We used to do a lot of microbatch, but I've never come across something like this before!
I agree with Pat that Cd++ ions can't dissolve in oil because they're far too polar. I think there is a subtle effect related to protein concentration and/or water activity. Do you have a (protein) skin on the vapor diffusion drops? If so, the protein concentration in the vapor diffusion setup may be much lower. I would not use Al's Oil for this because it complicates things - I would try to find the microbatch conditions with paraffin oil that are equivalent to the vapor diffusion condition. This depends a bit on whether the main precipitant is a salt, which causes significant dehydration during equilibration, or, for example, PEG, where little dehydration occurs, or if it does, it's a slow process. What was the main precipitant, and how long did the crystals take to grow in vapor diffusion? I would try to establish seeding conditions, which would give you much more control. You may need to dilute the seedstock significantly to get the crystal size that you want. A mini phase diagram might be helpful - say 6 microbatch wells (with paraffin) where you vary the ratio of protein to crystallization cocktail. Then another 6 wells with a small volume of diluent eg water added. Ideally, you would do it with and without seed to establish the metastable zone of the phase diagram. We're working on a couple of scripts to do similar phase diagrams automatically! Good luck Patrick On Tue, Apr 22, 2025 at 2:41 PM Patrick Loll <pjl...@gmail.com> wrote: > I’m skeptical that the oil is acting as a reservoir for the metal, as > shouldn’t the metal be too hydrophilic to partition into the oil phase? > This is testable, at least. > > Another (to me, more plausible) explanation is that there are subtle > differences in water activity in your two crystallization setups. Many > years ago (doi: 10.1021/bi00013a021) I saw a metal change position owing to > crystal dehydration and concomitant subtle shifts in atomic positions; > perhaps this is what’s going on? > > FWIW, > > Pat > > --------------------------------------------------------------------------------------- > Patrick J. Loll, Ph. D. (he, him, his) > Professor of Biochemistry & Molecular Biology > Drexel University College of Medicine > Room 10-102 New College Building > 245 N. 15th St., Mailstop 497 > Philadelphia, PA 19102 USA > > (215) 762-7706 > pjl...@gmail.com > pj...@drexel.edu > > > On Apr 22, 2025, at 5:03 AM, Flavio Di Pisa <dipi...@gmail.com> wrote: > > > > Dear community, > > I’ve observed differences when crystallizing the same protein using two > different setups: microbatch under (Al’s) oil and vapor diffusion sitting > drop. > > The protein crystallizes in a condition containing 50 mM cadmium as one > of the precipitating agents. In the sitting drop setup, I observe 2 > well-defined cadmium ions at the so-called mineralization site (please see > PDB entry 5lg8 and the related paper: > https://www.pnas.org/doi/10.1073/pnas.1614302114), with occupancies close > to 1, as confirmed by the anomalous signal, plus other "anomalous blob" > near to this site. > > However, in the microbatch under oil setup, I never observe these > cadmium ions. Instead, I consistently detect only one cadmium ion with high > occupancy, and occasionally a second one with lower occupancy. > > In summary, crystallizing the same protein using these two setups > results in different metal-binding behavior. > > My question is: could it be that in microbatch under oil, ions might > diffuse away from the mineralization site? Could this account for the > reduced number of cadmium ions observed? Additionally, and more > importantly, could the crystallization setup influence the soaking > efficiency of other metals, such as iron (the natural substrate of this > protein)? > > I’ve attached two screenshots: > > • One (orange blob) represents the protein crystallized via vapor > diffusion, showing two well-defined anomalous peaks. > > • The other shows the same site in a crystal grown via microbatch, > where only one anomalous signal (in white) is visible. > > I hope I’ve been clear. Thank you in advance, and I wish you all a great > day! > > Best regards, > > Flavio > > > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <Schermata del 2025-04-22 10-54-23.png><Schermata del 2025-04-22 > 10-54-51.png> > > Patrick Loll > pjl...@gmail.com > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a > mailing list hosted by www.jiscmail.ac.uk, terms & conditions are > available at https://www.jiscmail.ac.uk/policyandsecurity/ > -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
