Scalepack help

Zbyszek Otwinowski zbyszek at mix.swmed.edu
Wed Oct 9 20:05:14 EST 1996


Tim Allison wrote:
> 
> I'm having trouble in Scalepack and hope someone can help.
> 
> My limited experience has been that, at least in initial rounds,
> before tinkering with error values, the value of Chi^2 increases with
> increasing resolution.  That is, if I set "estimated error" to 0.04
> and "error scale factor" to 1.3, Chi^2 may be 1 or 2 for low
> resolution shells and increase up to 4,5, maybe 10 for the highest
> resolution shell.

This is sign that something wrong is in the experiment or in data
reduction. I would expect Chi^2 values to be in the range 0.8-2.0
when experiment is done well and the crystal is single, does not
change due to radiation damage etc.

> 
> I'm currently reducing and scaling SeMet MAD data from the NSLS (Fuji
> scanner).  Instead of the situation I describe above, I'm finding that
> my highest resolution shell (1.66-1.6A) has Chi^2 of around 1.0 and my low
> resolution shells are around 3 or 4.  I've tried an array of error
> scale factors, estimated errors, and rejection probabilities but can't
> seem to lower the Chi^2 of the low res shells to below 2.0.
> 

Use option "scale anomalous". Otherwise anomalous signal contributes
to Chi^2. Without scale anomalous, one should expect _large_ Chi^2 at
low resolution in MAD experiment. Otherwise there there is no measurable
anomalous signal.

> Included below are sample scalepack inputs.
> 
> So, my question is this:  Is my experience simply too limited; is this
> a normal phenomenon?  Is this indicative of something related to my
> use of denzo?  Overloads?  Mis-indexing?  Spot area?  Anomalous signal?
> 
> I've scaled with "anomalous" off, on and with "scale anomalous" on;
> all give the same result.
> 
> Additionally, what are reasonable values for Chi^2?  Do most people
> try to refine them down to 1.0 or do they not worry about it and
> accept higher values?
> 
> Thanks for any help and/or advice you can offer,
> Tim
> 
> --------------------------------------------------------------
> Tim Allison                             Dept of Biochemistry
> tja2f at dayhoff.med.virginia.edu          University of Virginia
> --------------------------------------------------------------
> 
> -----------
> se1a.scpk:
> /net/rule1/tja2f/denzo.1.5.4/scalepack <<stopinp > se1a.splog
> number of zones 10
> estimated error 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06
> error scale factor 1.5
> rejection probability 0.0001
> ignore overloads
> [@reject]
> write rejection file 0.9
> space group P21
> resolution 100 1.6
> sigma cutoff -3.0
> reference batch 01
> add partials 01 to 80
> postrefine 10
> fit crystal a* 01 to 80
> fit crystal b* 01 to 80
> fit crystal c* 01 to 80
> fit crystal beta* 01 to 80
> fit batch rotx 01 to 80
> fit batch roty 01 to 80
> fit crystal mosaicity 1 to 80
> anomalous
> format DENZO_york1
> sector 01 to 80
> file 'se1_a0##.y2'
> output file 'se1a.spout'
> stopinp
> echo 'Scalepack DONE'
> 
> #I then rerun with @reject uncommented and "write rejection file 0.5"
> 
> ----------
> typical output (using error scale factor 1.3 and estimated error of
> 0.06 for all shells:
> 
>      Shell            Summary of observation redundancies by shells:
>   Lower Upper      No. of reflections with given No. of observations
>   limit limit     0     1     2     3     4   5-6   7-8  9-12 13-19   >19  total
>  100.00  3.45    23     8   226   494   646   210    10     0     0     0   1594
>    3.45  2.74    10     0   143   449   712   251     5     0     0     0   1560
>    2.74  2.39     5     1   126   321   823   286     0     0     0     0   1557
>    2.39  2.17     2     1   104   271   866   309     0     0     0     0   1551
>    2.17  2.02     3     1   111   255   871   331     0     0     0     0   1569
>    2.02  1.90     4     2    93   221   880   355     0     0     0     0   1551
>    1.90  1.80     2     2    99   208   876   369     0     0     0     0   1554
>    1.80  1.72     3    22   179   506   591   245     0     0     0     0   1543
>    1.72  1.66     0    49   291   668   395   172     0     0     0     0   1575
>    1.66  1.60    30   204   415   487   292   109     0     0     0     0   1507
>  All hkl         82   290  1787  3880  6952  2637    15     0     0     0  15561
> 
>      Shell             Summary of observation redundancies:
>   Lower Upper      % of reflections with given No. of observations
>   limit limit     0     1     2     3     4   5-6   7-8  9-12 13-19   >19  total
>  100.00  3.45   1.4   0.5  14.0  30.6  40.0  13.0   0.6   0.0   0.0   0.0   98.6
>    3.45  2.74   0.6   0.0   9.1  28.6  45.4  16.0   0.3   0.0   0.0   0.0   99.4
>    2.74  2.39   0.3   0.1   8.1  20.6  52.7  18.3   0.0   0.0   0.0   0.0   99.7
>    2.39  2.17   0.1   0.1   6.7  17.5  55.8  19.9   0.0   0.0   0.0   0.0   99.9
>    2.17  2.02   0.2   0.1   7.1  16.2  55.4  21.1   0.0   0.0   0.0   0.0   99.8
>    2.02  1.90   0.3   0.1   6.0  14.2  56.6  22.8   0.0   0.0   0.0   0.0   99.7
>    1.90  1.80   0.1   0.1   6.4  13.4  56.3  23.7   0.0   0.0   0.0   0.0   99.9
>    1.80  1.72   0.2   1.4  11.6  32.7  38.2  15.8   0.0   0.0   0.0   0.0   99.8
>    1.72  1.66   0.0   3.1  18.5  42.4  25.1  10.9   0.0   0.0   0.0   0.0  100.0
>    1.66  1.60   2.0  13.3  27.0  31.7  19.0   7.1   0.0   0.0   0.0   0.0   98.0
>  All hkl        0.5   1.9  11.4  24.8  44.4  16.9   0.1   0.0   0.0   0.0   99.5
> 
>      Shell            I/Sigma in resolution shells:
>   Lower Upper      No. of reflections with I / Sigma less than
>   limit limit     0     1     2     3     5    10    20   >20  total
>  100.00  3.45     1     3     8    13    22    48   130  1464   1594
>    3.45  2.74     3    10    23    33    58   107   309  1251   1560
>    2.74  2.39     7    19    37    60   111   224   589   968   1557
>    2.39  2.17     8    30    67   119   184   375   830   721   1551
>    2.17  2.02    12    56   119   197   296   538  1004   565   1569
>    2.02  1.90    37   120   214   312   460   743  1218   333   1551
>    1.90  1.80    77   206   342   481   701  1033  1403   151   1554
>    1.80  1.72   115   299   490   652   882  1171  1482    61   1543
>    1.72  1.66   190   429   665   844  1054  1347  1555    20   1575
>    1.66  1.60   188   501   762   937  1129  1376  1502     5   1507
>  All hkl        638  1673  2727  3648  4897  6962 10022  5539  15561
> 
>      Shell            I/Sigma in resolution shells:
>   Lower Upper      % of reflections with I / Sigma less than
>   limit limit     0     1     2     3     5    10    20   >20  total
>  100.00  3.45   0.1   0.2   0.5   0.8   1.4   3.0   8.0  90.5   98.6
>    3.45  2.74   0.2   0.6   1.5   2.1   3.7   6.8  19.7  79.7   99.4
>    2.74  2.39   0.4   1.2   2.4   3.8   7.1  14.3  37.7  62.0   99.7
>    2.39  2.17   0.5   1.9   4.3   7.7  11.8  24.1  53.4  46.4   99.9
>    2.17  2.02   0.8   3.6   7.6  12.5  18.8  34.2  63.9  35.9   99.8
>    2.02  1.90   2.4   7.7  13.8  20.1  29.6  47.8  78.3  21.4   99.7
>    1.90  1.80   4.9  13.2  22.0  30.9  45.1  66.4  90.2   9.7   99.9
>    1.80  1.72   7.4  19.3  31.7  42.2  57.1  75.7  95.9   3.9   99.8
>    1.72  1.66  12.1  27.2  42.2  53.6  66.9  85.5  98.7   1.3  100.0
>    1.66  1.60  12.2  32.6  49.6  61.0  73.5  89.5  97.7   0.3   98.0
>  All hkl        4.1  10.7  17.4  23.3  31.3  44.5  64.1  35.4   99.5
> 
>                Summary of reflections intensities and R-factors by shells
>      R linear = SUM ( ABS(I - <I>)) / SUM (I)
>      R square = SUM ( (I - <I>) ** 2) / SUM (I ** 2)
>      Chi**2   = SUM ( (I - <I>) ** 2) / (Error ** 2 * N / (N-1) ) )
>      In all sums single measurements are excluded
> 
>  Shell Lower Upper Average      Average    Norm.  Linear Square
>  limit    Angstrom       I   error   stat. Chi**2  R-fac  R-fac
>      100.00   3.45 32741.9  1156.6   362.1  3.738  0.086  0.113
>        3.45   2.74 14355.0   504.8   177.1  3.561  0.090  0.115
>        2.74   2.39  5703.3   232.4   123.1  3.412  0.104  0.121
>        2.39   2.17  4151.1   192.5   120.5  3.053  0.120  0.140
>        2.17   2.02  3216.4   172.0   121.7  2.722  0.129  0.141
>        2.02   1.90  2003.5   141.6   115.5  2.439  0.160  0.168
>        1.90   1.80  1043.6   115.9   105.8  1.771  0.207  0.198
>        1.80   1.72   704.9   113.2   107.4  1.572  0.243  0.230
>        1.72   1.66   503.8   116.9   113.2  1.238  0.280  0.246
>        1.66   1.60   387.1   125.2   122.6  1.092  0.317  0.281
>   All reflections   6564.0   289.7   147.5  2.537  0.102  0.114
> 
>      Intensities of systematic absences
>       h   k   l  Intensity     Sigma   I/Sigma
> 
>       0   1   0       0.6       1.1       0.6
>       0   3   0      28.4      18.2       1.6
>       0   5   0      33.9      27.9       1.2
>       0   7   0     -19.3      39.8      -0.5
>       0   9   0      72.3      34.6       2.1
>       0  11   0      57.6      43.1       1.3
>       0  13   0     166.6      48.5       3.4
>       0  15   0     143.7      65.1       2.2
>       0  17   0     101.4      59.2       1.7
>       0  19   0     -87.8      55.2      -1.6
>       0  21   0     -31.5      61.9      -0.5
>       0  23   0     -90.3      74.0      -1.2
>       0  25   0      20.6      94.1       0.2
>       0  27   0      98.8      92.8       1.1
>       0  29   0      -8.8      92.6      -0.1
>       0  31   0      27.2      87.8       0.3
>       0  33   0     -68.5      89.1      -0.8
> -----------
> output for esf of 1.3 and ee of .14 for all shells:
> 
>  Shell Lower Upper Average      Average    Norm.  Linear Square
>  limit    Angstrom       I   error   stat. Chi**2  R-fac  R-fac
>      100.00   3.45 33071.8  2430.3   335.6  1.855  0.134  0.169
>        3.45   2.74 14530.9  1033.7   164.5  1.833  0.137  0.171
>        2.74   2.39  5790.0   435.7   116.5  1.786  0.144  0.174
>        2.39   2.17  4204.6   333.6   116.0  1.598  0.149  0.176
>        2.17   2.02  3279.2   278.9   118.1  1.495  0.156  0.181
>        2.02   1.90  2045.0   203.5   113.9  1.443  0.178  0.195
>        1.90   1.80  1066.2   144.6   105.5  1.234  0.217  0.215
>        1.80   1.72   721.0   132.2   107.8  1.153  0.248  0.238
>        1.72   1.66   508.1   129.8   114.4  0.957  0.282  0.254
>        1.66   1.60   392.5   135.7   124.5  0.900  0.316  0.280
>   All reflections   6679.4   534.1   142.4  1.471  0.143  0.170
> 
> --
> --------------------------------------------------------------
> Tim Allison                             Dept of Biochemistry
> tja2f at dayhoff.med.virginia.edu          University of Virginia


You may have very strong MAD signal!


-- 
  Zbyszek Otwinowski                     |        zbyszek at chop.swmed.edu
  University of Texas                    |        tel : (214)-648-5098 
  Southwestern Medical Center            |        fax : (214)-648-5095 
  5323 Harry Hines Boulevard  Dallas, Texas  75235-9038
[EOB]




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