Dear Markus, I believe the strong assumption in the community is that a clear single peak of appropriate Mw is a clear indication of pure protein, worthy intensive crystallization efforts. Whether it is active is another question and this should be measured.
For your analysis, it is not important in which buffers the protein is not active, but whether the protein you purified is active in the buffer (maybe without precipitant) you used for crystallization. A single apo structure is usually not enough to determine the catalytic mechanism of an enzyme, you usually need some substrate-, transition state- product- (analog) structures as well. If your protein is active in the crystallization buffer and the ligand complexes make chemical sense, you can be pretty sure that you have crystallized the right conformation. If your protein is not active in the crystallization buffer, you must critically analyze the structure, if it makes chemical sense and if you can explain the absence of activity (e.g. pH far from optimum; inhibitor bound in the active site). I am currently working on an enzyme who's active site loves all kinds of substituted and unsubstituted phosphates, sulfates etc. so it is not active in a wide range of buffers like phosphate, MES, MOPS, HEPES etc. However, the crystal structures still represent the active conformation, the active site is just blocked by some buffer component. Other proteins (proteases) can only be crystallized in an inactive form, since active they chew themselves to pieces. Hopes this helps, Herman -----Ursprüngliche Nachricht----- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Markus Heckmann Gesendet: Donnerstag, 11. Oktober 2018 12:59 An: CCP4BB@JISCMAIL.AC.UK Betreff: [EXTERNAL] [ccp4bb] Assumptions on protein purification 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://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwIFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=SbuuxlB2mE9bVksAMl3Nzwk-usfxWFkrtiQ1zLhkP4U&s=riaEgq4_yVcB9guEOuuXXoPNkfIwfub2SQwhBfNfZto&e= ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
