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I presume you refine non-H and non-water atoms as isotropic and the rest as anisotropic.

Acutually I refined everything anisotropic. Previously I refined waters isotropically but my Rwork and Rfree increased. Is this a bad idea? BTW Thanks for the tip on iding clashes with the phenix GUI.)

Actually, at 1.37A resolution it might be a valid thing to do. Pavel.

I just calculated unique reflection/parameter ratio including waters = 9.11 329525/36160 so I think I am safe. Thanks for all of your help.

It doesn't make much sense since you did not account for the restrains, which count as "observations". So I would waste time on this -:) At 1.37A you are safe to do the refinement strategy you mentioned. Pavel.

On Thu, Aug 19, 2010 at 10:16 AM, wrote:

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I know that the defualt is "riding-model" so that the hydrogen atoms are not refined. However, I thought that the program would be able to avoid steric clashes. Is there an aditional command I need to turn on to account for this? I am currently fixing every hydrogen that clashes on an individual basis in coot. There are also a handfull of hydrogens that exist out in space that I decided to delete.

I'm a little confused by this: are you saying that refining with hydrogens actually added clashes that weren't there before? To clarify, when you calculate clashes, either in Phenix, Coot, or the Molprobity server, this is *always* done with explicit hydrogens, which is the entire point of the exercise. So if adding hydrogens is making the clashes worse, there's something weird going on.*

No, I didn't previously have hydgrogens. This is the first time I looked at close contacts with hydrogens present. I submitted my pdb to the the PDB auto Deposition input tool which defines "close contacts" as less then 2.2 A After looking at these I could see that many were between hydroens of one side chain and usually an amid or sometimes CA of the mainchain. Sometimes they appeard hydrogen bonded but the hydrogens had to be rotated away. All I had to do was rotate the hydrogens. I was just suprised that this would not have been done in automatically in the refinement.

(* The one exception is that Reduce doesn't always take heteroatoms

into account when adding hydrogens, so it's possible, albeit unusual, for hydrogens to run into, say, an iron sulfur cluster because they weren't positioned well.)

-Nat _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

On Thu, Aug 19, 2010 at 3:46 PM, wrote:

No, I didn't previously have hydgrogens.  This is the first time I looked at close contacts with hydrogens present.  I submitted my pdb to the the PDB auto Deposition input tool which defines "close contacts" as less then 2.2 A After looking at these I could see that many were between hydroens of one side chain and usually an amid or sometimes CA of the mainchain. Sometimes they appeard hydrogen bonded but the hydrogens had to be rotated away.  All I had to do was rotate the hydrogens.  I was just suprised that this would not have been done in automatically in the refinement.

If you didn't have explicit hydrogens, there's no way for phenix.refine (or any other refinement program) to tell that there's a clash. Minimization algorithms are "dumb", in the sense that they know nothing about chemistry, only restraints, and rotating around a bond often requires passing over large energy barriers (a major reason why simulated annealing is used). Recent versions of phenix.refine can optionally re-fit sidechain rotamers and flip N/Q/H residues to minimize bad contacts, and more features like this will be added over time, but it's tricky. It's very suspicious that hydrogens are bumping into the C-alpha if there is space for them elsewhere; the tools in Phenix should almost never do this unless there is something else wrong with the model. But it's difficult to guess without seeing the structure. -Nat

Hi Yarrow, I could comment on this more specifically if you send me (to my email address, not to the whole phenixbb) the data and model files, and the exact phenix.refine command that you used to obtain your final model that has clashes you are mentioning. Also, it will save me some time if you outline the residues in question. Pavel. On 8/20/10 10:20 AM, [email protected] wrote:

On Thu, Aug 19, 2010 at 3:46 PM, wrote:

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No, I didn't previously have hydgrogens. This is the first time I looked at close contacts with hydrogens present. I submitted my pdb to the the PDB auto Deposition input tool which defines "close contacts" as less then 2.2 A After looking at these I could see that many were between hydroens of one side chain and usually an amid or sometimes CA of the mainchain. Sometimes they appeard hydrogen bonded but the hydrogens had to be rotated away. All I had to do was rotate the hydrogens. I was just suprised that this would not have been done in automatically in the refinement. If you didn't have explicit hydrogens, there's no way for phenix.refine (or any other refinement program) to tell that there's a clash. Minimization algorithms are "dumb", in the sense that they know nothing about chemistry, only restraints, and rotating around a bond often requires passing over large energy barriers (a major reason why simulated annealing is used). Recent versions of phenix.refine can optionally re-fit sidechain rotamers and flip N/Q/H residues to minimize bad contacts, and more features like this will be added over time, but it's tricky.

It's very suspicious that hydrogens are bumping into the C-alpha if there is space for them elsewhere; the tools in Phenix should almost never do this unless there is something else wrong with the model. But it's difficult to guess without seeing the structure.

-Nat I think I may have over-reacted (this being my first structure and all)

By "clash" I meant contacts closer then 2.2A (most 1-1.5A). There are only about 28 of these involving side chain Hydrogens out of over 1600 residues. Only one involves the CA. The rest are basically hydrogen bonds where the hydrogens should be rotated away from the heroatom. Most of the other 50 involved contacts with misplaced waters I still need to clean-up. I think this may be normal. However, I still can't acount for the four residues that had hydrogens off in space. Thanks for all your help.

-Yarrow

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