[phenixbb] helix/sheet outliers

[Oleg Sobolev]

Hi Casper,


> I am doing real space refinement on a protein model build from a cryo-em
> map. I run the refinement with Amber gradients turned on and with secondary
> structure restraints (otherwise, more or less default settings).
>
Amber gradients should not be relevant to the issue. SS filtration is based
on input model geometry before any coordinate refinement was done.


> I have some issues with helix and sheet outliers.
>
> Every time I run the refinement (after making sure that the bond distance
> in the helices are within the 3.5 Å)
>
I get a lot of bad annotation remarks, but with outliers just above 3.5 Å
> (see .log below):
>
>
>
> …
>
> removed outlier: 3.576A  pdb=" N   VAL A 179 " --> pdb=" O   LEU A 175 "
> (cutoff:3.500A)
>
>       Proline residue:  A 180  - end of helix
>
>       removed outlier: 3.681A  pdb=" N   PHE A 188 " --> pdb=" O   LEU A
> 184 " (cutoff:3.500A)
>
>       removed outlier: 3.628A  pdb=" N   PHE A 189 " --> pdb=" O   ILE A
> 185 " (cutoff:3.500A)
>
>       removed outlier: 3.521A  pdb=" N   ILE A 190 " --> pdb=" O   ALA A
> 186 " (cutoff:3.500A)
>
>     Processing helix  chain 'A' and resid 219 through 228
>
>       removed outlier: 3.737A  pdb=" N   MET A 227 " --> pdb=" O   GLU A
> 223 " (cutoff:3.500A)
>
> …
>
>
>
> Is this something that I just need to fix in several rounds of refinement
> or is there an explanation?
>
If you believe the distances you see in the log are wrong, I'm happy to
investigate. Please send me the model file (off-list) with an explanation
why do you think so. The distances (even 3.5) suggest a rather poor helix
geometry since the target distance is 2.9A. If you are sure your
annotations are correct, you may try to relax the threshold by setting
parameter "distance_cut_n_o" to some larger value than default 3.5. Then
the restraints will be imposed and hopefully the geometry of the helix will
become good during refinement.


> In addition, I have some bad sheet annotations such as:
>
>
>
> …
>
> removed outlier: 6.987A  pdb=" N   VAL B 433 " --> pdb=" O   LEU B 440 "
> (cutoff:3.500A)
>
>       removed outlier: 4.659A  pdb=" N   VAL B 442 " --> pdb=" O   LEU B
> 431 " (cutoff:3.500A)
>
>       removed outlier: 6.296A  pdb=" N   LEU B 431 " --> pdb=" O   VAL B
> 442 " (cutoff:3.500A)
>
>       removed outlier: 5.062A  pdb=" N   ALA B 444 " --> pdb=" O   THR B
> 429 " (cutoff:3.500A)
>
>       removed outlier: 6.129A  pdb=" N   THR B 429 " --> pdb=" O   ALA B
> 444 " (cutoff:3.500A)
>
> …
>
>
>
> When I look at the model, the residues are definitely a part of the sheet
> – the long distance between the N-O atoms is due to the O being “flipped”
> (hope it makes sense).
>
> Is this because of wrong annotation?
>
If you believe the residues are part of the sheet - then the annotation is
correct, the model is wrong. If you believe the O needs to be "flipped" -
go ahead and flip it. Do any other adjustments you deem necessary to
improve the model and hydrogen-bonding pattern. This may result in proper
distance between N and O and established restraint. Such big model changes
are better done manually rather than during refinement with restraints.

Some relevant tutorials are:
https://www.youtube.com/watch?v=9dCkAdR1RDk&feature=youtu.be
https://www.youtube.com/watch?v=qB8W_6yuw5k&feature=youtu.be

Relevant documentation and links in it:
https://www.phenix-online.org/documentation/reference/secondary_structure.html

Please let us know if you have more questions.

Best regards,
Oleg Sobolev.


>
>
>
>
> Best
>
> Casper
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