[phenixbb] How to locate and refine a ligand using anomalousscattering

Schubert, Carsten [PRDUS] CSCHUBER at its.jnj.com
Wed Feb 9 05:09:36 PST 2011

Just a quick note from somebody who does this for a living....

-If the compound in question retains its bromine it will practically
scream at you when you look at the fofc maps. No need for elaborate
anomalous difference maps, unless you like to go through the motions,
which is commendable.

-Beware that if the part of the compound were the bromine is bound is
dangling, i.e. not well ordered, you will lose the anomalous signal, so
use common sense to decide if the compound is bound or not. 

-Always good to collect an intrinsic control to make sure that you have
the procedure under control or experiment with a high redundancy (>10x)
lysozyme dataset collected at home to get comfortable with the
procedure. The sulfurs in the lysozyme dataset light up in an anomalous
map, when the collection was done right.

-I usually only use anomalous maps in the context of sulfur containing
compounds, since it allows me to determine the orientation of thiazole
rings and such. Everything with more electrons is usually easily
detected in the fofc maps. 

-Make sure you collect a high redundancy dataset (> x6) on or a bit
beyond the Br edge. Don't overkill it, radiation damage is you enemy. 


Good luck.





From: phenixbb-bounces at phenix-online.org
[mailto:phenixbb-bounces at phenix-online.org] On Behalf Of Jason
Sent: Tuesday, February 08, 2011 1:14 PM
To: phenixbb at phenix-online.org
Subject: [phenixbb] How to locate and refine a ligand using


Hello everyone,


I have a few crystals to be x-rayed next week. Before that I hope to get
a clear idea about what I am doing (I am new to anomalous scattering). 



1.	The crystal is a protein co-crystallized with a ligand
2.	The protein structure is known.
3.	The ligand has a heavy atom bromide (absorption K-egde=13.47Kev)
4.	Data resolution is ~3 angstrom



1.	Locate the bromide position
2.	Locate and refine the ligand



1.	Do I need to carry out MAD experiment at 3 wavelengths or there
is some other easier way since the protein structure is known (I am not
expecting big change of the protein structure     itself)?
2.	Assuming I have the MAD data, what should I do next using phenix
to achieve the two goals listed above? Here are some thoughts:

	*	Using phenix.hyss to locate the anomalous scatterers 
	*	Using phenix.autobuild to build the protein model (which
data set to use?)
	*	Using coot to add ligands to the protein structure (Is
the relative position between the protein and the ligand known based on
phenix.hyss and phenix.autobuild?)
	*	Using phenix.refine to refine the ligand+protein complex


Thank you all for reading this.




Structural Biology Department

University of Pittsburgh



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