[phenixbb] Ca occupancy in multiple soaked structures {go slow}

Steven Herron sherron_AAC at yahoo.com
Wed May 22 10:50:24 PDT 2013

I did something similar back in 2003 [see: JBC 278(14):12271-12277 {2003})

The occupancy and the B-factors are going to affect each other in the 
refinement.  Refining both the occupancy and the B-factor parameters at 
the same time is what you want to avoid.  The refinement programs want 
to believe you know what you are doing (that you know the occupancy in 
this case).  Thus, you need to refine in cycles.  First refine the 
occupancy, then refine the B-factor, then the occupancy, and then the 
B-Factor of each individual Ca2+ atom.  Don't try to rush this by doing 
multiple atoms or automating the runs, you spent how many hours 
collecting all this data, go slow and really study each Calcium site.  
Once you think you have the right B-Factor and Occupancy value, try 
nudging one of the values up or down to see if you are in a local minima 
or if you really have the answer you are looking for.

Steven Herron
sherron_AAC at yahoo.com

On 5/22/2013 11:10 AM, Daniel Slade wrote:
> Hi Everyone,
> I have a complex case of data analysis I would love some opinions on.  I have a group of structures of a protein that binds 6 calciums, and we want to know the order/concentration at which they fill.  The crystals were soaked with calcium from 0-10 mM and all are 1.6-2.1 A in resolution.  I determine the order of the calcium binding, and now I am working on the percent occupancy at each concentration.  To do this, I have removed the calciums, and then performed simulated annealing for each structure from 2.1-30A resolution to 'normalize' them.  I then look at the omit map and scroll out to find the peak sigma intensity of where the calciums should be.
> The curves fit very well for each calcium, and the numbers correspond well with other solution studies we have. My thought is that I need to normalize this in another way, so I thought choosing a water molecule that is present in all of the structures and removing it, then performing simulated annealing would be a good idea to normalize the peak intensity for each calcium soak concentration, since this water molecule shouldn't change occupancy.  Is this the proper way to do this, or can you guys suggest a better way to go about this.  I also looked at B factor and e/A3, but that isn't as consistent as peak sigma.
> Thanks in advance.
> Dan
> Daniel J. Slade, Ph.D.
> Research Associate
> Thompson Lab
> The Scripps Research Institute
> 130 Scripps Way
> Jupiter, FL 33458
> (561) 228-3213
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