is calcuated to have a MW of 144kD. There is a minor peak at 10.0 ml. In an effort to make nice looking pictures, I rerun the experiement using the same setup (column, buffer, protein, speed) and again the major peak is at 11.4 ml but it is calcuated to have a MW of 80KD. I rerun the sample multiple times with the same result. I tried to reprocess the old data and the calcuated MW is still 144KD. I am puzzled about what I have done wrong and which data I should trust. Well, I am inclined to trust the later experiment because I took extra caution and had multiple data sets. However, I should be able to reprocess the old data to have a MW close to 80KD if I can find out what is wrong and correct the mistake, which I have not been able to. Any suggestions? Zhen Zhang
Re: [ccp4bb] Puzzle with MALS
Hi Zhen -was concentration higher in old run? Might be monomer-dimer equilibrium, for recent paper, see Benfield et al. (JBC, in press) -we have a miniDAWN with a 'low-high-medium' setting in the back of the setting. Inadvertently changing the setting scales the MW values up and down. Have you run a BSA standard, to insure system is OK? -Amir From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of zhen zhang [zz2...@columbia.edu] Sent: Thursday, April 21, 2011 5:32 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Puzzle with MALS Dear All, I am having a consistency issue processing my SEC-MALS data. The sample is a 80KD protein which may form a dimer. The experiment I run a year ago shows a major peak at 11.4 ml (GE S200 column), which is calcuated to have a MW of 144kD. There is a minor peak at 10.0 ml. In an effort to make nice looking pictures, I rerun the experiement using the same setup (column, buffer, protein, speed) and again the major peak is at 11.4 ml but it is calcuated to have a MW of 80KD. I rerun the sample multiple times with the same result. I tried to reprocess the old data and the calcuated MW is still 144KD. I am puzzled about what I have done wrong and which data I should trust. Well, I am inclined to trust the later experiment because I took extra caution and had multiple data sets. However, I should be able to reprocess the old data to have a MW close to 80KD if I can find out what is wrong and correct the mistake, which I have not been able to. Any suggestions? Zhen Zhang
Re: [ccp4bb] Puzzle with MALS
Hi Amir, Ade, Mark and Tom, Thanks a lot for your replies. Checking with BSA is a great idea and I should've done it earlier. The BSA run for recent experiment is OK. I cannot find a standard profile for my experiment one year ago. So I guess that there was something wrong with the machine one year ago. Besides, as Tom pointed out, 144KD is way off the MW of dimer. So I am quite confident that I should use the new data. BTW, the protein concentration is same. And it is a monomer-dimer equilibrium (11.4ml and 10.0ml). Zhen Quoting Amir Khan amirr...@tcd.ie: Hi Zhen -was concentration higher in old run? Might be monomer-dimer equilibrium, for recent paper, see Benfield et al. (JBC, in press) -we have a miniDAWN with a 'low-high-medium' setting in the back of the setting. Inadvertently changing the setting scales the MW values up and down. Have you run a BSA standard, to insure system is OK? -Amir From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of zhen zhang [zz2...@columbia.edu] Sent: Thursday, April 21, 2011 5:32 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Puzzle with MALS Dear All, I am having a consistency issue processing my SEC-MALS data. The sample is a 80KD protein which may form a dimer. The experiment I run a year ago shows a major peak at 11.4 ml (GE S200 column), which is calcuated to have a MW of 144kD. There is a minor peak at 10.0 ml. In an effort to make nice looking pictures, I rerun the experiement using the same setup (column, buffer, protein, speed) and again the major peak is at 11.4 ml but it is calcuated to have a MW of 80KD. I rerun the sample multiple times with the same result. I tried to reprocess the old data and the calcuated MW is still 144KD. I am puzzled about what I have done wrong and which data I should trust. Well, I am inclined to trust the later experiment because I took extra caution and had multiple data sets. However, I should be able to reprocess the old data to have a MW close to 80KD if I can find out what is wrong and correct the mistake, which I have not been able to. Any suggestions? Zhen Zhang
Re: [ccp4bb] Map correlation coefficient
Hi Everybody, I posted a question earlier on the bulletin regarding how to calculate the map correlation coefficient using Overlapamp or any other program? My follow up question is, does anybody know how to calculate the map correlation coefficient for the main chain and side chain separately? Thank you Maher On Wed, Feb 23, 2011 at 11:40 PM, Maher Alayyoubi maher.alayyo...@gmail.com wrote: Hi Everybody, I am a new user on the ccp4 bulletin, I have a question on how to calculate the map correlation coefficient using Overlapamp or any other program? Thank You, Maher
Re: [ccp4bb] Map correlation coefficient
Hi Maher, I posted a question earlier on the bulletin regarding how to calculate the map correlation coefficient using Overlapamp *or * *any other program*? My follow up question is, does anybody know how to calculate the map correlation coefficient for the main chain and side chain separately? depending on resolution, the command phenix.model_vs_data data.mtz model.pdb comprehensive=true reports map CC either per atom or per residue. If it reports map CC per atom, then knowing which atoms belong to main/side chains you can extract the information you need. However, if the resolution of your data is low enough so it outputs map CC computed per residue - then this doesn't answer your question. Good luck! Pavel.
Re: [ccp4bb] Map correlation coefficient
Hi Maher My version of Overlapmap reports the CC for main chain and for side chain in the log-file (per residue and overall). Have you looked there? -Bjørn On 2011-04-21 14:25, Maher Alayyoubi wrote: Hi Everybody, I posted a question earlier on the bulletin regarding how to calculate the map correlation coefficient using Overlapamp or any other program? My follow up question is, does anybody know how to calculate the map correlation coefficient for the main chain and side chain separately? Thank you Maher On Wed, Feb 23, 2011 at 11:40 PM, Maher Alayyoubi maher.alayyo...@gmail.com wrote: Hi Everybody, I am a new user on the ccp4 bulletin, I have a question on how to calculate the map correlation coefficient using Overlapamp or any other program? Thank You, Maher
