Dear all,

 

We are currently working on a small GTPase. The structure has been solved to 1.4 A with two molecules in the ASU. In the difference electron density we can clearly see difference density (in one monomer) attached to a Cys residue.

 

The protein has been expressed in E. coli. For crystallization experiments the GTPase was incubated with GMPPNP, that had been dissolved in 20 mM HEPES pH 7.5 and 50 mM NaCl. Prior to crystallization

the protein was stored in a buffer composed of 20 mM HEPES pH 7.5, 200 mM NaCl, 5 mM, Mg acetate, 2 mM DTT and 2% (v/v) glycerol.

 

The protein crystallized under the following conditions:

28% (v/v) PEG 200, 5% (w/v) PEG 3000 and 100 mM MES buffer at pH 6.0

 

The anomalous signal is too weak to judge the positions of the sulphur atoms. We have performed MS analysis on the protein before crystallization and on dissolved protein crystals. MS revealed a mass difference of about 135 Da, indicating that some chemistry must have went on in the crystallization drop.

 

The extra electron density has a planar shape and is quite symmetric. We have placed some dummy water molecules in the density. Distances are given in A in the PNG file.

 

Attached files

 

coot1.png: 2FoFc electron density map (blue) @ sigma=1 and FoFc electron density map (blue) @ sigma=+3 after phenix.refine

coot2.png: same electron densities as in coot1.png, with dummy atoms placed

coot3.png: same electron densities as in coot1.png, side view

 

Thanks for your time and efforts.

 

Cheers,

 

Bernhard

-- 
Dr. Bernhard Loll
Freie Universitaet Berlin
Fachbereich Biologie, Chemie, Pharmazie
Institut fuer Chemie und Biochemie
AG Strukturbiochemie
Takustr. 6
D-14195 Berlin
Germany

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