and others..
Wow what a response, seems I started several discussions too.
Good to see the experts views, thanks.
I will try and respond individually but for now a bit background (many questions asking for details and suggesting similar things done already - glad to see I've been on the right track).
So project 'x' (teehee) has an active site with 2 metals, supposed to be di-zinc but XRF anaysis (and sample colour) suggest a mix... mostly iron and zinc. Seems the protein is quite promiscuous and scavanges any metal around during production.. Of cours ethat has opened adifferent debate with regards 'native' states.
Have managed to produce spliting rod/needles at neutral pH (I had the notion that lower pHs may be an issue, for a related protein I have structures from crystals grown at pHs 5-6.5 and as mentioned by Ronny at 5 metals were never there). Data colledctions have been done using a helical strategy with data quality lasting throughout collection (approx 2.3Å) . An XRF scan directly after the last point already demonstrated that metals have vanished.
One complication: crystals are often not simply split but are intergrown with the ends having diffeent cell parameters, but thats a different problem :0)
One suggestion that I had not thought of, and will... is move away from the absorption edge (thanks Klaus). Funny normaly one wants the anom signal and it sort of goes against the grain to collect where you essentially minimise it, but makes absolute sense in this case.
I will also try a much reduced dose regime.. get a modest (but complete) structure... qu. is hard and fast better than low dose and slow?
Anyway thaks for all the comments... nice to hear from names from my past too - hi, hope all is well.
:0)
D.
________________________________
From: Helland Ronny [[log in to unmask]]
Sent: 01 May 2014 23:57
To: Dean Derbyshire
Subject: RE: metals disapear
Hi Dean,
What is the metal and what is the pH where you crystallized your protein? We had a Mg binding protein (DNA binding protein) which crystallized both at at pH 5 and pH 7. At pH 7 the Mg was there, at pH 5 it was not. Although the conditions were slightly different, we suspected that the acidic groups binding the metal might be protonated at pH 5 thus reducing the affinity for the metal. We never tested this further so it is still only speculations.
Regards,
Ronny
*******************************************
Ronny Helland
Department of Chemistry, NorStruct
Research Park 3
Faculty of Science and Technology
UiT – The Arctic University of Norway
9037 Tromsø
Norway
Mail: [log in to unmask]<mailto:[log in to unmask]>
Phone: +47 77646474
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Dean Derbyshire
Sent: 30. april 2014 12:33
To: [log in to unmask]
Subject: [ccp4bb] metals disapear
Hi all,
Has anyone experienced catalytic metal ions disappearing during data collection ?
If so, is there a way of preventing it?
D.
Dean Derbyshire
Senior Research Scientist
[cid:image001.jpg@01CF6598.8312B770]
Box 1086
SE-141 22 Huddinge
SWEDEN
Visit: Lunastigen 7
Direct: +46 8 54683219
www.medivir.com<http://www.medivir.com>
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