(I am new to ccp4bb and it is nice to read all those insightful discussions!)
Dr. Cusack, Regarding your problem, we have observed a similar phenomenon in the past with a protein expressed in E. coli and purified using Ni-NTA resin. We later discovered that the density in the structure was indeed a metal which was coordinated with histine from the recombinant protein. The metal was identified to be nickel that likely leached out from the NTA resin. This protein was not previously known to bind metal ions. We are now investigating this using other biophysical methods. To purify the protein, we used some resin in bulk that we loaded ourselves with nickel. This could be the source of metal ions. Hope this helps! Norm -- Normand Cyr, PhD Postdoctoral Fellow in Structural Biology Dept of Biochemistry and Molecular Medicine, Université de Montréal +1-514-343-6111 ext. 5154 https://www.normandcyr.com ________________________________________ De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Stephen Cusack [cus...@embl.fr] Envoyé : 9 octobre 2017 11:40 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] help identifying unknown density Dear All, I am refining the crystal structure of an E. coli expressed protein at 2.65 A resolution. The crystals grew in 0.1 M MES pH6, 0.7 M sodium formate pH 6. Ni-NTA was used in the purification. There are four molecules in the asymmetric unit. A particular histidine in each molecular has clear, strong extra density which looks like a metal bonded to an imidazole nitrogen (distance approx 2 A) with three other co-ordinating atoms in a triangular planar arrangement (see attached screen shot for unbiased extra density, atoms are only put in the density for illustrative purposes). Has anyone seen anything like this ? Any suggestions ? thanks very much Stephen -- ********************************************************************** Dr. Stephen Cusack, FRS Head of Grenoble Outstation of the European Molecular Biology Laboratory (EMBL) Group leader in structural biology of protein-RNA complexes and viral proteins ********************************************************************** Email: cus...@embl.fr Website: http://www.embl.fr Tel: (33) 4 76 20 7238 Secretary (33) 4 76 20 7123 Fax: (33) 4 76 20 7199 Postal address: EMBL Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, 71 Avenue des Martyrs, 38000 Grenoble, France **********************************************************************
