Incorrect secondary structure assignment in a .pdb

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Maciej Jagielnicki

28 Jun 2018 28 Jun '18

4:23 p.m.

Hi All, I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next. When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants... Regards, Maciej J. -- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

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Pavel Afonine

28 Jun 28 Jun

4:38 p.m.

Hi Maciej, please have a look at item #10 and Notes section here: http://www.phenix-online.org/documentation/reference/real_space_refine.html One sentence summary is: Make sure you use secondary structure (SS) restraints in refinement, also making sure you provide SS annotations as HELIX/SHEET records in input PDB that are as accurate as possible; this is critical for successful refinement. If you have more questions please get back to us! Good luck! Pavel On 6/28/18 09:23, Maciej Jagielnicki wrote:

...

Hi All,

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next.

When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants...

Regards, Maciej J.

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

Oleg Sobolev

5:36 p.m.

Hi Maciej, I would add that you can obtain recreated SS annotations for your model using phenix.secondary_structure_restraints <your model> ignore_annotation_in_file=True format=pdb More on this: https://www.phenix-online.org/documentation/reference/secondary_structure_re... Best regards, Oleg Sobolev. On Thu, Jun 28, 2018 at 9:38 AM, Pavel Afonine wrote:

...

Hi Maciej,

please have a look at item #10 and Notes section here:

http://www.phenix-online.org/documentation/reference/real_ space_refine.html

One sentence summary is: Make sure you use secondary structure (SS) restraints in refinement, also making sure you provide SS annotations as HELIX/SHEET records in input PDB that are as accurate as possible; this is critical for successful refinement.

If you have more questions please get back to us!

Good luck! Pavel

On 6/28/18 09:23, Maciej Jagielnicki wrote:

Hi All,

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next.

When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants...

Regards, Maciej J.

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

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

Maciej Jagielnicki

6:02 p.m.

Thank you for the suggestion. Nothing changes when I run phenix real space refinement with SS restraints turned on. I can either input a .pdb file with correct SS information in the header or a .pdb file with no SS information at all and the end result (new refined .pdb) is the same file with the same SS information in a new header. The information stays the same and 'correct'. The main problem in my original question is that when I (or the pdb extract tool that converts a .pdb file to a .cif file for submission to PDB) remove the header SS information altogether (why you would do that for PDB deposition is unknown to me), that resulting .pdb file still shows SS elements in different pdb viewers but SS info is no longer correct. How can you have SS elements displayed if it description is missing from the header? Maciej On Thu, Jun 28, 2018 at 12:38 PM, Pavel Afonine wrote:

...

Hi Maciej,

please have a look at item #10 and Notes section here:

http://www.phenix-online.org/documentation/reference/real_ space_refine.html

One sentence summary is: Make sure you use secondary structure (SS) restraints in refinement, also making sure you provide SS annotations as HELIX/SHEET records in input PDB that are as accurate as possible; this is critical for successful refinement.

If you have more questions please get back to us!

Good luck! Pavel

On 6/28/18 09:23, Maciej Jagielnicki wrote:

Hi All,

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next.

When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants...

Regards, Maciej J.

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

Ricardo Righetto

8:10 p.m.

Hi Maciej, If the PDB file has no secondary structure assignments defined in the header and you open it in Chimera, it automatically runs ksdssp in the background to assign helices and sheets, which may or may not be correct and might make you think that you have secondary structure records in the header when you actually have nothing: https://plato.cgl.ucsf.edu/chimera/docs/UsersGuide/midas/ksdssp.html If in turn you save the PDB from Chimera with such automatically generated assignments, the records corresponding to this assignment will be written to the header. And if it's written in the header, by default phenix.real_space_refine will use these restraints. Bottom line is that throughout modelling and refinement you should always inspect your output PDB file from whatever program you use (PHENIX, Coot, Chimera, etc) to make sure that the information you think should be in the header is indeed there. I agree that some programs might unnecessarily "get rid" of the secondary structure records in the header and this is a bit annoying. Hope this helps, -- Ricardo Diogo Righetto 2018-06-28 20:02 GMT+02:00 Maciej Jagielnicki :

...

Thank you for the suggestion.

Nothing changes when I run phenix real space refinement with SS restraints turned on. I can either input a .pdb file with correct SS information in the header or a .pdb file with no SS information at all and the end result (new refined .pdb) is the same file with the same SS information in a new header. The information stays the same and 'correct'.

The main problem in my original question is that when I (or the pdb extract tool that converts a .pdb file to a .cif file for submission to PDB) remove the header SS information altogether (why you would do that for PDB deposition is unknown to me), that resulting .pdb file still shows SS elements in different pdb viewers but SS info is no longer correct. How can you have SS elements displayed if it description is missing from the header? Maciej

On Thu, Jun 28, 2018 at 12:38 PM, Pavel Afonine wrote:

...
Hi Maciej,

please have a look at item #10 and Notes section here:

http://www.phenix-online.org/documentation/reference/real_sp ace_refine.html

One sentence summary is: Make sure you use secondary structure (SS) restraints in refinement, also making sure you provide SS annotations as HELIX/SHEET records in input PDB that are as accurate as possible; this is critical for successful refinement.

If you have more questions please get back to us!

Good luck! Pavel

On 6/28/18 09:23, Maciej Jagielnicki wrote:

Hi All,

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next.

When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants...

Regards, Maciej J.

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

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

Pavel Afonine

8:16 p.m.

The point is that no automated secondary structure (SS) annotation software can assign SS fully correctly simply because it relies on input model geometry, and models that are subject to refinement are rarely perfect for these software to do a great job. This is why the Manual (see previous email) recommends getting initial automated assignment first using phenix.secondary_structure_restraints (which uses various annotation methods, including ksdssp) and then manually checking and editing it if necessary. Tools within Phenix always preserve HELIX/SHEET records in PDB files. Let us know if you have an example where this is not the case. Pavel On 6/28/18 13:10, Ricardo Righetto wrote:

...

Hi Maciej,

If the PDB file has no secondary structure assignments defined in the header and you open it in Chimera, it automatically runs ksdssp in the background to assign helices and sheets, which may or may not be correct and might make you think that you have secondary structure records in the header when you actually have nothing: https://plato.cgl.ucsf.edu/chimera/docs/UsersGuide/midas/ksdssp.html

If in turn you save the PDB from Chimera with such automatically generated assignments, the records corresponding to this assignment will be written to the header. And if it's written in the header, by default phenix.real_space_refine will use these restraints.

Bottom line is that throughout modelling and refinement you should always inspect your output PDB file from whatever program you use (PHENIX, Coot, Chimera, etc) to make sure that the information you think should be in the header is indeed there. I agree that some programs might unnecessarily "get rid" of the secondary structure records in the header and this is a bit annoying.

Hope this helps,

-- Ricardo Diogo Righetto

2018-06-28 20:02 GMT+02:00 Maciej Jagielnicki mailto:[email protected]>:

Thank you for the suggestion.

Nothing changes when I run phenix real space refinement with SS restraints turned on. I can either input a .pdb file with correct SS information in the header or a .pdb file with no SS information at all and the end result (new refined .pdb) is the same file with the same SS information in a new header. The information stays the same and 'correct'.

The main problem in my original question is that when I (or the pdb extract tool that converts a .pdb file to a .cif file for submission to PDB) remove the header SS information altogether (why you would do that for PDB deposition is unknown to me), that resulting .pdb file still shows SS elements in different pdb viewers but SS info is no longer correct. How can you have SS elements displayed if it description is missing from the header? Maciej

On Thu, Jun 28, 2018 at 12:38 PM, Pavel Afonine mailto:[email protected]> wrote:

Hi Maciej,

please have a look at item #10 and Notes section here:

http://www.phenix-online.org/documentation/reference/real_space_refine.html http://www.phenix-online.org/documentation/reference/real_space_refine.html

One sentence summary is: Make sure you use secondary structure (SS) restraints in refinement, also making sure you provide SS annotations as HELIX/SHEET records in input PDB that are as accurate as possible; this is critical for successful refinement.

If you have more questions please get back to us!

Good luck! Pavel

On 6/28/18 09:23, Maciej Jagielnicki wrote:

...
Hi All,

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next.

When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants...

Regards, Maciej J.

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

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

Maciej Jagielnicki

8:26 p.m.

Thank you for clarification Ricardo and Pavel. It still amazes me though that different pdb viewers would make such ridiculous SS predictions/assignments at times for a file that does not come with that information in the header. And it seems that during a deposition to PDB I have to manually add the location of helices/sheets into the .cif files generated by the deposition system...otherwise I go for deposition somewhat blind (this has of course nothing to do with Phenix, but adds more to my confusion). Maciej On Thu, Jun 28, 2018 at 4:16 PM, Pavel Afonine wrote:

...

The point is that no automated secondary structure (SS) annotation software can assign SS fully correctly simply because it relies on input model geometry, and models that are subject to refinement are rarely perfect for these software to do a great job. This is why the Manual (see previous email) recommends getting initial automated assignment first using phenix.secondary_structure_restraints (which uses various annotation methods, including ksdssp) and then manually checking and editing it if necessary.

Tools within Phenix always preserve HELIX/SHEET records in PDB files. Let us know if you have an example where this is not the case.

Pavel

On 6/28/18 13:10, Ricardo Righetto wrote:

Hi Maciej,

If the PDB file has no secondary structure assignments defined in the header and you open it in Chimera, it automatically runs ksdssp in the background to assign helices and sheets, which may or may not be correct and might make you think that you have secondary structure records in the header when you actually have nothing: https://plato.cgl.ucsf.edu/chimera/docs/UsersGuide/midas/ksdssp.html

If in turn you save the PDB from Chimera with such automatically generated assignments, the records corresponding to this assignment will be written to the header. And if it's written in the header, by default phenix.real_space_refine will use these restraints.

Bottom line is that throughout modelling and refinement you should always inspect your output PDB file from whatever program you use (PHENIX, Coot, Chimera, etc) to make sure that the information you think should be in the header is indeed there. I agree that some programs might unnecessarily "get rid" of the secondary structure records in the header and this is a bit annoying.

Hope this helps,

-- Ricardo Diogo Righetto

2018-06-28 20:02 GMT+02:00 Maciej Jagielnicki :

...
Thank you for the suggestion.

Nothing changes when I run phenix real space refinement with SS restraints turned on. I can either input a .pdb file with correct SS information in the header or a .pdb file with no SS information at all and the end result (new refined .pdb) is the same file with the same SS information in a new header. The information stays the same and 'correct'.

The main problem in my original question is that when I (or the pdb extract tool that converts a .pdb file to a .cif file for submission to PDB) remove the header SS information altogether (why you would do that for PDB deposition is unknown to me), that resulting .pdb file still shows SS elements in different pdb viewers but SS info is no longer correct. How can you have SS elements displayed if it description is missing from the header? Maciej

On Thu, Jun 28, 2018 at 12:38 PM, Pavel Afonine wrote:

...
Hi Maciej,

please have a look at item #10 and Notes section here:

http://www.phenix-online.org/documentation/reference/real_sp ace_refine.html

One sentence summary is: Make sure you use secondary structure (SS) restraints in refinement, also making sure you provide SS annotations as HELIX/SHEET records in input PDB that are as accurate as possible; this is critical for successful refinement.

If you have more questions please get back to us!

Good luck! Pavel

On 6/28/18 09:23, Maciej Jagielnicki wrote:

Hi All,

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next.

When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants...

Regards, Maciej J.

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

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

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

Ricardo Righetto

8:43 p.m.

Dear Pavel, An example of Phenix tool that does not preserve HELIX/SHEET records is phenix.ready_set (I have version 1.13-2998). Is this a bug or normal behavior? (apologies for getting slightly off topic) And thanks for clarifying about the behavior of secondary structure restraints in phenix.real_space_refine! Best wishes, -- Ricardo Diogo Righetto 2018-06-28 22:26 GMT+02:00 Maciej Jagielnicki :

...

Thank you for clarification Ricardo and Pavel.

It still amazes me though that different pdb viewers would make such ridiculous SS predictions/assignments at times for a file that does not come with that information in the header. And it seems that during a deposition to PDB I have to manually add the location of helices/sheets into the .cif files generated by the deposition system...otherwise I go for deposition somewhat blind (this has of course nothing to do with Phenix, but adds more to my confusion).

Maciej

On Thu, Jun 28, 2018 at 4:16 PM, Pavel Afonine wrote:

...
The point is that no automated secondary structure (SS) annotation software can assign SS fully correctly simply because it relies on input model geometry, and models that are subject to refinement are rarely perfect for these software to do a great job. This is why the Manual (see previous email) recommends getting initial automated assignment first using phenix.secondary_structure_restraints (which uses various annotation methods, including ksdssp) and then manually checking and editing it if necessary.

Tools within Phenix always preserve HELIX/SHEET records in PDB files. Let us know if you have an example where this is not the case.

Pavel

On 6/28/18 13:10, Ricardo Righetto wrote:

Hi Maciej,

If the PDB file has no secondary structure assignments defined in the header and you open it in Chimera, it automatically runs ksdssp in the background to assign helices and sheets, which may or may not be correct and might make you think that you have secondary structure records in the header when you actually have nothing: https://plato.cgl.ucsf.edu/chimera/docs/UsersGuide/midas/ksdssp.html

If in turn you save the PDB from Chimera with such automatically generated assignments, the records corresponding to this assignment will be written to the header. And if it's written in the header, by default phenix.real_space_refine will use these restraints.

Bottom line is that throughout modelling and refinement you should always inspect your output PDB file from whatever program you use (PHENIX, Coot, Chimera, etc) to make sure that the information you think should be in the header is indeed there. I agree that some programs might unnecessarily "get rid" of the secondary structure records in the header and this is a bit annoying.

Hope this helps,

-- Ricardo Diogo Righetto

2018-06-28 20:02 GMT+02:00 Maciej Jagielnicki :

...
Thank you for the suggestion.

Nothing changes when I run phenix real space refinement with SS restraints turned on. I can either input a .pdb file with correct SS information in the header or a .pdb file with no SS information at all and the end result (new refined .pdb) is the same file with the same SS information in a new header. The information stays the same and 'correct'.

The main problem in my original question is that when I (or the pdb extract tool that converts a .pdb file to a .cif file for submission to PDB) remove the header SS information altogether (why you would do that for PDB deposition is unknown to me), that resulting .pdb file still shows SS elements in different pdb viewers but SS info is no longer correct. How can you have SS elements displayed if it description is missing from the header? Maciej

On Thu, Jun 28, 2018 at 12:38 PM, Pavel Afonine wrote:

...
Hi Maciej,

please have a look at item #10 and Notes section here:

http://www.phenix-online.org/documentation/reference/real_sp ace_refine.html

One sentence summary is: Make sure you use secondary structure (SS) restraints in refinement, also making sure you provide SS annotations as HELIX/SHEET records in input PDB that are as accurate as possible; this is critical for successful refinement.

If you have more questions please get back to us!

Good luck! Pavel

On 6/28/18 09:23, Maciej Jagielnicki wrote:

Hi All,

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next.

When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants...

Regards, Maciej J.

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

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

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 https://maps.google.com/?q=480+Ray+C+Hunt+Drive+Rm+320+Charlottesville,+VA&entry=gmail&source=g Charlottesville, VA https://maps.google.com/?q=480+Ray+C+Hunt+Drive+Rm+320+Charlottesville,+VA&entry=gmail&source=g Lab Phone: 434-243-2520

Pavel Afonine

8:49 p.m.

It is a bug. Thanks for letting us know! Pavel On 6/28/18 13:43, Ricardo Righetto wrote:

...

Dear Pavel,

An example of Phenix tool that does not preserve HELIX/SHEET records is phenix.ready_set (I have version 1.13-2998). Is this a bug or normal behavior? (apologies for getting slightly off topic)

And thanks for clarifying about the behavior of secondary structure restraints in phenix.real_space_refine!

Best wishes,

-- Ricardo Diogo Righetto

2018-06-28 22:26 GMT+02:00 Maciej Jagielnicki mailto:[email protected]>:

Thank you for clarification Ricardo and Pavel.

It still amazes me though that different pdb viewers would make such ridiculous SS predictions/assignments at times for a file that does not come with that information in the header. And it seems that during a deposition to PDB I have to manually add the location of helices/sheets into the .cif files generated by the deposition system...otherwise I go for deposition somewhat blind (this has of course nothing to do with Phenix, but adds more to my confusion).

Maciej

On Thu, Jun 28, 2018 at 4:16 PM, Pavel Afonine mailto:[email protected]> wrote:

The point is that no automated secondary structure (SS) annotation software can assign SS fully correctly simply because it relies on input model geometry, and models that are subject to refinement are rarely perfect for these software to do a great job. This is why the Manual (see previous email) recommends getting initial automated assignment first using phenix.secondary_structure_restraints (which uses various annotation methods, including ksdssp) and then manually checking and editing it if necessary.

Tools within Phenix always preserve HELIX/SHEET records in PDB files. Let us know if you have an example where this is not the case.

Pavel

On 6/28/18 13:10, Ricardo Righetto wrote:

...
Hi Maciej,

If the PDB file has no secondary structure assignments defined in the header and you open it in Chimera, it automatically runs ksdssp in the background to assign helices and sheets, which may or may not be correct and might make you think that you have secondary structure records in the header when you actually have nothing: https://plato.cgl.ucsf.edu/chimera/docs/UsersGuide/midas/ksdssp.html https://plato.cgl.ucsf.edu/chimera/docs/UsersGuide/midas/ksdssp.html

If in turn you save the PDB from Chimera with such automatically generated assignments, the records corresponding to this assignment will be written to the header. And if it's written in the header, by default phenix.real_space_refine will use these restraints.

Bottom line is that throughout modelling and refinement you should always inspect your output PDB file from whatever program you use (PHENIX, Coot, Chimera, etc) to make sure that the information you think should be in the header is indeed there. I agree that some programs might unnecessarily "get rid" of the secondary structure records in the header and this is a bit annoying.

Hope this helps,

-- Ricardo Diogo Righetto

2018-06-28 20:02 GMT+02:00 Maciej Jagielnicki mailto:[email protected]>:

Thank you for the suggestion.

Nothing changes when I run phenix real space refinement with SS restraints turned on. I can either input a .pdb file with correct SS information in the header or a .pdb file with no SS information at all and the end result (new refined .pdb) is the same file with the same SS information in a new header. The information stays the same and 'correct'.

The main problem in my original question is that when I (or the pdb extract tool that converts a .pdb file to a .cif file for submission to PDB) remove the header SS information altogether (why you would do that for PDB deposition is unknown to me), that resulting .pdb file still shows SS elements in different pdb viewers but SS info is no longer correct. How can you have SS elements displayed if it description is missing from the header? Maciej

On Thu, Jun 28, 2018 at 12:38 PM, Pavel Afonine mailto:[email protected]> wrote:

Hi Maciej,

please have a look at item #10 and Notes section here:

http://www.phenix-online.org/documentation/reference/real_space_refine.html http://www.phenix-online.org/documentation/reference/real_space_refine.html

One sentence summary is: Make sure you use secondary structure (SS) restraints in refinement, also making sure you provide SS annotations as HELIX/SHEET records in input PDB that are as accurate as possible; this is critical for successful refinement.

If you have more questions please get back to us!

Good luck! Pavel

On 6/28/18 09:23, Maciej Jagielnicki wrote:

...
Hi All,

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next.

When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants...

Regards, Maciej J.

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

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

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 https://maps.google.com/?q=480+Ray+C+Hunt+Drive+Rm+320+Charlottesville,+VA&entry=gmail&source=g Charlottesville, VA https://maps.google.com/?q=480+Ray+C+Hunt+Drive+Rm+320+Charlottesville,+VA&entry=gmail&source=g Lab Phone: 434-243-2520

Christian Roth

8:47 p.m.

I wouldn't necessarily say they are ridiculous. If you choose to show a cartoon, some viewers run in the background a secondary structure analysis program (dssp and variants thereof) and depending on the threshold will assign automatically secondary structure elements. None of these automatic routines is perfect and even if you do it manually, opinions between different people will give a spread in terms of when to draw a helix or how long it should be for example. However, no one has to use the provided ss definitions in a file. If someone downloads a file from the pdb and interprets the structrue might disagree with the boundaries and definitions of the secondary structure provided and amend them. Cheers Christian On Thu, Jun 28, 2018 at 9:27 PM Maciej Jagielnicki wrote:

...

Thank you for clarification Ricardo and Pavel.

It still amazes me though that different pdb viewers would make such ridiculous SS predictions/assignments at times for a file that does not come with that information in the header. And it seems that during a deposition to PDB I have to manually add the location of helices/sheets into the .cif files generated by the deposition system...otherwise I go for deposition somewhat blind (this has of course nothing to do with Phenix, but adds more to my confusion).

Maciej

On Thu, Jun 28, 2018 at 4:16 PM, Pavel Afonine wrote:

...
The point is that no automated secondary structure (SS) annotation software can assign SS fully correctly simply because it relies on input model geometry, and models that are subject to refinement are rarely perfect for these software to do a great job. This is why the Manual (see previous email) recommends getting initial automated assignment first using phenix.secondary_structure_restraints (which uses various annotation methods, including ksdssp) and then manually checking and editing it if necessary.

Tools within Phenix always preserve HELIX/SHEET records in PDB files. Let us know if you have an example where this is not the case.

Pavel

On 6/28/18 13:10, Ricardo Righetto wrote:

Hi Maciej,

If the PDB file has no secondary structure assignments defined in the header and you open it in Chimera, it automatically runs ksdssp in the background to assign helices and sheets, which may or may not be correct and might make you think that you have secondary structure records in the header when you actually have nothing: https://plato.cgl.ucsf.edu/chimera/docs/UsersGuide/midas/ksdssp.html

If in turn you save the PDB from Chimera with such automatically generated assignments, the records corresponding to this assignment will be written to the header. And if it's written in the header, by default phenix.real_space_refine will use these restraints.

Bottom line is that throughout modelling and refinement you should always inspect your output PDB file from whatever program you use (PHENIX, Coot, Chimera, etc) to make sure that the information you think should be in the header is indeed there. I agree that some programs might unnecessarily "get rid" of the secondary structure records in the header and this is a bit annoying.

Hope this helps,

-- Ricardo Diogo Righetto

2018-06-28 20:02 GMT+02:00 Maciej Jagielnicki :

...
Thank you for the suggestion.

Nothing changes when I run phenix real space refinement with SS restraints turned on. I can either input a .pdb file with correct SS information in the header or a .pdb file with no SS information at all and the end result (new refined .pdb) is the same file with the same SS information in a new header. The information stays the same and 'correct'.

The main problem in my original question is that when I (or the pdb extract tool that converts a .pdb file to a .cif file for submission to PDB) remove the header SS information altogether (why you would do that for PDB deposition is unknown to me), that resulting .pdb file still shows SS elements in different pdb viewers but SS info is no longer correct. How can you have SS elements displayed if it description is missing from the header? Maciej

On Thu, Jun 28, 2018 at 12:38 PM, Pavel Afonine wrote:

...
Hi Maciej,

please have a look at item #10 and Notes section here:

http://www.phenix-online.org/documentation/reference/real_space_refine.html

One sentence summary is: Make sure you use secondary structure (SS) restraints in refinement, also making sure you provide SS annotations as HELIX/SHEET records in input PDB that are as accurate as possible; this is critical for successful refinement.

If you have more questions please get back to us!

Good luck! Pavel

On 6/28/18 09:23, Maciej Jagielnicki wrote:

Hi All,

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next.

When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants...

Regards, Maciej J.

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

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

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

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

Maciej Jagielnicki

29 Jun 29 Jun

3:07 p.m.

Agreed. Just wanted to make sure I am providing my interpretation of SS along with just atom coordinates. And got even more confused by the pdb - to - cif conversions... Maciej On Thu, Jun 28, 2018 at 4:47 PM, Christian Roth wrote:

...

I wouldn't necessarily say they are ridiculous. If you choose to show a cartoon, some viewers run in the background a secondary structure analysis program (dssp and variants thereof) and depending on the threshold will assign automatically secondary structure elements. None of these automatic routines is perfect and even if you do it manually, opinions between different people will give a spread in terms of when to draw a helix or how long it should be for example. However, no one has to use the provided ss definitions in a file. If someone downloads a file from the pdb and interprets the structrue might disagree with the boundaries and definitions of the secondary structure provided and amend them.

Cheers Christian

On Thu, Jun 28, 2018 at 9:27 PM Maciej Jagielnicki wrote:

...
Thank you for clarification Ricardo and Pavel.

It still amazes me though that different pdb viewers would make such ridiculous SS predictions/assignments at times for a file that does not come with that information in the header. And it seems that during a deposition to PDB I have to manually add the location of helices/sheets into the .cif files generated by the deposition system...otherwise I go for deposition somewhat blind (this has of course nothing to do with Phenix, but adds more to my confusion).

Maciej

On Thu, Jun 28, 2018 at 4:16 PM, Pavel Afonine wrote:

...
The point is that no automated secondary structure (SS) annotation software can assign SS fully correctly simply because it relies on input model geometry, and models that are subject to refinement are rarely perfect for these software to do a great job. This is why the Manual (see previous email) recommends getting initial automated assignment first using phenix.secondary_structure_restraints (which uses various annotation methods, including ksdssp) and then manually checking and editing it if necessary.

Tools within Phenix always preserve HELIX/SHEET records in PDB files. Let us know if you have an example where this is not the case.

Pavel

On 6/28/18 13:10, Ricardo Righetto wrote:

Hi Maciej,

If the PDB file has no secondary structure assignments defined in the header and you open it in Chimera, it automatically runs ksdssp in the background to assign helices and sheets, which may or may not be correct and might make you think that you have secondary structure records in the header when you actually have nothing: https://plato.cgl.ucsf.edu/chimera/docs/UsersGuide/midas/ksdssp.html

If in turn you save the PDB from Chimera with such automatically generated assignments, the records corresponding to this assignment will be written to the header. And if it's written in the header, by default phenix.real_space_refine will use these restraints.

Bottom line is that throughout modelling and refinement you should always inspect your output PDB file from whatever program you use (PHENIX, Coot, Chimera, etc) to make sure that the information you think should be in the header is indeed there. I agree that some programs might unnecessarily "get rid" of the secondary structure records in the header and this is a bit annoying.

Hope this helps,

-- Ricardo Diogo Righetto

2018-06-28 20:02 GMT+02:00 Maciej Jagielnicki :

...
Thank you for the suggestion.

Nothing changes when I run phenix real space refinement with SS restraints turned on. I can either input a .pdb file with correct SS information in the header or a .pdb file with no SS information at all and the end result (new refined .pdb) is the same file with the same SS information in a new header. The information stays the same and 'correct'.

The main problem in my original question is that when I (or the pdb extract tool that converts a .pdb file to a .cif file for submission to PDB) remove the header SS information altogether (why you would do that for PDB deposition is unknown to me), that resulting .pdb file still shows SS elements in different pdb viewers but SS info is no longer correct. How can you have SS elements displayed if it description is missing from the header? Maciej

On Thu, Jun 28, 2018 at 12:38 PM, Pavel Afonine wrote:

...
Hi Maciej,

please have a look at item #10 and Notes section here:

http://www.phenix-online.org/documentation/reference/real_ space_refine.html

One sentence summary is: Make sure you use secondary structure (SS) restraints in refinement, also making sure you provide SS annotations as HELIX/SHEET records in input PDB that are as accurate as possible; this is critical for successful refinement.

If you have more questions please get back to us!

Good luck! Pavel

On 6/28/18 09:23, Maciej Jagielnicki wrote:

Hi All,

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next.

When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants...

Regards, Maciej J.

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

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

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 https://maps.google.com/?q=480+Ray+C+Hunt+Drive+Rm+320+Charlottesville,+VA&entry=gmail&source=g Charlottesville, VA https://maps.google.com/?q=480+Ray+C+Hunt+Drive+Rm+320+Charlottesville,+VA&entry=gmail&source=g Lab Phone: 434-243-2520

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

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

Pavel Afonine

28 Jun 28 Jun

8:23 p.m.

...

Nothing changes when I run phenix real space refinement with SS restraints turned on. I can either input a .pdb file with correct SS information in the header or a .pdb file with no SS information at all and the end result (new refined .pdb) is the same file with the same SS information in a new header. The information stays the same and 'correct'.

Did you actually enable using SS restraints in refinement? Just providing HELIX/SHEET records in input PDB is NOT sufficient, you actually need to tell the program to use them! If you did tell the program to use SS restraints (as it seems the case!) and still there is no effect, then please send us all files and we will investigate. Check the log file -- does it say anything about using SS restraints? It should be very verbose about it.

...

The main problem in my original question is that when I (or the pdb extract tool that converts a .pdb file to a .cif file for submission to PDB) remove the header SS information altogether (why you would do that for PDB deposition is unknown to me), that resulting .pdb file still shows SS elements in different pdb viewers but SS info is no longer correct. How can you have SS elements displayed if it description is missing from the header?

You should not remove HELIX/SHEET records from PDB file yourself. Phenix tools will not remove HELIX/SHEET records from PDB (if you find a tools that does it, please send us a bug report). We can't control what other (non-Phenix) software do. Pavel

Oleg Sobolev

8:37 p.m.

Hi Maciej, The main problem in my original question is that when I (or the pdb extract

...

tool that converts a .pdb file to a .cif file for submission to PDB)

Can you describe more specifically what exactly the tool you are using? One way around it would be to convert your pdb to mmcif using phenix.pdb_as_cif tool which will preserve SS information.

...

How can you have SS elements displayed if it description is missing from the header?

As pointed by Ricardo, visualization programs usually can display SS without any header. On top of that, whether they will use the header if it is present - also depend on particular program. Best regards, Oleg Sobolev.

...

On Thu, Jun 28, 2018 at 12:38 PM, Pavel Afonine wrote:

...
Hi Maciej,

please have a look at item #10 and Notes section here:

http://www.phenix-online.org/documentation/reference/real_sp ace_refine.html

One sentence summary is: Make sure you use secondary structure (SS) restraints in refinement, also making sure you provide SS annotations as HELIX/SHEET records in input PDB that are as accurate as possible; this is critical for successful refinement.

If you have more questions please get back to us!

Good luck! Pavel

On 6/28/18 09:23, Maciej Jagielnicki wrote:

Hi All,

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next.

When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants...

Regards, Maciej J.

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

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

Maciej Jagielnicki

29 Jun 29 Jun

3:05 p.m.

Thanks Oleg, I was using the 'pdb extract' online tool provided by the PDB website. This creates a .cif file but removes SS info. Your way of doing things creates .cif files with SS info in them (at least in my hands phenix works better here). The fact that Chimera does not read SS info from cif files is another story, but cif support is limited. I think this is as good as it gets. Most importantly, my pdbs are in order, so I should now be in the clear - regardless what individual viewers display. Thank you for the explanation! Maciej On Thu, Jun 28, 2018 at 4:37 PM, Oleg Sobolev wrote:

...

Hi Maciej,

The main problem in my original question is that when I (or the pdb

...
extract tool that converts a .pdb file to a .cif file for submission to PDB)

Can you describe more specifically what exactly the tool you are using? One way around it would be to convert your pdb to mmcif using phenix.pdb_as_cif tool which will preserve SS information.

...
How can you have SS elements displayed if it description is missing from the header?

As pointed by Ricardo, visualization programs usually can display SS without any header. On top of that, whether they will use the header if it is present - also depend on particular program.

Best regards, Oleg Sobolev.

...
On Thu, Jun 28, 2018 at 12:38 PM, Pavel Afonine wrote:

...
Hi Maciej,

please have a look at item #10 and Notes section here:

http://www.phenix-online.org/documentation/reference/real_sp ace_refine.html

One sentence summary is: Make sure you use secondary structure (SS) restraints in refinement, also making sure you provide SS annotations as HELIX/SHEET records in input PDB that are as accurate as possible; this is critical for successful refinement.

If you have more questions please get back to us!

Good luck! Pavel

On 6/28/18 09:23, Maciej Jagielnicki wrote:

Hi All,

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next.

When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants...

Regards, Maciej J.

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 https://maps.google.com/?q=480+Ray+C+Hunt+Drive+Rm+320+Charlottesville,+VA&entry=gmail&source=g Charlottesville, VA https://maps.google.com/?q=480+Ray+C+Hunt+Drive+Rm+320+Charlottesville,+VA&entry=gmail&source=g Lab Phone: 434-243-2520

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

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

Pavel Afonine

5:29 p.m.

If you have manual SS annotation (or semi-manual) that you believe is correct -- keep it. Likely, automatically generated SS annotation is going to be worse. But if you like jumping between tools then I afraid you are to live with the fact that all of them have different ideas about handling SS records. A tool that may be handy in this context is phenix.secondary_structure_validation which validates SS records. Pavel On 6/29/18 08:05, Maciej Jagielnicki wrote:

...

Thanks Oleg, I was using the 'pdb extract' online tool provided by the PDB website. This creates a .cif file but removes SS info. Your way of doing things creates .cif files with SS info in them (at least in my hands phenix works better here). The fact that Chimera does not read SS info from cif files is another story, but cif support is limited. I think this is as good as it gets. Most importantly, my pdbs are in order, so I should now be in the clear - regardless what individual viewers display. Thank you for the explanation! Maciej

On Thu, Jun 28, 2018 at 4:37 PM, Oleg Sobolev mailto:[email protected]> wrote:

Hi Maciej,

The main problem in my original question is that when I (or the pdb extract tool that converts a .pdb file to a .cif file for submission to PDB)

Can you describe more specifically what exactly the tool you are using? One way around it would be to convert your pdb to mmcif using phenix.pdb_as_cif tool which will preserve SS information.

How can you have SS elements displayed if it description is missing from the header?

As pointed by Ricardo, visualization programs usually can display SS without any header. On top of that, whether they will use the header if it is present - also depend on particular program.

Best regards, Oleg Sobolev.

On Thu, Jun 28, 2018 at 12:38 PM, Pavel Afonine mailto:[email protected]> wrote:

Hi Maciej,

please have a look at item #10 and Notes section here:

http://www.phenix-online.org/documentation/reference/real_space_refine.html http://www.phenix-online.org/documentation/reference/real_space_refine.html

One sentence summary is: Make sure you use secondary structure (SS) restraints in refinement, also making sure you provide SS annotations as HELIX/SHEET records in input PDB that are as accurate as possible; this is critical for successful refinement.

If you have more questions please get back to us!

Good luck! Pavel

On 6/28/18 09:23, Maciej Jagielnicki wrote:

...
Hi All,

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements, and I had to manually edit the header so that helices would show up in correct places when the .pdb file was viewed in Chimera or similar software. Not an ideal way of doing this which is likely related to what happened next.

When the header information is removed, however, as it happens when the pdb.extract tool prepares a .pdb file for submission by turning it into a .cif file, the secondary structure elements are still displayed when a .cif file is visualized (bizzare in itself) but also the secondary structure is taken straight from the starting .pdb file (the one I used as a base for my model to which there is no connection whatsoever). The question is this - where does the incorrect secondary information come from in the absence of the header? How to fix this? I cannot deposit a file that shows helices/sheets wherever it wants...

Regards, Maciej J.

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 https://maps.google.com/?q=480+Ray+C+Hunt+Drive+Rm+320+Charlottesville,+VA&entry=gmail&source=g Charlottesville, VA https://maps.google.com/?q=480+Ray+C+Hunt+Drive+Rm+320+Charlottesville,+VA&entry=gmail&source=g Lab Phone: 434-243-2520

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

-- Maciej Jagielnicki, MS. Graduate Student University of Virginia Molecular Physiology and Biological Physics 480 Ray C Hunt Drive Rm 320 Charlottesville, VA Lab Phone: 434-243-2520

Paul Emsley

28 Jun 28 Jun

9:14 p.m.

On 28/06/18 17:23, Maciej Jagielnicki wrote:

...

I used a combination of phenix real space refine and Coot to build a model into a cryoEM density map. I used an existing .pdb file as a starting point for my building. During the build, some helical elements became strands and vice versa (it was very clear to my eyes). Unfortunately, neither Phenix nor Coot would automatically update the header information for secondary structure elements

FYI, Coot indeed won't update the header information for secondary structure elements automatically. However you can use Coot to update or generate it add_header_secondary_structure_info(imol) Regards, Paul.

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Christian Roth
Maciej Jagielnicki
Oleg Sobolev
Paul Emsley
Pavel Afonine
Ricardo Righetto

Incorrect secondary structure assignment in a .pdb

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