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Sorry that I didn't explain the situation clearly. I used only one output.sca file from HLK2000. I ran the scalepack2mtz (in CCP4i, data reduction, import merged data) several times, on both linux Fedora and window XP, the same computer though.

For the next step refinement, I mean add H2O, ion, ligands, double check certain sidechains.... In those refmac runs, I always use the same mtz file, which I generated from the beginning. I am as surprised as you are the Rfree flags are identical from those different "first" runs.

Thanks

On Jan 23, 2008 9:50 AM, Zheng Zhou <[log in to unmask]> wrote:
Hi, All

Could any tell me how CCP4 handle free R flag? I know It is important to select the same FreeR reflections if I move to next step of refinement. But everytime I start from fresh, the freeR Flag remains unchanged. The Rwork and  Rfree of my models are fine ( 20.7% and 22.9%). I thought Rfree was randomly generated. I asked more experienced people in the lab and neighbor labs, and didn't get a straight answer. Did I do something wrong?

Thanks and sorry to bother others.

Zheng (Joe)
I cant understand this.
Do you mean you have multiple data sets and they all generate the same
FreeRs? If you have EXACTLY the same reflection list, and generate FreeR
flags on the same machine I guess they would be the same.. but it would
be surprising

Or if you are using the same file which already has FreeR flags then
they wont change of course - the default is always to use the
reflections flagged with FreeR = 0.  (Thats as it should be..)

By the by - your agreement between R and FreeR seems unusually low
unless you have very high resolution data or a great deal of
non-crystallographic symmetry.
Eleanor

Zheng  - I am not sure what you are doing, but as long as you work
with one dataset only, you generate the free reflections at the very
beginning when you import your data into CCP4. Then use the resulting
mtz file for all following refinement steps. There are situations
where one will have to deviate from this scheme, but those are rare.
If you feel you have such a case, then tell us more about it. Hope
that helps. Best - MM

Joe,


can you explain a bit clearer your problem?  what do you mean the 'next step of refinement'?  are you just doing another round of refinement?  what program are you using, REFMAC5?  also, R/Rfree of 20.7/22.9 is pretty good, depending on resolution.

yes, you are right, Rfree flags are generated randomly and can be anywhere from 5-10% of your reflections (your choice).  then, once the Rfree flagged reflections are selected, they will not change (nor be refined) throughout the rest of your structure determination.  they should have their own column in your *.mtz file called RfreeFlag or something like this (in CCP4 that is).  other programs handle this relatively the same, but may use various naming conventions.  one caveat is that CCP4 flags reflections using 0 by default (meaning 5% of your reflections are flagged with 0 and are the Rfree set).  Other programs such as CNS and PHENIX use 1 by default, so be careful when switching back and forth.  You can tell either program which to use for Rfree set, but needs to be set, since it is not default.

again, not exactly sure what the problem is, but i hope some of this helps.  feel free to email with further questions if needed.  best of luck!



cheers,
nick