Pavel, Thanks so much for the suggestions. They are really helpful! A few questions relative to Phenix. Is there a way to check with Phenix the thin resolution shells? I created Free Rs in Phenix using the thin resolution shells options but removed the 2000 limit and instead used 5%. Maybe I over-did it relative to your reply. Just wondering how to check each of these thin shells. Alternatively, does Phenix ensure one has an adequate number of test reflections in each "thin shell" if this option is set? If so, what should I used for the maximum number of reflections and/or %? Thanks again! Joe ___________________________________________________________ Joseph P. Noel, Ph.D. Investigator, Howard Hughes Medical Institute Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla, CA 92037 USA Phone: (858) 453-4100 extension 1442 Cell: (858) 349-4700 Fax: (858) 597-0855 E-mail: [email protected] Web Site (Salk): http://www.salk.edu/faculty/faculty_details.php?id=37 Web Site (HHMI): http://hhmi.org/research/investigators/noel.html ___________________________________________________________ On Aug 3, 2010, at 11:02 AM, [email protected] wrote:

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Today's Topics:

1. Re: Adequate size for Free R test set? (Pavel Afonine) 2. Re: follow up on message 2: phenix.maps --Message: not implemented ([email protected]) 3. Re: follow up on message 2: phenix.maps --Message: not implemented (Pavel Afonine)

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Message: 1 Date: Tue, 03 Aug 2010 10:38:57 -0700 From: Pavel Afonine To: [email protected] Cc: Joseph Noel Subject: Re: [phenixbb] Adequate size for Free R test set? Message-ID: <[email protected]> Content-Type: text/plain; charset="iso-8859-1"; Format="flowed"

Hi Joe,

I think almost every one has his/her own opinion on this... Here is what I think:

1) The test set should be such that each "relatively thin resolution shell" receives at least 50 reflections, and we empirically found that 150 is "good enough" withing phenix.refine framework. For "relatively thin resolution shell" definition see: Lunin & Skovoroda. Acta Cryst. (1995). A51, 880-887. "R-free likelihood-based estimates of errors for phases calculated from atomic models".

This basically defines how many test reflections you need.

2) It is customary to set aside either 5 or 10% for test set, with the total maximum 2000. These are all "magic numbers", that I presume more or less satisfy "1)" so they became widely used.

3) Presence of high-order NCS and selecting free-flags using "thin shells" algorithm is a different story (Acta Cryst. (2006). D62, 227--238). It is good to do that because it removes the cross-talk between test and work reflections due to NCS, but at the same time it invalidates the requirement "1)". So, this is a gray area (for me at least).

4) Some people believe that the final refinement run should be done using all reflections, arguing that taking away 5-10% of test reflections worsens the maps. There is some truth in this, yes, removing the data worsens the maps, but: a) it is noticeable (in a sense that it can reduce the interpretability of some parts of the map) only in extreme cases of somewhat low resolution or low completeness data, b) in most of all other cases it is simply negligible, c) removing reflections randomly has much smaller effect than removing them systematically (see page #40 here: http://www.phenix-online.org/presentations/latest/pavel_maps.pdf and some relevant references in 2010 PHENIX paper in Acta D). However, if you do that "final run", you will invalidate the final refinement statistics, Rfree and Rwork, and thus obtained final structure cannot have the Rfree associated with it anymore.

Pavel.

On 8/3/10 10:04 AM, Joseph Noel wrote:

...

Hi Folks,

Its been a while since I personally refined many structures. In the past, I used as a default, 5% of my unique reflections for the Free R test set. I have a high resolution structure with 150,000 unique reflections and noticed that Phenix defaults are 5% or 2000 reflections which ever is smaller. What is the current consensus on an adequate number of unique reflections to use for cross-validation?

Thanks! Joe

P.S. I really, really love Phenix. ___________________________________________________________ Joseph P. Noel, Ph.D. Investigator, Howard Hughes Medical Institute Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla, CA 92037 USA

Phone: (858) 453-4100 extension 1442 Cell: (858) 349-4700 Fax: (858) 597-0855 E-mail: [email protected] mailto:[email protected]

Web Site (Salk): http://www.salk.edu/faculty/faculty_details.php?id=37 Web Site (HHMI): http://hhmi.org/research/investigators/noel.html ___________________________________________________________

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