Thank you all for your helpful discussion.
Cheers,
Flavio
Il 22/04/25 19:27, Patrick Shaw Stewart ha scritto:
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
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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
>
>
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