Re: [ccp4bb] Deposition of riding H

[Ethan Merritt]

On Saturday, 12 May 2012, Yuri Pompeu wrote:
 If you used riding hydrogens throughout refinement and arrived at a final 
 model that you believe best describes your x-ray data to a certain level of 
 accuracy (Rvalues, geometry, map CC, etc...) would you not be invalidating 
 the whole refinement process by going in and removing the hydrogen atoms 
 right before deposition?

My view:

You are not removing hydrogen atoms at all. You are stating that the model
being deposited includes riding hydrodens.  The consumer of your model can
regenerate the individual hydrogen coordinates from that information if
needed, just as refmac does when you start a new refinement cycle with the
riding hydrogen model selected.  You don't need to output the individual 
hydrogen coordinates between cycles, or at deposition time, because they
are adequately described by the riding hydrogen model.

You might as well ask why do we remove all copies of the molecules in
the crystal except for those in a single asymmetric unit?
They are not really removed; they are implicit in the statement of
the crystallographic symmetry.

Ethan

Re: [ccp4bb] question on metal refinement in a protein structure

[David Roberts]

Thanks for the replies.  Yuri, yes, those were my exact concerns.  This 
is a molecular replacement structure, but that was done with an apo 
structure, so I am certain the metal is there.  My resolution is around 
2.3 angstroms, which I think is OK for determining geometry.


I have done omit maps as well as simply several rounds of refinement 
with no metal present.  It always comes back, and has a certain shape 
(tetrahedral).


I just was concerned that I was forcing a geometry by not specifying 
nickel properly in my pdb file.  I didn't know right off how one might 
specify different oxidation states in a pdb file (say Nickel I vs Nickel 
II; that is not what I am trying to do, but it is something I'd be 
curious about).  I haven't worked with a lot of metalloenzymes, so I 
just wanted to be sure.  I think you are right, and I am doing things 
properly (the programs are of course very good).


Thanks again

Dave


On 5/12/2012 2:49 PM, Yuri Pompeu wrote:

Hi Dave,
I sounds to me like you are worried about 2 separate things here.
A: Am I affecting the geometry of the coordination sites with a  restraint file 
that is innacurate?
B:Are my electron density maps biased, and what I am seeing is not really there?

AFAIU, if you have a restraint file that is innacurate, lets say it is defining the 
metal/ ligand angles to be those of a tetrahedron, that would influence the position of 
your atoms after refinement as the program will try to obey the restraint 
file.
The electron density maps, however do not directly take into account your 
restraints file.
With that being said, model bias can be a problem, yes. This is dependent on 
many factors, and if you have obtained your phases through a molecular 
replacement solution rather than experimentally (MAD, SAD, etc..) your maps 
will be particularly susceptible to bias. And if your dealing with a low 
resolution data set this can become even more of a problem (you dont mention 
your resolution). If you are working with a 1.2A data set, I would not lose 
sleep over it.  People have spent many hours of thinking and programming to 
develop ways of eliminating model bias and many programs can calculate electron 
density maps in a way that your bias is minimized. Always check your difference 
map (mFo-Dfc), you can calculate omit maps, averaged kick maps, and in the case 
of metals even an anomalous maps sometimes. All of these would help you put 
your mind at ease.
Hope this helps