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.

Carsten

From: [email protected] [mailto:[email protected]] On Behalf Of Jason
Sent: Tuesday, February 08, 2011 1:14 PM
To: [email protected]
Subject: [phenixbb] How to locate and refine a ligand using anomalousscattering

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). 

Facts:

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

Goals:

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

Questions:

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.

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Jason

Structural Biology Department

University of Pittsburgh

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