sca file & anomalous difference map: understanding the story from the scratch,
i am new user first Q?? with HKL2000 (with scale anomalous option: ON) the output.sca file is like that (for example): 1 -985 38.064 89.066 51.543 90.000 100.446 90.000 p21 0 0 2 3433.6 77.1 0 0 3 735.6 14.0 0 0 11 1564.7 25.4 0 0 12 5643.2 88.3 0 0 13 541.5 9.7 0 0 14 1701.0 27.4 0 1 1 2060.5 47.0 0 1 2 4115.3 75.3 4257.1 97.5 0 1 3 8782.0 280.6 7728.9 173.9 0 1 5 1760.8 28.4 1778.5 33.5 0 1 6 6650.2 122.2 6843.2 126.0 0 1 7 1935.8 36.6 1794.2 29.1 is that one is merged or unmerged????? if i compare with the output.sca with scale anomalous option (off): 0 0 2 3444.4 77.3 0 0 3 734.9 14.0 0 0 4 1705.0 27.5 0 0 5 2693.8 49.4 0 0 6 8383.1 153.9 ------------------------------------------------------- second Q? using output.sca (with scale anomalous option on) for molecular Replacment generate MR.mtz which shows F,SIGF in data labels under phenix.refine the generated (refine_data.mtz) shows: I-obs(+),SIGI-obs(+),I-obs(-),SIGI-obs(-) what is going on in first and second run? ----------------------------------------------------------------- third Q? i want to generate anomalous difference map using phenix.map but when i use output.sca (with scale anomalous option: ON) as reflections file the data label show ((i-obs,sigma)) how to convert this SCA file (if it is merged) to unmerged sca file to creat anomalous difference map for protein contains Br, Sn and Sulpher?? i want to see peaks for these 3 elements to confirm their presence. N.B: the wavelenght was 1.00900 N.B: resolution 1.78 A N.B: i use GUI phenix i am sorry for that long e-mail. thank you in advance haytham wahba biochemi UdeM Canada
Hi Haytham, It is important to differentiate between "unmerged" and "anomalous": unmerged means "each measurement of each reflection kept with the original indices" merged means "measurements mapped to the unique set and averaged" anomalous means "keep Bijvoet pairs separate, consider them separate reflections" non-anomalous means "consider Bijvoet pairs as repeat measurements of the same reflection" So your first data file is "merged" and is "anomalous". Your second I can't tell for sure but if you have anomalous off, it is probably merged non-anomalous. So for your anomalous difference map you can use a merged or an unmerged file, but it must be anomalous data. I hope that helps! -Tom T
i am new user
first Q?? with HKL2000 (with scale anomalous option: ON)
the output.sca file is like that (for example): 1 -985 38.064 89.066 51.543 90.000 100.446 90.000 p21 0 0 2 3433.6 77.1 0 0 3 735.6 14.0 0 0 11 1564.7 25.4 0 0 12 5643.2 88.3 0 0 13 541.5 9.7 0 0 14 1701.0 27.4 0 1 1 2060.5 47.0 0 1 2 4115.3 75.3 4257.1 97.5 0 1 3 8782.0 280.6 7728.9 173.9 0 1 5 1760.8 28.4 1778.5 33.5 0 1 6 6650.2 122.2 6843.2 126.0 0 1 7 1935.8 36.6 1794.2 29.1 is that one is merged or unmerged?????
if i compare with the output.sca with scale anomalous option (off): 0 0 2 3444.4 77.3 0 0 3 734.9 14.0 0 0 4 1705.0 27.5 0 0 5 2693.8 49.4 0 0 6 8383.1 153.9 ------------------------------------------------------- second Q? using output.sca (with scale anomalous option on) for molecular Replacment generate MR.mtz which shows F,SIGF in data labels under phenix.refine
the generated (refine_data.mtz) shows: I-obs(+),SIGI-obs(+),I-obs(-),SIGI-obs(-)
what is going on in first and second run? ----------------------------------------------------------------- third Q? i want to generate anomalous difference map using phenix.map
but when i use output.sca (with scale anomalous option: ON) as reflections file the data label show ((i-obs,sigma))
how to convert this SCA file (if it is merged) to unmerged sca file to creat anomalous difference map for protein contains Br, Sn and Sulpher?? i want to see peaks for these 3 elements to confirm their presence.
N.B: the wavelenght was 1.00900 N.B: resolution 1.78 A N.B: i use GUI phenix
i am sorry for that long e-mail. thank you in advance
haytham wahba biochemi UdeM Canada
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On Mon, Jun 13, 2011 at 5:28 AM, Haytham Wahba
second Q? using output.sca (with scale anomalous option on) for molecular Replacment generate MR.mtz which shows F,SIGF in data labels under phenix.refine
the generated (refine_data.mtz) shows: I-obs(+),SIGI-obs(+),I-obs(-),SIGI-obs(-)
what is going on in first and second run?
Two different things going on here: 1) in Phenix, the MR programs (AutoMR, or the Phaser GUI) will take any format input (amplitudes or intensities, anomalous or non-anomalous), but the output will always be non-anomalous amplitudes in MTZ format. Note: do not use MR.mtz for refinement!* The data are corrected for anisotropy, and thus are no longer the experimental observations. 2) When you use an anomalous Scalepack file for refinement, phenix.refine will output the same data (unmodified) along with your newly generated R-free flags in MTZ format, which is what you're seeing. You should hold on to this file and use it for all subsequent refinements, since you have now refined against that particular set of R-free flags. (A further note: the Phaser GUI also writes out a data file in MTZ format, which contains the original data, but modified to be non-anomalous amplitudes. This is a Phaser quirk and can be ignored, but the data in that file [which also ends in _data.mtz] are safe to use.) third Q?
i want to generate anomalous difference map using phenix.map
but when i use output.sca (with scale anomalous option: ON) as reflections file the data label show ((i-obs,sigma))
Unfortunately Phenix does not always treat labels the same between different reflection file formats. In MTZ files there is a strict correspondence between "column labels" and what you see in the various programs in Phenix, so anomalous intensities with sigmas will always appear as something like "I(+),SIGI(+),I(-),SIGI(-)". Scalepack files will always appear as "i_obs,sigma" whether or not they contain anomalous data. If they are genuinely unmerged (as Tom explained), they will appear as "i_obs,sigma,merged", which is a signal that Phenix will merge the data internally. how to convert this SCA file (if it is merged) to unmerged sca file to creat
anomalous difference map for protein contains Br, Sn and Sulpher??
You can use the refine_data.mtz file for this - you should use a file with R-free flags anyway. Alternately, use the French&Wilson program to convert the intensities to amplitudes (while preserving the anomalous signal) with correction for weak and negative values; it will also add R-free flags if present. However, if you have already run a round of refinement with the anomalous output.sca as input, phenix.refine should have output an anomalous difference map along with the other map coefficients, so there is no need to make it a separate step. (The GUI may not be automatically opening this map in Coot, however - so you might need to choose the "Open MTZ..." option in the first menu of Coot, and load the file manually. I'll double-check this when I get in to work.) A general recommendation: if you now have an MTZ file with your anomalous data and a set of R-free flags, start using this for everything you do in Phenix, and ignore output.sca from now on - it will prevent confusion and ensure that you don't end up with a new set of R-free flags by accident. -Nat
Hi Nat,
Unfortunately Phenix does not always treat labels the same between different reflection file formats. In MTZ files there is a strict correspondence between "column labels" and what you see in the various programs in Phenix, so anomalous intensities with sigmas will always appear as something like "I(+),SIGI(+),I(-),SIGI(-)". Scalepack files will always appear as "i_obs,sigma" whether or not they contain anomalous data.
This is really easy to change. What labels would be best? "i_obs,sigma(+/-)" ? Ralf
On Mon, Jun 13, 2011 at 10:16 AM, Ralf Grosse-Kunstleve < [email protected]> wrote:
Unfortunately Phenix does not always treat labels the same between different reflection file formats. In MTZ files there is a strict correspondence between "column labels" and what you see in the various programs in Phenix, so anomalous intensities with sigmas will always appear as something like "I(+),SIGI(+),I(-),SIGI(-)". Scalepack files will always appear as "i_obs,sigma" whether or not they contain anomalous data.
This is really easy to change. What labels would be best? "i_obs,sigma(+/-)"
"i_obs(+),sigma(+),i_obs(-),sigma(-)" would be closer to the equivalent in MTZ files - but we could also just use "I(+),SIGI(+),I(-),SIGI(-)". Using a different number of comma-separated fields is dangerous, especially in the reflection file editor (although I guess adding ",merged" to unmerged data is probably necessary right now). d*TREK files have the same problem. -Nat
Hi,
third Q? i want to generate anomalous difference map using phenix.map
if your input data file contains anomalous fobs+ and fobs- (or iobs+ and iobs-) then phenix.maps (as well as phenix.refine) will generate anomalous difference map automatically. All you need to do is: 1) Run phenix.maps to create a parameter file; 2) Edit that parameter file: specify input data file and labels, PDB file, etc; 3) Run it: phenix.maps maps.params This is all explained in great details here: http://phenix-online.org/documentation/phenix_maps.htm Alternatively, you can use PHENIX GUI to do the same thing: it is very easy and may be a better option for someone who is uncomfortable with running things in the command line, editing parameter files, etc. Pavel.
Dear Prof. Dr. Nathaniel Echols
thank you very much for this comprhensive explanation.
you payed my attention to something i did
in first round of refinment after molecular replacment
i used
1- output.sca
2- MR.1.pdb
3- MR.1.mtz
as input files
then i proceded with the output of this round of refinment to other
because you sayed ((do not use MR.mtz for refinement!*))
1- in this case should i start agian from the begining or it does not matter because .sca was included.
2- how can i know form the output files of this first run, if both .sca and MR.1.mtz were used or only one was used in refinment
thank you for your time...
Haytham
UdeM
Canada
Message: 10
Date: Mon, 13 Jun 2011 07:26:04 -0700
From: Nathaniel Echols
second Q? using output.sca (with scale anomalous option on) for molecular Replacment generate MR.mtz which shows F,SIGF in data labels under phenix.refine
the generated (refine_data.mtz) shows: I-obs(+),SIGI-obs(+),I-obs(-),SIGI-obs(-)
what is going on in first and second run?
Two different things going on here: 1) in Phenix, the MR programs (AutoMR, or the Phaser GUI) will take any format input (amplitudes or intensities, anomalous or non-anomalous), but the output will always be non-anomalous amplitudes in MTZ format. Note: do not use MR.mtz for refinement!* The data are corrected for anisotropy, and thus are no longer the experimental observations. 2) When you use an anomalous Scalepack file for refinement, phenix.refine will output the same data (unmodified) along with your newly generated R-free flags in MTZ format, which is what you're seeing. You should hold on to this file and use it for all subsequent refinements, since you have now refined against that particular set of R-free flags. (A further note: the Phaser GUI also writes out a data file in MTZ format, which contains the original data, but modified to be non-anomalous amplitudes. This is a Phaser quirk and can be ignored, but the data in that file [which also ends in _data.mtz] are safe to use.) third Q?
i want to generate anomalous difference map using phenix.map
but when i use output.sca (with scale anomalous option: ON) as reflections file the data label show ((i-obs,sigma))
Unfortunately Phenix does not always treat labels the same between different reflection file formats. In MTZ files there is a strict correspondence between "column labels" and what you see in the various programs in Phenix, so anomalous intensities with sigmas will always appear as something like "I(+),SIGI(+),I(-),SIGI(-)". Scalepack files will always appear as "i_obs,sigma" whether or not they contain anomalous data. If they are genuinely unmerged (as Tom explained), they will appear as "i_obs,sigma,merged", which is a signal that Phenix will merge the data internally. how to convert this SCA file (if it is merged) to unmerged sca file to creat
anomalous difference map for protein contains Br, Sn and Sulpher??
You can use the refine_data.mtz file for this - you should use a file with R-free flags anyway. Alternately, use the French&Wilson program to convert the intensities to amplitudes (while preserving the anomalous signal) with correction for weak and negative values; it will also add R-free flags if present. However, if you have already run a round of refinement with the anomalous output.sca as input, phenix.refine should have output an anomalous difference map along with the other map coefficients, so there is no need to make it a separate step. (The GUI may not be automatically opening this map in Coot, however - so you might need to choose the "Open MTZ..." option in the first menu of Coot, and load the file manually. I'll double-check this when I get in to work.) A general recommendation: if you now have an MTZ file with your anomalous data and a set of R-free flags, start using this for everything you do in Phenix, and ignore output.sca from now on - it will prevent confusion and ensure that you don't end up with a new set of R-free flags by accident. -Nat
On Mon, Jun 13, 2011 at 8:59 AM, Haytham Wahba
you payed my attention to something i did
in first round of refinment after molecular replacment
i used 1- output.sca 2- MR.1.pdb 3- MR.1.mtz
as input files
then i proceded with the output of this round of refinment to other
because you sayed ((do not use MR.mtz for refinement!*))
1- in this case should i start agian from the begining or it does not matter because .sca was included.
2- how can i know form the output files of this first run, if both .sca and MR.1.mtz were used or only one was used in refinment
I just tried this, and if you added the Scalepack file first (which, FYI, would get done for you automatically if you clicked the "Run phenix.refine" button in the AutoMR or Phaser GUIs), then MR.1.mtz will be used as experimental phase restraints, but nothing else. This isn't appropriate (you should only use genuine experimental phases for this, not model-based phases), but it doesn't negate the results of the refinement. However, you really need to look at the log file from refinement to be certain - it will state which parameters each reflection file were used for. Could you please send the log to [email protected] (not the entire list)? -Nat
participants (5)
-
Haytham Wahba -
Nathaniel Echols -
Pavel Afonine -
Ralf Grosse-Kunstleve -
Thomas C. Terwilliger