Thank you Tom and Oleg. I tried refinement as you suggested. I did local refinement after I did the rigid body. However, the first couple of cycles, the clashscore and everything (statistics) seems improved, but then getting worse after more cycles. I did both 5 and 10 cycles, the final outputs are all worse than what I started with, which means only after rigid body fitting. Is this normal? Best, Yangqi On Tue, Jan 21, 2020 at 5:32 PM Oleg Sobolev wrote:

Hi Yangqi,

Yes, phenix.simple_ncs_from_pdb is the tool to search for NCS in models. Documentation:

https://www.phenix-online.org/documentation/reference/simple_ncs_from_pdb.ht...

Also, it is available in GUI: "Select atoms" -> "NCS" -> "Find NCS" but with less options available. For matrices you'll have to run command-line.

Best regards, Oleg Sobolev.

On Tue, Jan 21, 2020 at 1:17 PM BuddySphinx wrote:

...

Also, another question. Could I find ncs from my pdb model? I tried with map, but it failed to find the correct symmetry. Can I use phenix.simple.ncs to generate the ncs operator file?

Best,

Yangqi

Sent from Mail https://go.microsoft.com/fwlink/?LinkId=550986 for Windows 10

*From: *Tom Terwilliger *Sent: *Tuesday, January 21, 2020 9:18 AM *To: *Yangqi Gu ; Thomas Charles Terwilliger *Cc: *PHENIX user mailing list *Subject: *Re: [phenixbb] How critical is ncs operator for real space refinement

Hi Yangqi,

You don't actually need the NCS relationships in order to refine with NCS restraints because the default is to use torsion-angle restraints which only use local relationships, not overall NCS matrices.

So you can go ahead and use your rigid-body-refined starting model with torsion-angle restraints in phenix.real_space_refine. That will not impose perfect helical restraints, but I am guessing that your map already has near-perfect helical symmetry so that should not matter very much.

Also you might first run with just one monomer which will be much quicker and give you almost the same answer.

Let us know if that does not do it!

All the best,

Tom T

On Mon, Jan 20, 2020 at 9:33 PM Yangqi Gu wrote:

Dear PHENIX developers,

I have a question about NCS operator. Since I have a cryo-EM map and I want to refine my filament coordinates within the map. I generated the coordinate map manually in Chimera by building multiple chains with rigid body refinement. Since I did not generate the NCS operator file (I tried by doing map symmetry, it did not give me the right helical symmetry), I did not refine with ncs restraints. I am wondering in this case, how critical it is to refine a model with helical symmetry applied. Is it a must or can I go with the way I did? Hope to hear from you soon!

Best,

Yangqi

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

--

Thomas C Terwilliger

Laboratory Fellow, Los Alamos National Laboratory

Senior Scientist, New Mexico Consortium

100 Entrada Dr, Los Alamos, NM 87544

Email: [email protected]

Tel: 505-431-0010

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

-- Yangqi Gu Graduate Student Malvankar Lab Yale University, West Campus

Hi Pavel, I sent you a separate email with all the files. Thank you very much for your help! Best, Yangqi On Tue, Jan 21, 2020 at 8:31 PM Pavel Afonine wrote:

Hi Yangqi,

it depends what you call "worse" and also keep in mind that clashscore is not the only metric of success. Some metrics get get worse and some better, and changes in some may be just fine and some may indicate problems. It's hard to comment meaningfully based upon rather scarce amount of information you provide. I can have a look if you share (off list!) map, models (input and output) and the log file from refinement.

Pavel

On 1/21/20 17:19, Yangqi Gu wrote:

Thank you Tom and Oleg. I tried refinement as you suggested. I did local refinement after I did the rigid body. However, the first couple of cycles, the clashscore and everything (statistics) seems improved, but then getting worse after more cycles. I did both 5 and 10 cycles, the final outputs are all worse than what I started with, which means only after rigid body fitting. Is this normal? Best, Yangqi

On Tue, Jan 21, 2020 at 5:32 PM Oleg Sobolev wrote:

...

Hi Yangqi,

Yes, phenix.simple_ncs_from_pdb is the tool to search for NCS in models. Documentation:

https://www.phenix-online.org/documentation/reference/simple_ncs_from_pdb.ht...

Also, it is available in GUI: "Select atoms" -> "NCS" -> "Find NCS" but with less options available. For matrices you'll have to run command-line.

Best regards, Oleg Sobolev.

On Tue, Jan 21, 2020 at 1:17 PM BuddySphinx wrote:

...

Also, another question. Could I find ncs from my pdb model? I tried with map, but it failed to find the correct symmetry. Can I use phenix.simple.ncs to generate the ncs operator file?

Best,

Yangqi

Sent from Mail https://go.microsoft.com/fwlink/?LinkId=550986 for Windows 10

*From: *Tom Terwilliger *Sent: *Tuesday, January 21, 2020 9:18 AM *To: *Yangqi Gu ; Thomas Charles Terwilliger *Cc: *PHENIX user mailing list *Subject: *Re: [phenixbb] How critical is ncs operator for real space refinement

Hi Yangqi,

You don't actually need the NCS relationships in order to refine with NCS restraints because the default is to use torsion-angle restraints which only use local relationships, not overall NCS matrices.

So you can go ahead and use your rigid-body-refined starting model with torsion-angle restraints in phenix.real_space_refine. That will not impose perfect helical restraints, but I am guessing that your map already has near-perfect helical symmetry so that should not matter very much.

Also you might first run with just one monomer which will be much quicker and give you almost the same answer.

Let us know if that does not do it!

All the best,

Tom T

On Mon, Jan 20, 2020 at 9:33 PM Yangqi Gu wrote:

Dear PHENIX developers,

I have a question about NCS operator. Since I have a cryo-EM map and I want to refine my filament coordinates within the map. I generated the coordinate map manually in Chimera by building multiple chains with rigid body refinement. Since I did not generate the NCS operator file (I tried by doing map symmetry, it did not give me the right helical symmetry), I did not refine with ncs restraints. I am wondering in this case, how critical it is to refine a model with helical symmetry applied. Is it a must or can I go with the way I did? Hope to hear from you soon!

Best,

Yangqi

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

--

Thomas C Terwilliger

Laboratory Fellow, Los Alamos National Laboratory

Senior Scientist, New Mexico Consortium

100 Entrada Dr, Los Alamos, NM 87544

Email: [email protected]

Tel: 505-431-0010

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

--

Yangqi Gu Graduate Student Malvankar Lab Yale University, West Campus

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

-- Yangqi Gu Graduate Student Malvankar Lab Yale University, West Campus