No, I disagree strongly with that suggestion about pruning the data! The conversion of intensities to amplitudes loses information that cannot be recovered, so the intensities should *always* be included in the deposition. Depositing unmerged intensities (now possible, as Gerard says) is even better and, if you do that, I won’t complain about only depositing amplitudes in the merged data. Removing Bijvoet pairs when you’re sure there is no anomalous signal is fine (but if anyone disagrees and you’ve deposited unmerged intensities, they can get them back). Best wishes, Randy Read

On 12 Jan 2022, at 15:03, Tanner, John J. wrote:

This is a common problem. I use ccp4i CAD to remove all the columns from the .mtz file except for F, SIGF, and FreeR_flag, and then use this new .mtz file in refinement before depositing. For the next structure, remember to do this in the early stages.

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From: [email protected] on behalf of Viney Singh Date: Wednesday, January 12, 2022 at 6:52 AM To: [email protected] Subject: [phenixbb] regarding number of unique reflections

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Dear all,

First of all sorry for the novice question.

I processed one of my datasets using xds. Since I was not expecting an anomalous signal, I kept Friedel's Law = True.

The number of unique reflections I got: 17,000

I used xds_ASCII.HKL after correct.LP and converted it to mtz using Phenix with 10% reflections for Rfree calculations.

Now, when I am trying to refine the structure in Phenix, the structure is being refined against 32,300 reflections with 3230 reflections for Rfree calculation. Looks like refinement is treating Friedel's pair as two different reflections.

When I am trying to upload PDB on rcsb, on the refinement tab, the number of reflections used for refinement and Rfree calculations are shown as 32,300 and 3220 respectively, while in the validation report, no. of reflections used for Rfree calculation are shown as 1700.

I would really appreciate if someone can guide me to resolve this discrepancy.

Thanks in advance.

Viney

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----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 The Keith Peters Building Fax: +44 1223 336827 Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk

On 1/13/22 5:21 AM, Randy John Read wrote:

No, I disagree strongly with that suggestion about pruning the data!

The Original Poster's question arises from a manifestation of phenix.refine usurping user intent over which data to use during refinement. Pruning the data - which I do routinely - is a reaction to phenix.refine's very ill-advised behavior to pull intensities rather than structure factors from a MTZ file that contains both. It seems to then convert the intensities to F's and refine against those F's. phenix.refine also has an unhealthy tendency of grabbing the anomalous data and using that in refinement, even when there's little present. This ignores user intent as it is a conscious additional step to convert I to F after data processing. As long as phenix.refine maintains that default behavior, I will remove intensities from the reflection file prior to refinement. Often (as, I imagine, today) this will result in me not depositing intensities since I invariably deposit the files used in refinement. I can specify the reflection data used, but my command line for that program is already long, and the default behavior should not be to ignore user intent. I would expect that this is a significant source of discrepancy upon PDB deposition, and frequently at odds with what the user had intended to do. I don't think BUSTER behaves like that. I doubt that's true for REFMAC but I don't often run it from the command line. Fix the program to avoid second-guessing the experimenter, and you'll probably get more intensities deposited in PDB. Phil Jeffrey Princeton

Hi Pavel, hi Viney, you should NOT go back to data processing. Data processing should result in unmerged data, including I+ and I-. The refinement program (possibly under guidance of the user) should decide whether and how data should be merged for refinement purposes. This decision should also be reflected during data deposition when counting reflections, however, it is best practice to deposit unmerged data. Whether or not the PDB support deposition of unmerged data may be a different question. Cheers, Tim On Wed, 12 Jan 2022 09:19:35 -0800 Pavel Afonine wrote:

Hi Viney,

if your data are anomalous then you expect to have Fobs+, Fobs- and singletons. If your data are not anomalous, you should not have +/- mates in your data set. So then the real question is: since you do not expect any anomalous signal and thus assume your data set isn't anomalous (as you said), then how come you ended up with a data set that contains Fobs+, Fobs- and singletons? Perhaps you should go back to data processing step and make sure you don't get anomalous data set in the first place.

Now as to counting... Fobs+ and Fobs- are the two entries in the array of float values, so they are counted as two, not one (in Phenix).

Pavel

On 1/12/22 04:41, Viney Singh wrote:

...

Dear all,

First of all sorry for the novice question.

I _processed_ one of my datasets using xds. Since I was not expecting an anomalous signal, I kept Friedel's Law = True. The number of unique reflections I got: *17,000* I used xds_ASCII.HKL after correct.LP and converted it to mtz using Phenix with 10% reflections for Rfree calculations.

Now, when I am trying to_refine _the structure in Phenix, the structure is being refined against *32,300* reflections with *3230* reflections for Rfree calculation. Looks like refinement is treating Friedel's pair as two different reflections.

When I am trying to upload PDB on rcsb, on the refinement tab, the number of reflections used for refinement and Rfree calculations are shown as 32,300 and 3220 respectively, while in the validation report, no. of reflections used for Rfree calculation are shown as *1700*.

I would really appreciate if someone can guide me to resolve this discrepancy.

Thanks in advance.

Viney

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-- -- Tim Gruene Head of the Centre for X-ray Structure Analysis Faculty of Chemistry University of Vienna Phone: +43-1-4277-70202 GPG Key ID = A46BEE1A

Dear Tim and others, Thanks for your valuable insights. Will it be okay if I treat Friedel's pair as separate reflections, even if there is no anomalous signal for structure refinement. Can such reflections (Friedel's pair) be considered unique reflections in that case? If I treat them separately I still have a multiplicity of 6 and completion of more than 95%. Thanks again. On Thu, Jan 13, 2022, 00:24 Tim Gruene wrote:

Hi Pavel, hi Viney,

you should NOT go back to data processing. Data processing should result in unmerged data, including I+ and I-. The refinement program (possibly under guidance of the user) should decide whether and how data should be merged for refinement purposes. This decision should also be reflected during data deposition when counting reflections, however, it is best practice to deposit unmerged data. Whether or not the PDB support deposition of unmerged data may be a different question.

Cheers, Tim

On Wed, 12 Jan 2022 09:19:35 -0800 Pavel Afonine wrote:

...

Hi Viney,

if your data are anomalous then you expect to have Fobs+, Fobs- and singletons. If your data are not anomalous, you should not have +/- mates in your data set. So then the real question is: since you do not expect any anomalous signal and thus assume your data set isn't anomalous (as you said), then how come you ended up with a data set that contains Fobs+, Fobs- and singletons? Perhaps you should go back to data processing step and make sure you don't get anomalous data set in the first place.

Now as to counting... Fobs+ and Fobs- are the two entries in the array of float values, so they are counted as two, not one (in Phenix).

Pavel

On 1/12/22 04:41, Viney Singh wrote:

...

Dear all,

First of all sorry for the novice question.

I _processed_ one of my datasets using xds. Since I was not expecting an anomalous signal, I kept Friedel's Law = True. The number of unique reflections I got: *17,000* I used xds_ASCII.HKL after correct.LP and converted it to mtz using Phenix with 10% reflections for Rfree calculations.

Now, when I am trying to_refine _the structure in Phenix, the structure is being refined against *32,300* reflections with *3230* reflections for Rfree calculation. Looks like refinement is treating Friedel's pair as two different reflections.

When I am trying to upload PDB on rcsb, on the refinement tab, the number of reflections used for refinement and Rfree calculations are shown as 32,300 and 3220 respectively, while in the validation report, no. of reflections used for Rfree calculation are shown as *1700*.

I would really appreciate if someone can guide me to resolve this discrepancy.

Thanks in advance.

Viney

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Phone: +43-1-4277-70202

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