Hi Oleg, Thanks for the response. 1. Yes, I am using the command line version 2. The run.eff file that I had included in my e-mail includes the lines you sent. I would appreciate the help in converting the old-style into the new style. ? Best wishes, Reza Reza Khayat, PhD Associate Professor City College of New York Department of Chemistry and Biochemistry New York, NY 10031 ________________________________ From: Oleg Sobolev Sent: Tuesday, September 8, 2020 7:43 PM To: Reza Khayat Cc: PHENIX user mailing list Subject: [EXTERNAL] Re: [phenixbb] Improving Ramachandran plot Hi Reza, I assume that you run Phenix via command line. We have a new enhanced "ramachandran_plot_restraints" scope to define these (similar) parameters. The previous one is there (for a short while) for backward compatibility (so users can restore old GUI jobs). The new scope looks like: ramachandran_plot_restraints { enabled = True favored = *oldfield emsley emsley8k allowed = *oldfield emsley emsley8k outlier = *oldfield emsley emsley8k selection = None inject_emsley8k_into_oldfield_favored = True oldfield { weight = 0. weight_scale = 0.01 distance_weight_min = 2.0 distance_weight_max = 10.0 plot_cutoff = 0.027 } emsley { weight = 1.0 scale_allowed = 1.0 } emsley8k { weight_favored = 5.0 weight_allowed = 10.0 weight_outlier = 10.0 } } So please use this one. Notable change is that now you can restrain favored/allowed/outliers separately (or not restrain one or another). Please let me know if you need to know exactly how to convert old-style parameters into new ones. In case you are going to perform a similar study as you are mentioning, I would like to suggest adding the Rama-Z metric to the set of validation metrics. It was included particularly to judge the distribution of residues on Ramachandran plot: https://doi.org/10.1016/j.str.2020.08.005https://urldefense.proofpoint.com/v2/url?u=https-3A__doi.org_10.1016_j.str.2020.08.005&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=CC98DLroSZABXnlwA93ckm5Bfu3Zx893dguNRHGVqJE&s=ptg6ZgZREaTRrW9ujd0LnZ-8kkjQnOgvn0w4NVpj8jI&e= Let me know if you have any questions. Best regards, Oleg Sobolev. On Sun, Sep 6, 2020 at 9:12 PM Reza Khayat mailto:[email protected]> wrote: Hi, I'm trying to follow the protocol described in White et al., Structural principles of SNARE complex recognition by the AAA+ protein NSF e-life 2018 https://elifesciences.org/articles/38888https://urldefense.proofpoint.com/v2/url?u=https-3A__elifesciences.org_articles_38888&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=CC98DLroSZABXnlwA93ckm5Bfu3Zx893dguNRHGVqJE&s=AL0jOsHeTv1ypmAgBkrS4fg6oj-WdYhsXDJ4qWcKh50&e= Here is the pertinent paragraph: "To improve the Ramachandran statistics and geometry of the models, a two-parameter grid search of real space refinements was performed in which 3-5 macrocycles of global minimization and local grid search were performed in the presence of secondary structure restraints. First, a grid refinement search was performed for both target functions with around 1000 refinements each. For the emsley target function, rama_weight and scale_allowed were varied from 0.01 to 300; for the oldfield target function, a grid of refinements was performed over plot_cutoff values from 0.01 to 1.0 and weight_scale values from 0.1 to 300. Results were judged empirically and based primarily on a balance between CCmask and a minimal fraction of residues flagged by the program CaBLAM (Richardson et al., 2018https://urldefense.proofpoint.com/v2/url?u=https-3A__elifesciences.org_articles_38888-23bib37&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=CC98DLroSZABXnlwA93ckm5Bfu3Zx893dguNRHGVqJE&s=A-IYsxXhwe3dvl7oZ8JRr0rxyZ4W29522VR7YCPRmKg&e=) because focusing on the fraction of residues with favored CCmask and Ramachandran statistics alone often resulted in unrealistic models with serious problems (Figure 12https://urldefense.proofpoint.com/v2/url?u=https-3A__elifesciences.org_articles_38888-23fig12&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=CC98DLroSZABXnlwA93ckm5Bfu3Zx893dguNRHGVqJE&s=e7ESAeeMjGInzQkzBfSgzzZPRTccskQn25ODvsw4nlQ&e=, Table 2https://urldefense.proofpoint.com/v2/url?u=https-3A__elifesciences.org_articles_38888-23table2&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=CC98DLroSZABXnlwA93ckm5Bfu3Zx893dguNRHGVqJE&s=lgVCnWzCNio89ZUuhejZ7-iD0MwdtumGbIm5-yMuYLw&e=)." There are no differences in the refined structures regardless of what parameters I change. Attached is the input file I use for phenix.real_space_refine (1.18.2?). Altering the oldfield weight_scale (0.1 to 300) and plot_cutoff (0.1 to 0.5) makes no difference. Any help is highly appreciated. Best wishes, Reza Reza Khayat, PhD Associate Professor City College of New York Department of Chemistry and Biochemistry New York, NY 10031 _______________________________________________ phenixbb mailing list [email protected]mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbbhttps://urldefense.proofpoint.com/v2/url?u=http-3A__phenix-2Donline.org_mailman_listinfo_phenixbb&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=CC98DLroSZABXnlwA93ckm5Bfu3Zx893dguNRHGVqJE&s=D4qwIgec6F5jI3fbAP4ttSzC7wuUt5qTb1uJJ_kC9LM&e= Unsubscribe: [email protected]mailto:[email protected]

Hi Reza, If you have the new scope in the .eff file, it will be used at all times. The behavior that you observed confirms it. You have the new scope, but was putting weight_scale=300 into the old one, which was ignored. I believe you should be able to remove the old scope from the .eff file to avoid any confusion. For the *emsley* target function, *rama_weight* and *scale_allowed* These are moved to ramachandran_plot_restraints.emsley subscope and named "weight" and "scale_allowed". the *oldfield* target function, a grid of refinements was performed over

*plot_cutoff* values from 0.01 to 1.0 and *weight_scale*

These are moved to ramachandran_plot_restraints.oldfield subscope and the names are the same. Best regards, Oleg Sobolev. On Wed, Sep 9, 2020 at 8:37 AM Reza Khayat wrote:

Hi Oleg,

I figured it out, but please correct me if I'm wrong. Setting

peptide_link { ramachandran_restraints = False

and ramachandran_plot_restraints { enabled = True

imposes the new enhanced protocol. Setting both to true does not impose the new protocol.

Best wishes, Reza

Reza Khayat, PhD Associate Professor City College of New York Department of Chemistry and Biochemistry New York, NY 10031 ------------------------------ *From:* [email protected] < [email protected]> on behalf of Reza Khayat < [email protected]> *Sent:* Wednesday, September 9, 2020 9:21 AM *To:* Oleg Sobolev *Cc:* PHENIX user mailing list *Subject:* Re: [phenixbb] [EXTERNAL] Re: Improving Ramachandran plot

Hi Oleg,

Thanks for the response.

1. Yes, I am using the command line version

2. The run.eff file that I had included in my e-mail includes the lines you sent. I would appreciate the help in converting the old-style into the new style. ​

Best wishes, Reza

Reza Khayat, PhD Associate Professor City College of New York Department of Chemistry and Biochemistry New York, NY 10031 ------------------------------ *From:* Oleg Sobolev *Sent:* Tuesday, September 8, 2020 7:43 PM *To:* Reza Khayat *Cc:* PHENIX user mailing list *Subject:* [EXTERNAL] Re: [phenixbb] Improving Ramachandran plot

Hi Reza,

I assume that you run Phenix via command line. We have a new enhanced "ramachandran_plot_restraints" scope to define these (similar) parameters. The previous one is there (for a short while) for backward compatibility (so users can restore old GUI jobs). The new scope looks like:

ramachandran_plot_restraints { enabled = True favored = *oldfield emsley emsley8k allowed = *oldfield emsley emsley8k outlier = *oldfield emsley emsley8k selection = None inject_emsley8k_into_oldfield_favored = True oldfield { weight = 0. weight_scale = 0.01 distance_weight_min = 2.0 distance_weight_max = 10.0 plot_cutoff = 0.027 } emsley { weight = 1.0 scale_allowed = 1.0 } emsley8k { weight_favored = 5.0 weight_allowed = 10.0 weight_outlier = 10.0 } } So please use this one. Notable change is that now you can restrain favored/allowed/outliers separately (or not restrain one or another). Please let me know if you need to know exactly how to convert old-style parameters into new ones.

In case you are going to perform a similar study as you are mentioning, I would like to suggest adding the Rama-Z metric to the set of validation metrics. It was included particularly to judge the distribution of residues on Ramachandran plot: https://doi.org/10.1016/j.str.2020.08.005 https://urldefense.proofpoint.com/v2/url?u=https-3A__doi.org_10.1016_j.str.2020.08.005&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=CC98DLroSZABXnlwA93ckm5Bfu3Zx893dguNRHGVqJE&s=ptg6ZgZREaTRrW9ujd0LnZ-8kkjQnOgvn0w4NVpj8jI&e=

Let me know if you have any questions.

Best regards, Oleg Sobolev.

On Sun, Sep 6, 2020 at 9:12 PM Reza Khayat wrote:

...

Hi,

I'm trying to follow the protocol described in White et al., Structural principles of SNARE complex recognition by the AAA+ protein NSF e-life 2018

https://elifesciences.org/articles/38888 https://urldefense.proofpoint.com/v2/url?u=https-3A__elifesciences.org_articles_38888&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=CC98DLroSZABXnlwA93ckm5Bfu3Zx893dguNRHGVqJE&s=AL0jOsHeTv1ypmAgBkrS4fg6oj-WdYhsXDJ4qWcKh50&e=

Here is the pertinent paragraph:

"To improve the Ramachandran statistics and geometry of the models, a two-parameter grid search of real space refinements was performed in which 3–5 macrocycles of global minimization and local grid search were performed in the presence of secondary structure restraints. First, a grid refinement search was performed for both target functions with around 1000 refinements each. For the *emsley* target function, *rama_weight* and *scale_allowed* were varied from 0.01 to 300; for the *oldfield* target function, a grid of refinements was performed over *plot_cutoff* values from 0.01 to 1.0 and *weight_scale* values from 0.1 to 300. Results were judged empirically and based primarily on a balance between *CCmask* and a minimal fraction of residues flagged by the program CaBLAM (Richardson et al., 2018 https://urldefense.proofpoint.com/v2/url?u=https-3A__elifesciences.org_articles_38888-23bib37&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=CC98DLroSZABXnlwA93ckm5Bfu3Zx893dguNRHGVqJE&s=A-IYsxXhwe3dvl7oZ8JRr0rxyZ4W29522VR7YCPRmKg&e=) because focusing on the fraction of residues with favored *CCmask* and Ramachandran statistics alone often resulted in unrealistic models with serious problems (Figure 12 https://urldefense.proofpoint.com/v2/url?u=https-3A__elifesciences.org_articles_38888-23fig12&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=CC98DLroSZABXnlwA93ckm5Bfu3Zx893dguNRHGVqJE&s=e7ESAeeMjGInzQkzBfSgzzZPRTccskQn25ODvsw4nlQ&e= , Table 2 https://urldefense.proofpoint.com/v2/url?u=https-3A__elifesciences.org_articles_38888-23table2&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=CC98DLroSZABXnlwA93ckm5Bfu3Zx893dguNRHGVqJE&s=lgVCnWzCNio89ZUuhejZ7-iD0MwdtumGbIm5-yMuYLw&e= )."

There are no differences in the refined structures regardless of what parameters I change. Attached is the input file I use for phenix.real_space_refine (1.18.2​). Altering the oldfield weight_scale (0.1 to 300) and plot_cutoff (0.1 to 0.5) makes no difference. Any help is highly appreciated.

Best wishes, Reza

Reza Khayat, PhD Associate Professor City College of New York Department of Chemistry and Biochemistry New York, NY 10031 _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb https://urldefense.proofpoint.com/v2/url?u=http-3A__phenix-2Donline.org_mailman_listinfo_phenixbb&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=CC98DLroSZABXnlwA93ckm5Bfu3Zx893dguNRHGVqJE&s=D4qwIgec6F5jI3fbAP4ttSzC7wuUt5qTb1uJJ_kC9LM&e= Unsubscribe: [email protected]