Dear all, Not directly a ccp4 question. I am working on a multi-domain protein with multiple catalytic centres. Purifying in gel-filtration (Äkta) with different buffers gives a clear distinct peak indicating pure protein. We could even crystallise it and determine 3D structure to about 2.5 A.
Why is there a strong assumption in the community (or at least in my limited experience) that a clear single peak of appropriate Mw is indication of *active and folded* protein that upon crystallisation/structure determination can describe the working of the enzyme? When I tested the activity of the protein using assays, I found 3 out of 4 buffers give very poor product turnover? Could we discount the possibility that some 3D-structures in PDB are inactive (differently folded) and hence may not represent active state? Are there any best practices? Or is this dependent on the protein, especially multi-domain proteins show such weird behaviour? Thanks for your comments, Markus ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
