Hi Chad,
Re SV: the way I understand it, there’s a pitfall to mass determination in the case of interacting systems (including oligomers) – the accurate determination of the S value for a complex is going to be affected by its on and off rate and the concentration at which it is analyzed (and hence, any derivation of mass?). From what I understand from one resource, even 10-fold above the Kd for a rapid association, the highest peak in a c(S) distribution may not be the actual complex S value. No doubt, though, a well-behaved single species should provide a good determination of mass. Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine <BLOCKED::mailto:kgu...@stwing.upenn.edu> kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 From: Chad Brautigam [mailto:cabrautc...@yahoo.com] Sent: Thursday, June 02, 2011 2:38 PM To: Kushol Gupta Cc: ccp4bb@jiscmail.ac.uk Subject: Re: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation Dear Kushol & Jerry, I have to take exception to Kushol's contention about SV. As long as the buffer and protein parameters are correct and the sample is well behaved (i.e. not undergoing dynamic rearrangement on the time scale of the SV experiment, not aggregated, homogeneous), one can derive very accurate molar masses from SV, and it is far superior in this respect than SEC. However, as you aptly point out, the problem may lie in the sample's behavior. If the protein is populating multiple oligomeric states, or if it is undergoing a fast interconversion of such states, or if it is not pure, spurious masses may be calculated. If such pathologies are observed, it's not clear to me that a sedimentation equilibrium experiment would help. For a complicated interaction model, SE data (and AUC data in general) can be difficult to analyze, and some model assumptions are almost always present. So, the questions are: 1. Did Jerry run SV or SE? 2. If the former, then did he see a nice, single boundary, or a big smear? 3. If the latter, how were the data analyzed to come to his conclusion? Hopefully he can comment on these aspects. Cheerio, Chad _____ From: Kushol Gupta <kushol.gu...@gmail.com> To: CCP4BB@JISCMAIL.AC.UK Sent: Fri, May 27, 2011 9:51:04 AM Subject: Re: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation Hi Jerry – By AUC, do you mean sedimentation velocity (SV)? Both gel filtration and SV are not terribly great ways to determine precise molecular mass, especially if the macromolecule of interest is anisotropic in shape. In your SV values, do you see a large f/fo, or a broad distribution? Can you run a sedimentation equilibrium experiment? If you run HYDROPRO on your prospective oligomer structure, do you arrive at theoretical S and Rs values that jive with your solution data? A nice crosscheck you could do with the data in hand (if your measurements from both approaches were performed in the same buffer at the same temperature) is calculate the mass using the Siegel and Monty equation (Siegel, L. M., and Monty, K. J. (1966) Biochim. Biophys. Acta 112, 346–362), where the mass of the particle is calculated from Rs (from gel filtration) and the S(t,b) value from sedimentation velocity. Hope this helps, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jerry McCully Sent: Friday, May 27, 2011 10:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation Dear ALL; I am sorry for this off-topic question about analytic ultracentrifugation (AUC). We recently solved one structure from crystals grown out of PEG4000 plus buffer. Since the crystal was grown from PEG, we think the protein would maintain its native oligomerization state as in the solution. Indeed, the crystal packing clearly shows a tetramer of this protein. However, both the gel-filtration and AUC showed larger molecular weight, roughly around 6-mer or 7-mer. IN the crystal lattice, we could not find any 6-mer or 7-mer state. Could anyone give some comments on this discrepancy? Thanks a lot and have a nice weekend! Jerry McCully
