Hi Tatiana,

 Your problem is most reminiscent to the problem that Max Perutz faced when he dealt with deoxy-haemoglobin crystals, but in those days only mounting in capillaries was the way to bring the crystals to the beam, so he used dithionite, just like you, but mounted the crystals in (specially made for him at LMB) glove box under Nitrogen atmosphere. I guess you're now freezing your crystals for data collection, right? I'm not sure how to do this in a glove box nor am I sure whether after freezing your crystals is protected against oxidation. Maybe. But perhaps you can also consider using Parthon oil (or something similar) as cryo-protectant so it will "coat" your crystal in the glove box  and will also reduce oxidation? 

Good luck,

               Boaz 


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: [log in to unmask]
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710





________________________________________
From: CCP4 bulletin board [[log in to unmask]] on behalf of ISABET Tatiana [[log in to unmask]]
Sent: Thursday, October 02, 2014 11:22 AM
To: [log in to unmask]
Subject: [ccp4bb] Question about enzyme behavior

Dear all,

Sorry for a non purely crystallographic question.

I am working on an enzyme which binds Fe2+ cations to catalyzes an FeII-dependent hydroxylation reaction.

Because of fast oxidation in presence of the enzyme, it is very difficult to soak Fe2+ ions into the crystals. We succeed only under anaerobic conditions (glove box). I use a combination of dithionite as a reducing agent and Fe2+SO4 or (NH4)2Fe(SO4)2 as Fe2+ source. Despite these precautions, the Fe2+ is most often disordered in the active site.

When I add Fe2+ under aerobic conditions, Fe2+ oxidizes immediately upon contact with the protein solution (despite 1mM Dithionite for 5mM Fe2+ and protein concentration = 230uM). Furthermore, the hydroxyl donor molecule, which should bind Fe2+ (before the substrate) and one residue of the protein, is not seen in the electron-density maps in the active site. I have tried several soaking conditions. When I try a co-crystallization approach, adding Fe2+ and this hydroxyl donor molecule directly to the protein solution under anaerobic conditions, the protein precipitates.
Does anybody have an idea or experience with this type of results? or how to fix the molecule to such a site? What type of phenomena could occur at the active site preventing the binding of the product?

Thanks for your help

Best regards

Tatiana