Dear all,

Sorry for my delayed reply.

Here are answers to all suggestions and clarification for all confusions.

1> Generate an anomalous map and look for peaks. Many metals would generate anomalous.

A: I have checked the anomalous map and found no signal for these 7 sites (I/sigma < 3 in contrast to 20-40 for Se sites).

2> What was certain beyond doubt is that it was somehow caused by the presence of cobalt ions. Do you, by chance, also have cobalt or similar ions present in your crystallization condition?

A: There was no cobalt during purification. Purification buffers contain only Tris, NaCl, Glycerol, BME/DTT (Ni-HiTrap -> GF). Crystallization condition contains only Tris, LiCl & PEG4K.

3> Do you have Molybdenum present in the system? Molybdenum can form Mo6 cluster.

A: Nope.

4> Probably a typical magnesium + 6 waters, also considering your 2Fo-Fc peak is the same height for the metal and waters.

A: …

5>By chance, P222 is your space group? It seems to be three perpendicular two-fold axes are passing through your central atom which makes them all fall in the same plane. From the density it looks more like a water in the center but anomalous density map should help.

A: Space group is P21 (49.9, 90.2, 72.4; 110.2).

6> Did you check the distance between the center one and others. If the distance is close to 2A rather than 2.8A, it is a metal in the middle. I had a Na in my case.

A: Distance between the central one and the surrounding 6 are all ~2.8A.

7>It may be three water related by non-crystallographic two fold symmetry.

Then the middle water is on the axis... If there is no residual density in Fo-Fc after putting water, then it is likely to be metal..

&

8> I would expect most metal ions to be in an octahedral, not a hexagonal, coordination environment. (Include protein ligands, e.g. carbonyl oxygens, etc.) In addition, if this density was due to transition metal ions, I would expect you would still have some positive difference density after modeling with waters. (Maybe not for Na or Mg.) Bond distances can also be a clue. Metal-water bonded distances will typically be quite a bit shorter (2.0-2.6 A depending on the metal ion) than water-water hydrogen bonding interactions (2.6-3.2 A or so).

A: Following shows both 2Fo-Fc and Fo-Fc at I/sigma of 2.5 (all 7 waters are placed). There is a ripple of positive densities (red arrows) around each of the two side waters (yellow circles). Could anyone explain what this means?

Thank you all!!!

Gang

From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Gang Dong
Sent: Monday, February 04, 2013 1:40 PM
To: [log in to unmask]
Subject: [ccp4bb] electron density assignment

Dear all,

Here are some “hexmeric” densities we observed in our 1.6-A resolution 2Fo-Fc map. They are located in between two dimers. Although 7 waters would fit nicely in the densities, we are not sure whether they might be something else (metals?). Any suggestions are welcome. Thanks! Gang

__________________________________

Gang Dong, PhD

Junior Group Leader

Max F. Perutz Laboratories (MFPL)

Dr. Bohrgasse 9/3

A-1030 Vienna, Austria

Phone: +43-1-4277-61625

FAX: +43-1-4277-9616

http://www.mfpl.ac.at/mfpl-group/group/dong.html