Difference between revisions of "Simulated-1g1c"

From XDSwiki
m
m
 
Line 575: Line 575:
 
but it traces only about 62 residues. The density looks somewhat reasonable, though.
 
but it traces only about 62 residues. The density looks somewhat reasonable, though.
  
The files [ftp://{{SERVERNAME}}/pub/xds-datared/1g1c/xds-simulated-1g1c-I.mtz xds-simulated-1g1c-I.mtz] and [ftp://{{SERVERNAME}}/pub/xds-datared/1g1c/xds-simulated-1g1c-F.mtz xds-simulated-1g1c-F.mtz] are available.
+
The files [https://{{SERVERNAME}}/pub/xds-datared/1g1c/xds-simulated-1g1c-I.mtz xds-simulated-1g1c-I.mtz] and [https://{{SERVERNAME}}/pub/xds-datared/1g1c/xds-simulated-1g1c-F.mtz xds-simulated-1g1c-F.mtz] are available.
  
 
I refined against 1g1c.pdb:
 
I refined against 1g1c.pdb:
Line 623: Line 623:
 
showing that the anomalous completeness, and even the quality of the anomalous signal, can indeed be increased. I doubt, however, that going to three or more frames would improve things even more.
 
showing that the anomalous completeness, and even the quality of the anomalous signal, can indeed be increased. I doubt, however, that going to three or more frames would improve things even more.
  
The MTZ files are at [ftp://{{SERVERNAME}}/pub/xds-datared/1g1c/xds-simulated-1g1c-F-2frames.mtz] and [ftp://{{SERVERNAME}}/pub/xds-datared/1g1c/xds-simulated-1g1c-I-2frames.mtz], respectively. They were of course obtained with XDSCONV.INP:
+
The MTZ files are at [https://{{SERVERNAME}}/pub/xds-datared/1g1c/xds-simulated-1g1c-F-2frames.mtz] and [https://{{SERVERNAME}}/pub/xds-datared/1g1c/xds-simulated-1g1c-I-2frames.mtz], respectively. They were of course obtained with XDSCONV.INP:
 
  INPUT_FILE=temp.ahkl
 
  INPUT_FILE=temp.ahkl
 
  OUTPUT_FILE=temp.hkl CCP4_I
 
  OUTPUT_FILE=temp.hkl CCP4_I
Line 638: Line 638:
 
</pre>
 
</pre>
  
Using the default (see above) phenix.refine job, I obtain against the [ftp://{{SERVERNAME}}/pub/xds-datared/1g1c/xds-simulated-1g1c-F-2frames.mtz MTZ file with amplitudes]:
+
Using the default (see above) phenix.refine job, I obtain against the [https://{{SERVERNAME}}/pub/xds-datared/1g1c/xds-simulated-1g1c-F-2frames.mtz MTZ file with amplitudes]:
 
  Start R-work = 0.3434, R-free = 0.3540
 
  Start R-work = 0.3434, R-free = 0.3540
 
  Final R-work = 0.2209, R-free = 0.2479
 
  Final R-work = 0.2209, R-free = 0.2479
and against the [ftp://{{SERVERNAME}}/pub/xds-datared/1g1c/xds-simulated-1g1c-I-2frames.mtz MTZ file with intensities]
+
and against the [https://{{SERVERNAME}}/pub/xds-datared/1g1c/xds-simulated-1g1c-I-2frames.mtz MTZ file with intensities]
 
  Start R-work = 0.3492, R-free = 0.3606
 
  Start R-work = 0.3492, R-free = 0.3606
 
  Final R-work = 0.2244, R-free = 0.2504
 
  Final R-work = 0.2244, R-free = 0.2504
Line 754: Line 754:
 
     total      165799  42025    43003      97.7%      11.7%    12.3%  162399  10.07    13.5%    14.8%    17%  0.908  16219
 
     total      165799  42025    43003      97.7%      11.7%    12.3%  162399  10.07    13.5%    14.8%    17%  0.908  16219
  
Using these data (stored in [ftp://{{SERVERNAME}}/pub/xds-datared/1g1c/xscale.oldversion]), I was finally able to solve the structure (see screenshot below) - SHELXE traced 160 out of 198 residues. All files produced by SHELXE are in [ftp://{{SERVERNAME}}/pub/xds-datared/1g1c/shelx].
+
Using these data (stored in [https://{{SERVERNAME}}/pub/xds-datared/1g1c/xscale.oldversion]), I was finally able to solve the structure (see screenshot below) - SHELXE traced 160 out of 198 residues. All files produced by SHELXE are in [https://{{SERVERNAME}}/pub/xds-datared/1g1c/shelx].
  
 
[[File:1g1c-shelxe.png]]
 
[[File:1g1c-shelxe.png]]

Latest revision as of 13:14, 24 March 2020

This is an exercise, devised by James Holton, which deals with merging of datasets that were obtained in the presence of strong radiation damage.

The datasets were actually simulated using his program MLFSOM. There are 100 of them, and they are in random orientations wrt each other. Each dataset consists of 15 frames of 1 degree rotation.

The goal of data processing is to obtain a good and complete dataset. In this case, it is tempting to think about the possibility of only using the first frame of each dataset. This has three advantages:

  1. radiation damage does not lower the resolution
  2. the completeness should be adequate if the symmetry is at least orthorhombic
  3. a successful procedure could also serve for processing data from a X-ray Free Electron Laser (see the recent Nature paper at [1])

Preparation

From visual inspection (using adxv) we realize that the first frame of each dataset looks good (diffraction to 2 A), the last bad (10 A), and there is an obvious degradation from each frame to the next.

We have to get some idea about possible spacegroups first. This means processing some of the datasets. Let's choose "xtal100", the last one.

generate_XDS.INP "../../Illuin/microfocus/xtal100_1_0??.img"

To maximize the number of reflections that should be used for spacegroup determination, the only changes to XDS.INP are:

TEST_RESOLUTION_RANGE= 50 0 ! default is 10 4 ; we want all reflections instead
DATA_RANGE= 1 1             ! R-factors involving more than 1 frame are meaningless 
                            ! with such strong radiation damage

We run "xds" and, after a few seconds, can inspect IDXREF.LP and CORRECT.LP. It turns out that the primitve cell is 38.3, 79.2, 79.2, 90, 90, 90 which is compatible with tetragonal spacegroups, or those with lower symmetry:

 LATTICE-  BRAVAIS-   QUALITY  UNIT CELL CONSTANTS (ANGSTROEM & DEGREES)    REINDEXING TRANSFORMATION
CHARACTER  LATTICE     OF FIT      a      b      c   alpha  beta gamma

*  31        aP          0.0      38.3   79.2   79.2  90.0  90.0  90.0    1  0  0  0  0  1  0  0  0  0  1  0
*  44        aP          0.1      38.3   79.2   79.2  90.0  90.0  90.0   -1  0  0  0  0 -1  0  0  0  0  1  0
*  35        mP          0.4      79.2   38.3   79.2  90.0  90.0  90.0    0  1  0  0  1  0  0  0  0  0 -1  0
*  33        mP          0.9      38.3   79.2   79.2  90.0  90.0  90.0   -1  0  0  0  0 -1  0  0  0  0  1  0
*  34        mP          1.1      38.3   79.2   79.2  90.0  90.0  90.0    1  0  0  0  0  0 -1  0  0  1  0  0
*  32        oP          1.2      38.3   79.2   79.2  90.0  90.0  90.0   -1  0  0  0  0 -1  0  0  0  0  1  0
*  20        mC          1.2     112.0  111.9   38.3  90.0  90.0  90.0    0  1  1  0  0  1 -1  0 -1  0  0  0
*  23        oC          1.4     111.9  112.0   38.3  90.0  90.0  90.0    0 -1  1  0  0  1  1  0 -1  0  0  0
*  25        mC          1.4     111.9  112.0   38.3  90.0  90.0  90.0    0 -1  1  0  0  1  1  0 -1  0  0  0
*  21        tP          2.2      79.2   79.2   38.3  90.0  90.0  90.0    0 -1  0  0  0  0  1  0 -1  0  0  0
   37        mC        249.8     162.9   38.3   79.2  90.0  90.0  76.4   -1  0  2  0 -1  0  0  0  0 -1  0  0

This table exists in both IDXREF.LP and CORRECT.LP. The next table in CORRECT.LP tells us the Rmeas of the starred (*) lattices:

SPACE-GROUP         UNIT CELL CONSTANTS            UNIQUE   Rmeas  COMPARED  LATTICE-
  NUMBER      a      b      c   alpha beta gamma                            CHARACTER

      5     112.0  111.9   38.3  90.0  90.0  90.0     973     0.0        0    20 mC
     75      79.2   79.2   38.3  90.0  90.0  90.0     961    93.5       12    21 tP
     89      79.2   79.2   38.3  90.0  90.0  90.0     946    30.9       27    21 tP
     21     111.9  112.0   38.3  90.0  90.0  90.0     965    31.6        8    23 oC
      5     111.9  112.0   38.3  90.0  90.0  90.0     970    77.9        3    25 mC
      1      38.3   79.2   79.2  90.0  90.0  90.0     973     0.0        0    31 aP
     16      38.3   79.2   79.2  90.0  90.0  90.0     954     6.8       19    32 oP
      3      79.2   38.3   79.2  90.0  90.0  90.0     968     5.4        5    35 mP
      3      38.3   79.2   79.2  90.0  90.0  90.0     966     5.2        7    33 mP
      3      38.3   79.2   79.2  90.0  90.0  90.0     966    10.7        7    34 mP
      1      38.3   79.2   79.2  90.0  90.0  90.0     973     0.0        0    44 aP

Obviously the tetragonal lattices seem unfavourable, whereas orthorhombic is good. We repeat this procedure with a few other datasets, and observe that the "orthorhombic hypothesis" is confirmed. E.g. with xtal001 we obtain:

SPACE-GROUP         UNIT CELL CONSTANTS            UNIQUE   Rmeas  COMPARED  LATTICE-
  NUMBER      a      b      c   alpha beta gamma                            CHARACTER

      5     111.9  111.9   38.3  90.0  90.0  90.0     939   119.8        5    20 mC
     75      79.1   79.1   38.3  90.0  90.0  90.0     939    47.0        5    21 tP
     89      79.1   79.1   38.3  90.0  90.0  90.0     865    21.6       79    21 tP
     21     111.9  111.9   38.3  90.0  90.0  90.0     939   119.8        5    23 oC
      5     111.9  111.9   38.3  90.0  90.0  90.0     939   119.8        5    25 mC
      1      38.3   79.1   79.1  90.0  90.0  90.0     944     0.0        0    31 aP
     16      38.3   79.1   79.1  90.0  90.0  90.0     875     6.3       69    32 oP
      3      79.1   38.3   79.1  90.0  90.0  90.0     944     0.0        0    35 mP
      3      38.3   79.1   79.1  90.0  90.0  90.0     875     6.3       69    33 mP
      3      38.3   79.1   79.1  90.0  90.0  90.0     944     0.0        0    34 mP
      1      38.3   79.1   79.1  90.0  90.0  90.0     944     0.0        0    44 aP

devising a bootstrap procedure

We have to realize that, since the b and c axes are equal, we can index each dataset in two non-equivalent ways. This is the same situation as occurs e.g. for spacegroups P3(x) and P4(x), and means that we'll have to use a REFERENCE_DATA_SET to get the right setting for each of the 100 datasets.

However, we cannot expect that all of the datasets have enough reflections in common with a given dataset. Thus, we have to update and enlarge the REFERENCE_DATA_SET after the first round, using those datasets that have reflections in common with the old REFERENCE_DATA_SET. Then in a second round, we can hopefully identify the correct setting for all datasets. After that, we can scale everything together.

first round of bootstrap

We choose xtal100 as the first reference, and move its XDS_ASCII.HKL to bootstrap/reference.ahkl. A script that goes through all datasets, produces XDS.INP, and runs xds is the following (note that we only REFINE(IDXREF)= ORIENTATION BEAM , and the same for REFINE(INTEGRATE), since it may be useful to keep the b and c axis exactly the same):

#!/bin/csh -f
foreach f ( Illuin/microfocus/xtal*_1_001.img )
setenv x `echo $f | cut -c 19-25` 
echo processing $x
rm -rf bootstrap/$x
mkdir bootstrap/$x
cd bootstrap/$x
cat>XDS.INP<<EOF
JOB= XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT
ORGX= 1511.2 ORGY= 1553.1 ! ORGX=1507 ORGY=1570 if BEAM is not refined
DETECTOR_DISTANCE= 250
OSCILLATION_RANGE= 1
X-RAY_WAVELENGTH= 0.979338
NAME_TEMPLATE_OF_DATA_FRAMES=../../Illuin/microfocus/${x}_1_0??.img
DATA_RANGE=1 1
SPOT_RANGE=1 1
REFERENCE_DATA_SET=../reference.ahkl
TEST_RESOLUTION_RANGE= 50.0 2.0 ! for correlating with reference
SPACE_GROUP_NUMBER=16                   ! 0 if unknown
UNIT_CELL_CONSTANTS= 38.3 79.1 79.1  90 90 90 ! mean of CORRECT outputs
INCLUDE_RESOLUTION_RANGE=60 1.8  ! after CORRECT, insert high resol limit; re-run CORRECT
TRUSTED_REGION=0.00 1.  ! partially use corners of detectors; 1.41421=full use
VALUE_RANGE_FOR_TRUSTED_DETECTOR_PIXELS=7000. 30000. ! often 8000 is ok
MINIMUM_ZETA=0.05        ! integrate close to the Lorentz zone; 0.15 is default
STRONG_PIXEL=5           
MINIMUM_NUMBER_OF_PIXELS_IN_A_SPOT=3 ! default of 6 is sometimes too high
REFINE(INTEGRATE)=  ORIENTATION BEAM ! AXIS DISTANCE CELL 
REFINE(IDXREF)= ORIENTATION BEAM ! AXIS DISTANCE CELL 
! parameters specifically for this detector and beamline:
DETECTOR= ADSC MINIMUM_VALID_PIXEL_VALUE= 1 OVERLOAD= 65000
NX= 3072 NY= 3072  QX= 0.102539  QY= 0.102539 ! to make CORRECT happy if frames are unavailable
DIRECTION_OF_DETECTOR_X-AXIS=1 0 0
DIRECTION_OF_DETECTOR_Y-AXIS=0 1 0
INCIDENT_BEAM_DIRECTION=0 0 1 ! 0.00203 -0.0065 1.02107 ! mean of CORRECT outputs
ROTATION_AXIS=1 0 0    ! at e.g. SERCAT ID-22 this needs to be -1 0 0
FRACTION_OF_POLARIZATION=0.98   ! better value is provided by beamline staff!
POLARIZATION_PLANE_NORMAL=0 1 0
EOF
xds >& xds.log &
sleep 1
  cd ../..
end 

Running this script takes 2 minutes. After this, it's a good idea to check whether the cell parameters are really what we assumed they are:

grep UNIT_CELL_CO xtal0[01]*/XDS_ASCII.HKL | cut -c24- > CELLPARM.INP
cellparm
cat CELLPARM.LP

and obtain:

       A         B         C       ALPHA     BETA      GAMMA     WEIGHT
   38.311    79.096    79.107    90.000    90.000    90.000       1.0
   38.292    79.081    79.078    90.000    90.000    90.000       1.0
   38.285    79.021    79.048    90.000    90.000    90.000       1.0
   38.308    79.106    79.099    90.000    90.000    90.000       1.0
   38.298    79.096    79.084    90.000    90.000    90.000       1.0
   38.310    79.117    79.109    90.000    90.000    90.000       1.0
   38.317    79.120    79.124    90.000    90.000    90.000       1.0
   38.302    79.102    79.097    90.000    90.000    90.000       1.0
   38.309    79.119    79.134    90.000    90.000    90.000       1.0
   38.288    79.098    79.128    90.000    90.000    90.000       1.0
   38.294    79.102    79.119    90.000    90.000    90.000       1.0
   38.299    79.104    79.100    90.000    90.000    90.000       1.0
   38.296    79.113    79.058    90.000    90.000    90.000       1.0
   38.322    79.091    79.120    90.000    90.000    90.000       1.0
   38.284    79.082    79.094    90.000    90.000    90.000       1.0
   38.284    79.103    79.098    90.000    90.000    90.000       1.0
   38.303    79.109    79.111    90.000    90.000    90.000       1.0
   38.293    79.084    79.083    90.000    90.000    90.000       1.0
   38.300    79.095    79.101    90.000    90.000    90.000       1.0

   38.300    79.097    79.100    90.000    90.000    90.000      19.0

Why not use all datasets? The reason is that cellparm has a limit of 20 datasets! But it seems to confirm that the cell axes are really 38.3, 79.1, 79.1.

Now we run xscale with the following XSCALE.INP :

OUTPUT_FILE=temp.ahkl

INPUT_FILE=../xtal001/XDS_ASCII.HKL 
INPUT_FILE=../xtal002/XDS_ASCII.HKL 
INPUT_FILE=../xtal003/XDS_ASCII.HKL 
INPUT_FILE=../xtal004/XDS_ASCII.HKL
INPUT_FILE=../xtal005/XDS_ASCII.HKL
INPUT_FILE=../xtal006/XDS_ASCII.HKL
INPUT_FILE=../xtal007/XDS_ASCII.HKL
INPUT_FILE=../xtal008/XDS_ASCII.HKL
INPUT_FILE=../xtal009/XDS_ASCII.HKL
INPUT_FILE=../xtal010/XDS_ASCII.HKL
INPUT_FILE=../xtal011/XDS_ASCII.HKL
INPUT_FILE=../xtal012/XDS_ASCII.HKL
INPUT_FILE=../xtal013/XDS_ASCII.HKL
INPUT_FILE=../xtal014/XDS_ASCII.HKL
INPUT_FILE=../xtal015/XDS_ASCII.HKL
INPUT_FILE=../xtal016/XDS_ASCII.HKL
INPUT_FILE=../xtal017/XDS_ASCII.HKL
INPUT_FILE=../xtal018/XDS_ASCII.HKL
INPUT_FILE=../xtal019/XDS_ASCII.HKL
INPUT_FILE=../xtal020/XDS_ASCII.HKL
INPUT_FILE=../xtal021/XDS_ASCII.HKL
INPUT_FILE=../xtal022/XDS_ASCII.HKL
INPUT_FILE=../xtal023/XDS_ASCII.HKL
INPUT_FILE=../xtal024/XDS_ASCII.HKL
INPUT_FILE=../xtal025/XDS_ASCII.HKL
INPUT_FILE=../xtal026/XDS_ASCII.HKL
INPUT_FILE=../xtal027/XDS_ASCII.HKL
INPUT_FILE=../xtal028/XDS_ASCII.HKL
INPUT_FILE=../xtal029/XDS_ASCII.HKL
INPUT_FILE=../xtal030/XDS_ASCII.HKL
INPUT_FILE=../xtal031/XDS_ASCII.HKL
INPUT_FILE=../xtal032/XDS_ASCII.HKL
INPUT_FILE=../xtal033/XDS_ASCII.HKL
INPUT_FILE=../xtal034/XDS_ASCII.HKL
INPUT_FILE=../xtal035/XDS_ASCII.HKL
INPUT_FILE=../xtal036/XDS_ASCII.HKL
INPUT_FILE=../xtal037/XDS_ASCII.HKL
INPUT_FILE=../xtal038/XDS_ASCII.HKL
INPUT_FILE=../xtal039/XDS_ASCII.HKL
INPUT_FILE=../xtal040/XDS_ASCII.HKL
INPUT_FILE=../xtal041/XDS_ASCII.HKL
INPUT_FILE=../xtal042/XDS_ASCII.HKL
INPUT_FILE=../xtal043/XDS_ASCII.HKL
INPUT_FILE=../xtal044/XDS_ASCII.HKL
INPUT_FILE=../xtal045/XDS_ASCII.HKL
INPUT_FILE=../xtal046/XDS_ASCII.HKL
INPUT_FILE=../xtal047/XDS_ASCII.HKL
INPUT_FILE=../xtal048/XDS_ASCII.HKL
INPUT_FILE=../xtal049/XDS_ASCII.HKL
INPUT_FILE=../xtal050/XDS_ASCII.HKL
INPUT_FILE=../xtal051/XDS_ASCII.HKL
INPUT_FILE=../xtal052/XDS_ASCII.HKL
INPUT_FILE=../xtal053/XDS_ASCII.HKL
INPUT_FILE=../xtal054/XDS_ASCII.HKL
INPUT_FILE=../xtal055/XDS_ASCII.HKL
INPUT_FILE=../xtal056/XDS_ASCII.HKL
INPUT_FILE=../xtal057/XDS_ASCII.HKL
INPUT_FILE=../xtal058/XDS_ASCII.HKL
INPUT_FILE=../xtal059/XDS_ASCII.HKL
INPUT_FILE=../xtal060/XDS_ASCII.HKL
INPUT_FILE=../xtal061/XDS_ASCII.HKL
INPUT_FILE=../xtal062/XDS_ASCII.HKL
INPUT_FILE=../xtal063/XDS_ASCII.HKL
INPUT_FILE=../xtal064/XDS_ASCII.HKL
INPUT_FILE=../xtal065/XDS_ASCII.HKL
INPUT_FILE=../xtal066/XDS_ASCII.HKL
INPUT_FILE=../xtal067/XDS_ASCII.HKL
INPUT_FILE=../xtal068/XDS_ASCII.HKL
INPUT_FILE=../xtal069/XDS_ASCII.HKL
INPUT_FILE=../xtal070/XDS_ASCII.HKL
INPUT_FILE=../xtal071/XDS_ASCII.HKL
INPUT_FILE=../xtal072/XDS_ASCII.HKL
INPUT_FILE=../xtal073/XDS_ASCII.HKL
INPUT_FILE=../xtal074/XDS_ASCII.HKL
INPUT_FILE=../xtal075/XDS_ASCII.HKL
INPUT_FILE=../xtal076/XDS_ASCII.HKL
INPUT_FILE=../xtal077/XDS_ASCII.HKL
INPUT_FILE=../xtal078/XDS_ASCII.HKL
INPUT_FILE=../xtal079/XDS_ASCII.HKL
INPUT_FILE=../xtal080/XDS_ASCII.HKL
INPUT_FILE=../xtal081/XDS_ASCII.HKL
INPUT_FILE=../xtal082/XDS_ASCII.HKL
INPUT_FILE=../xtal083/XDS_ASCII.HKL
INPUT_FILE=../xtal084/XDS_ASCII.HKL
INPUT_FILE=../xtal085/XDS_ASCII.HKL
INPUT_FILE=../xtal086/XDS_ASCII.HKL
INPUT_FILE=../xtal087/XDS_ASCII.HKL
INPUT_FILE=../xtal088/XDS_ASCII.HKL
INPUT_FILE=../xtal089/XDS_ASCII.HKL
INPUT_FILE=../xtal090/XDS_ASCII.HKL
INPUT_FILE=../xtal091/XDS_ASCII.HKL
INPUT_FILE=../xtal092/XDS_ASCII.HKL
INPUT_FILE=../xtal093/XDS_ASCII.HKL
INPUT_FILE=../xtal094/XDS_ASCII.HKL
INPUT_FILE=../xtal095/XDS_ASCII.HKL
INPUT_FILE=../xtal096/XDS_ASCII.HKL
INPUT_FILE=../xtal097/XDS_ASCII.HKL
INPUT_FILE=../xtal098/XDS_ASCII.HKL
INPUT_FILE=../xtal099/XDS_ASCII.HKL
INPUT_FILE=../xtal100/XDS_ASCII.HKL

xscale writes XSCALE.LP which has the 5050 correlation coefficients of every dataset with every other dataset! The order of listing of the correlation coefficients is such that it turns out that is was a good choice to have xtal100 as the REFERENCE_DATA_SET, because we find this list:

     CORRELATIONS BETWEEN INPUT DATA SETS AFTER CORRECTIONS

DATA SETS  NUMBER OF COMMON  CORRELATION   RATIO OF COMMON   B-FACTOR
 #i   #j     REFLECTIONS     BETWEEN i,j  INTENSITIES (i/j)  BETWEEN i,j

with these final 99 lines:

   1  100          12           0.601            0.8200         0.0085
   2  100          24           0.998            0.9001         0.5637
   3  100          16           0.990            0.9216        -0.2983
   4  100          16           0.239            1.9141        -0.2253
   5  100          31           0.996            0.9231         0.3755
   6  100          22           0.997            0.9412         0.2726
   7  100          11           0.976            0.8848        -0.1225
   8  100           5           0.967            0.9166         0.0435
   9  100          34           0.160            1.2885         0.0774
  10  100          11           0.860            2.9740        -0.2614
  11  100           8           0.997            0.8732         0.6032
  12  100           8           0.998            1.0145        -0.4169
  13  100          22           1.000            0.9313         0.1664
  14  100           8           0.900            0.8040         0.2744
  15  100          10           0.986            0.9510         0.1738
  16  100           1           0.000            0.9685         0.0000
  17  100          14           0.991            0.8700         0.3395
  18  100           7           0.997            1.0546        -0.2113
  19  100          23           1.000            1.0451        -0.0246
  20  100          24           0.266            0.6392         0.1091
  21  100          20           0.995            0.8529         0.6281
  22  100          12           0.072            0.9376        -0.0406
  23  100          19           0.999            0.9366         0.0670
  24  100          14           0.998            1.0986        -0.7853
  25  100           4           0.939            1.0483        -0.0886
  26  100          26           0.993            0.9633         0.0813
  27  100          30           0.990            0.9782        -0.0191
  28  100          30           0.995            0.9124        -0.0781
  29  100          13           0.488            2.1279        -0.2548
  30  100          18           0.283            1.2442         0.0585
  31  100          23           0.995            0.9249         0.4751
  32  100          22           0.293            2.7799        -0.1715
  33  100           7           1.000            1.0706        -0.2011
  34  100           6           0.987            0.9888        -0.0007
  35  100           8           0.989            0.9895        -0.1751
  36  100          23           0.985            0.8494         0.3038
  37  100           8           0.966            0.7378        -0.0108
  38  100           7           1.000            1.1335        -0.0927
  39  100          11           0.982            0.9994        -0.5811
  40  100          16           0.994            0.7549         0.8741
  41  100          12           0.986            0.9478        -0.4168
  42  100          11           0.994            0.8285         0.7668
  43  100           9           0.997            0.9595        -0.2219
  44  100          15           1.000            0.8666         0.2884
  45  100          13           0.517            1.6433         0.0034
  46  100          13           0.296            1.4431        -0.0938
  47  100          18           0.857            0.9734         0.3337
  48  100          13           0.999            0.9627         0.2611
  49  100          22           0.991            0.8798         0.2976
  50  100          14           0.999            1.1206        -1.0748
  51  100          10           0.999            0.9296         0.5194
  52  100           8           0.899            1.3901         0.0190
  53  100          24           0.998            1.0383        -0.3979
  54  100           7           0.998            1.1332        -0.5519
  55  100           8           0.993            0.9258        -0.0688
  56  100          19           0.992            0.9138         0.0326
  57  100           5           0.994            0.9209        -0.2679
  58  100          22           0.996            0.8591         0.6813
  59  100           7           0.650            1.5471        -0.0597
  60  100          21           0.995            0.9013         0.0722
  61  100          16           0.998            0.8689         0.4326
  62  100           1           0.002            0.7717         0.0000
  63  100           6           0.995            0.9921         0.0243
  64  100          14           0.998            0.9398        -0.5243
  65  100          12           0.515            1.7489        -0.0858
  66  100          17           0.999            0.9457         0.0390
  67  100           9           0.840            0.7706         0.5165
  68  100           6           0.969            0.9477         0.0164
  69  100          12           0.999            0.9503        -0.1039
  70  100          10           0.949            0.8026        -0.1336
  71  100           4           0.689            2.0681         0.0039
  72  100          29           0.999            1.1291        -0.6696
  73  100           5          -0.316            0.4326         0.0269
  74  100          13          -0.233            1.4081        -0.0231
  75  100          21           0.991            0.9722        -0.0179
  76  100          27           0.996            0.9971        -0.7051
  77  100          26           0.090            0.9911         0.0042
  78  100          33           0.999            1.0320        -0.1129
  79  100          19           0.990            0.9761        -0.1856
  80  100           9          -0.405            0.6967         0.0026
  81  100          37           1.000            0.9449        -0.3532
  82  100          39           0.998            0.9688        -0.3311
  83  100          16           0.996            0.9339         0.3853
  84  100           4           0.999            0.8844         0.1728
  85  100           0           0.000            1.0000         0.0000
  86  100           4           1.000            1.0431        -0.8447
  87  100          20           0.998            0.9432         0.0283
  88  100          16           0.999            0.9415         0.2914
  89  100          39           0.995            0.9713        -0.2225
  90  100          15           0.992            1.0039         0.0773
  91  100           7           0.997            1.0149        -0.4369
  92  100          15           0.713            0.9845        -0.0447
  93  100          21           0.249            0.8322        -0.0360
  94  100          34           0.997            0.9991        -0.1059
  95  100           6           0.582            0.6511         0.1327
  96  100           8           0.988            0.8068         0.5740
  97  100          16           0.989            0.9331         0.4112
  98  100          13           0.974            0.9556         0.0624
  99  100          15           0.400            0.5817        -0.0325

We note that there are many datasets with high correlation coefficients. We use some of those to generate the REFERENCE_DATA_SET for the second round - XSCALE.INP is now

OUTPUT_FILE=../reference.ahkl
INPUT_FILE=../xtal002/XDS_ASCII.HKL 
INPUT_FILE=../xtal003/XDS_ASCII.HKL 
INPUT_FILE=../xtal005/XDS_ASCII.HKL
INPUT_FILE=../xtal006/XDS_ASCII.HKL
INPUT_FILE=../xtal007/XDS_ASCII.HKL
INPUT_FILE=../xtal008/XDS_ASCII.HKL
INPUT_FILE=../xtal011/XDS_ASCII.HKL
INPUT_FILE=../xtal012/XDS_ASCII.HKL
INPUT_FILE=../xtal013/XDS_ASCII.HKL
INPUT_FILE=../xtal015/XDS_ASCII.HKL
INPUT_FILE=../xtal017/XDS_ASCII.HKL
INPUT_FILE=../xtal018/XDS_ASCII.HKL
INPUT_FILE=../xtal019/XDS_ASCII.HKL
INPUT_FILE=../xtal100/XDS_ASCII.HKL

we could have included more datasets but it's pretty clear that these 14 already provide a completeness of 34.5% :

SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
RESOLUTION     NUMBER OF REFLECTIONS    COMPLETENESS R-FACTOR  R-FACTOR COMPARED I/SIGMA   R-meas  Rmrgd-F  Anomal  SigAno   Nano
  LIMIT     OBSERVED  UNIQUE  POSSIBLE     OF DATA   observed  expected                                      Corr
    8.05         111      92       304       30.3%       3.1%      4.2%       34   17.02     4.1%     3.7%     0%   0.000       0
    5.69         198     161       515       31.3%       3.5%      3.4%       70   16.78     4.8%     3.6%     0%   0.000       0
    4.65         289     230       639       36.0%       3.2%      3.5%      109   16.77     4.4%     3.8%     0%   0.000       0
    4.03         354     267       753       35.5%       3.4%      3.6%      151   18.70     4.5%     3.1%   -40%   1.012       2
    3.60         367     287       840       34.2%       2.4%      3.6%      147   17.35     3.2%     3.1%     0%   0.000       0
    3.29         408     326       919       35.5%       3.7%      3.6%      158   16.91     5.1%     4.0%     0%   0.000       0
    3.04         422     324       987       32.8%       3.8%      3.9%      180   14.95     5.1%     4.0%     0%   0.000       0
    2.85         498     387      1066       36.3%       5.2%      4.6%      212   12.72     7.1%     7.3%     0%   0.000       0
    2.68         523     402      1124       35.8%       5.5%      5.4%      219   11.28     7.4%     7.2%     0%   0.000       0
    2.55         512     399      1174       34.0%       5.8%      6.0%      210    9.98     7.9%     7.6%     0%   0.000       0
    2.43         558     426      1263       33.7%       8.7%      8.6%      237    8.37    11.7%    12.6%  -100%   0.829       2
    2.32         589     446      1287       34.7%       8.1%      9.0%      261    8.05    11.0%    14.0%    61%   0.690       3
    2.23         621     470      1350       34.8%       9.6%     10.4%      276    7.52    12.9%    16.8%     0%   0.000       0
    2.15         653     487      1380       35.3%       8.0%      8.8%      298    7.70    10.8%    13.5%    -2%   0.783       6
    2.08         624     493      1459       33.8%      11.6%     11.6%      247    6.57    16.0%    16.0%     0%   0.000       0
    2.01         660     510      1494       34.1%      11.3%     11.5%      271    6.16    15.0%    16.7%  -100%   0.382       2
    1.95         697     535      1546       34.6%      13.1%     13.8%      295    5.34    17.7%    22.9%     0%   0.000       0
    1.90         765     576      1571       36.7%      15.9%     16.3%      351    5.12    21.7%    23.9%     0%   0.000       0
    1.85         751     563      1635       34.4%      21.7%     22.0%      339    3.80    29.3%    35.2%     0%   0.000       0
    1.80         697     531      1660       32.0%      24.5%     25.5%      298    3.51    33.1%    40.5%   -11%   0.784       2
   total       10297    7912     22966       34.5%       5.6%      5.9%     4363    9.17     7.6%    11.5%    -9%   0.741      24

second round of bootstrap

Now we are ready to run our script "bootstrap.rc" a second time. Actually it would be enough to run the CORRECT step but since it only takes 2 minutes we don't bother to change the script. After this, we run xscale a third time, using the same XSCALE.INP (with all its 100 INPUT_FILE= lines) as the first time. The result is

SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
RESOLUTION     NUMBER OF REFLECTIONS    COMPLETENESS R-FACTOR  R-FACTOR COMPARED I/SIGMA   R-meas  Rmrgd-F  Anomal  SigAno   Nano
  LIMIT     OBSERVED  UNIQUE  POSSIBLE     OF DATA   observed  expected                                      Corr

    8.05         794     270       304       88.8%       4.4%      4.2%      729   23.94     5.1%     3.0%    76%   1.884      48
    5.69        1495     478       515       92.8%       4.6%      4.5%     1404   23.48     5.4%     3.3%    73%   1.633      80
    4.65        1936     598       639       93.6%       5.4%      5.3%     1827   24.31     6.3%     3.7%    66%   1.541     133
    4.03        2381     714       752       94.9%       4.5%      4.8%     2266   24.56     5.3%     3.2%    47%   1.157     151
    3.60        2536     786       841       93.5%       5.5%      5.8%     2409   23.59     6.6%     3.9%    46%   1.164     173
    3.29        2832     875       918       95.3%       5.5%      5.7%     2693   23.10     6.5%     3.8%    31%   1.013     189
    3.04        3132     916       987       92.8%       5.7%      5.9%     3014   21.78     6.7%     3.8%    19%   0.917     228
    2.85        3383    1014      1067       95.0%       7.1%      7.1%     3234   18.61     8.3%     5.7%    26%   0.963     233
    2.68        3688    1079      1126       95.8%       8.3%      8.2%     3545   16.88     9.7%     6.9%    16%   0.911     270
    2.55        3709    1109      1171       94.7%       9.6%      9.8%     3530   14.93    11.3%     8.5%    15%   0.855     252
    2.43        4037    1194      1266       94.3%      10.8%     11.5%     3855   12.86    12.7%    11.1%     9%   0.805     287
    2.32        4160    1217      1281       95.0%      11.7%     12.4%     3979   12.14    13.6%    10.1%    13%   0.886     312
    2.23        4349    1286      1354       95.0%      12.1%     12.9%     4181   11.73    14.3%    13.5%     8%   0.738     317
    2.15        4599    1324      1378       96.1%      13.6%     14.3%     4416   11.26    15.9%    12.9%     5%   0.841     341
    2.08        4726    1379      1459       94.5%      15.5%     16.6%     4548    9.98    18.1%    14.6%    -3%   0.784     352
    2.01        4729    1419      1500       94.6%      15.6%     16.5%     4521    9.46    18.3%    16.4%     6%   0.818     338
    1.95        4980    1480      1544       95.9%      20.3%     20.3%     4782    8.20    23.9%    21.1%    -2%   0.778     353
    1.90        5217    1511      1575       95.9%      22.7%     23.7%     5016    7.51    26.5%    23.6%    -4%   0.740     391
    1.85        5232    1555      1626       95.6%      29.8%     31.0%     5015    5.91    34.9%    28.6%     5%   0.813     359
    1.80        5024    1511      1669       90.5%      33.5%     34.6%     4790    5.25    39.4%    36.9%    -1%   0.767     347
   total       72939   21715     22972       94.5%       8.2%      8.5%    69754   13.36     9.7%    10.3%    16%   0.891    5154

so the data are practically complete, and actually quite good. The anomalous signal suggests that it may be possible to solve the structure from its anomalous signal.

We can find out the correct spacegroup (19 !) with "pointless xdsin temp.ahkl", and adjust our script accordingly.

Now we do another round, since the completeness is so good. We can then identify those few datasets which are still not indexed in the right setting, and fix those manually. It was only xtal085 which had a problem - it turned out that the indexing had not found the correct lattice, which was fixed with STRONG_PIXEL=6.

The final XSCALE.LP is then:

SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
RESOLUTION     NUMBER OF REFLECTIONS    COMPLETENESS R-FACTOR  R-FACTOR COMPARED I/SIGMA   R-meas  Rmrgd-F  Anomal  SigAno   Nano
  LIMIT     OBSERVED  UNIQUE  POSSIBLE     OF DATA   observed  expected                                      Corr

    8.05         804     276       316       87.3%       4.4%      4.2%      733   23.80     5.1%     3.1%    75%   1.899      49
    5.69        1509     481       520       92.5%       4.5%      4.4%     1416   23.61     5.2%     3.3%    75%   1.660      81
    4.65        1951     601       644       93.3%       4.3%      4.4%     1842   24.49     5.1%     3.3%    68%   1.579     134
    4.03        2402     715       755       94.7%       4.1%      4.4%     2289   24.75     4.8%     3.2%    44%   1.174     153
    3.60        2555     788       843       93.5%       4.0%      4.5%     2427   23.81     4.7%     3.2%    48%   1.169     179
    3.29        2862     877       921       95.2%       4.2%      4.7%     2724   23.35     5.0%     3.2%    31%   1.050     198
    3.04        3146     916       989       92.6%       5.0%      5.1%     3030   22.00     5.8%     4.0%    15%   0.897     231
    2.85        3399    1016      1070       95.0%       5.9%      6.1%     3251   18.75     7.0%     5.4%    28%   0.992     235
    2.68        3717    1081      1128       95.8%       7.2%      7.2%     3579   17.01     8.4%     7.1%    13%   0.883     274
    2.55        3724    1110      1174       94.5%       8.3%      8.6%     3543   15.03     9.7%     8.0%    15%   0.836     255
    2.43        4058    1196      1266       94.5%       9.9%     10.6%     3877   12.96    11.5%    10.3%     8%   0.811     291
    2.32        4190    1220      1283       95.1%      11.1%     11.8%     4013   12.21    12.9%    10.8%    11%   0.889     328
    2.23        4371    1288      1357       94.9%      11.5%     12.4%     4207   11.79    13.6%    12.6%     4%   0.757     318
    2.15        4626    1324      1378       96.1%      13.2%     13.9%     4444   11.33    15.4%    12.4%     8%   0.835     349
    2.08        4756    1383      1461       94.7%      15.2%     16.2%     4577   10.02    17.8%    14.2%    -4%   0.771     356
    2.01        4755    1423      1503       94.7%      15.4%     16.1%     4543    9.51    18.1%    15.2%     5%   0.817     342
    1.95        4995    1480      1544       95.9%      20.1%     19.9%     4794    8.24    23.6%    20.2%    -5%   0.787     359
    1.90        5242    1512      1577       95.9%      22.3%     23.2%     5034    7.55    26.1%    22.2%    -1%   0.772     400
    1.85        5261    1552      1626       95.4%      29.6%     30.6%     5054    5.95    34.6%    28.3%     6%   0.828     365
    1.80        5066    1514      1672       90.6%      33.4%     34.4%     4829    5.25    39.2%    35.7%    -1%   0.789     356
   total       73389   21753     23027       94.5%       7.4%      7.7%    70206   13.45     8.6%     9.8%    15%   0.898    5253

When inspecting the list of R-factors of each of the datasets it becomes clear that some of them are really good, but others are mediocre.

Optimizing the result

One method to improve XDS' knowledge of geometry would be to use all 15 frames for indexing, but still only to integrate frame 1. This is easily accomplished by changing in the script:

JOB=XYCORR INIT COLSPOT IDXREF DEFPIX
DATA_RANGE=1 15
SPOT_RANGE=1 15

and to use, instead of "xds >& xds.log &" the line "../../run_xds.rc &" with the following run_xds.rc :

#!/bin/csh -f
xds
egrep -v 'DATA_RANGE|JOB' XDS.INP >x
echo JOB=INTEGRATE CORRECT >XDS.INP
echo DATA_RANGE=1 1 >> XDS.INP
cat x >> XDS.INP
xds

Furthermore it seems good to change "sleep 1" to "sleep 5" because now each COLSPOT has to look at 15 frames, not one. Thus, this takes a little bit longer. Indeed the result is a bit better:

WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION

RESOLUTION     NUMBER OF REFLECTIONS    COMPLETENESS R-FACTOR  R-FACTOR COMPARED I/SIGMA   R-meas  Rmrgd-F  Anomal  SigAno   Nano
  LIMIT     OBSERVED  UNIQUE  POSSIBLE     OF DATA   observed  expected                                      Corr

    8.05         798     274       304       90.1%       4.4%      4.2%      726   23.88     5.2%     3.1%    71%   1.932      49
    5.69        1514     480       515       93.2%       4.5%      4.5%     1421   23.66     5.3%     3.4%    76%   1.670      83
    4.65        1951     599       639       93.7%       4.3%      4.4%     1845   24.57     5.0%     3.3%    67%   1.561     139
    4.03        2399     713       753       94.7%       4.1%      4.5%     2289   24.76     4.8%     3.1%    44%   1.176     154
    3.60        2546     786       840       93.6%       3.9%      4.5%     2417   23.78     4.6%     3.1%    46%   1.127     175
    3.29        2864     876       919       95.3%       4.2%      4.7%     2729   23.35     4.9%     3.2%    38%   1.018     199
    3.04        3154     918       987       93.0%       5.0%      5.2%     3037   21.98     5.8%     3.9%    18%   0.922     231
    2.85        3387    1015      1066       95.2%       5.9%      6.1%     3235   18.74     7.0%     5.2%    26%   0.992     235
    2.68        3724    1082      1126       96.1%       7.2%      7.2%     3583   17.03     8.4%     6.7%    15%   0.890     278
    2.55        3720    1111      1172       94.8%       8.3%      8.6%     3536   15.02     9.7%     8.1%    14%   0.857     255
    2.43        4079    1198      1267       94.6%       9.8%     10.6%     3898   12.96    11.5%    10.3%     9%   0.781     290
    2.32        4199    1221      1283       95.2%      11.1%     11.7%     4024   12.21    12.9%    10.8%    12%   0.911     331
    2.23        4365    1282      1350       95.0%      11.4%     12.2%     4205   11.87    13.4%    12.6%     3%   0.729     319
    2.15        4651    1332      1386       96.1%      13.3%     13.9%     4468   11.30    15.5%    12.5%     5%   0.821     354
    2.08        4745    1380      1455       94.8%      15.0%     16.0%     4569   10.04    17.6%    14.0%    -1%   0.760     358
    2.01        4744    1418      1496       94.8%      15.4%     16.0%     4531    9.50    18.1%    16.3%     5%   0.820     343
    1.95        5019    1487      1550       95.9%      19.6%     19.7%     4813    8.27    23.0%    19.7%    -1%   0.765     359
    1.90        5210    1504      1571       95.7%      21.9%     22.9%     5007    7.53    25.6%    22.8%    -6%   0.740     399
    1.85        5272    1561      1633       95.6%      29.1%     30.1%     5054    5.98    34.1%    28.8%     4%   0.801     366
    1.80        5054    1505      1659       90.7%      33.2%     34.1%     4822    5.25    38.9%    35.2%    -1%   0.790     354
   total       73395   21742     22971       94.6%       7.3%      7.7%    70209   13.46     8.6%     9.8%    16%   0.890    5271

but there does not appear a "magic bullet" that would produce much better data than with the quick bootstrap approach.

Trying to solve the structure

First, we repeat xscale after inserting FRIEDEL'S_LAW=FALSE into XSCALE.INP . This gives us

      NOTE:      Friedel pairs are treated as different reflections.

SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
RESOLUTION     NUMBER OF REFLECTIONS    COMPLETENESS R-FACTOR  R-FACTOR COMPARED I/SIGMA   R-meas  Rmrgd-F  Anomal  SigAno   Nano
  LIMIT     OBSERVED  UNIQUE  POSSIBLE     OF DATA   observed  expected                                      Corr

    8.05         804     382       476       80.3%       3.1%      3.4%      665   24.13     3.9%     2.7%    81%   2.507      50
    5.69        1527     723       882       82.0%       3.4%      3.6%     1251   22.48     4.2%     3.1%    85%   2.223      87
    4.65        1956     938      1136       82.6%       3.4%      3.6%     1602   22.73     4.3%     3.0%    72%   1.821     141
    4.03        2400    1136      1357       83.7%       3.5%      3.6%     1943   22.62     4.4%     3.2%    46%   1.347     154
    3.60        2549    1261      1533       82.3%       3.4%      3.7%     2053   21.53     4.3%     3.3%    51%   1.322     176
    3.29        2867    1393      1694       82.2%       3.7%      3.9%     2347   21.22     4.7%     3.5%    35%   1.159     199
    3.04        3154    1507      1830       82.3%       4.5%      4.3%     2607   19.33     5.7%     4.5%    17%   1.016     231
    2.85        3389    1649      1979       83.3%       5.3%      5.2%     2761   16.37     6.7%     6.0%    27%   1.054     235
    2.68        3724    1757      2104       83.5%       6.5%      6.1%     3088   14.63     8.1%     7.8%    15%   0.962     278
    2.55        3720    1813      2197       82.5%       7.3%      7.6%     2999   12.84     9.2%     9.1%    16%   0.896     255
    2.43        4079    1933      2384       81.1%       9.0%      9.5%     3352   11.01    11.3%    12.5%     9%   0.840     290
    2.32        4199    2006      2420       82.9%      10.0%     10.5%     3474   10.17    12.7%    13.8%    14%   0.939     331
    2.23        4363    2099      2551       82.3%      10.6%     11.0%     3595    9.91    13.4%    14.5%     5%   0.790     319
    2.15        4651    2203      2634       83.6%      12.2%     12.5%     3827    9.29    15.3%    15.7%     7%   0.856     354
    2.08        4745    2248      2758       81.5%      14.2%     14.7%     3945    8.32    18.0%    18.7%    -2%   0.822     358
    2.01        4744    2287      2843       80.4%      14.3%     14.6%     3896    7.92    18.1%    19.2%     7%   0.868     343
    1.95        5019    2429      2945       82.5%      18.5%     18.3%     4079    6.76    23.3%    24.6%     0%   0.789     359
    1.90        5210    2484      3000       82.8%      20.4%     21.0%     4282    6.06    25.6%    27.9%    -4%   0.757     399
    1.85        5272    2569      3119       82.4%      27.8%     28.0%     4272    4.77    35.0%    36.5%     4%   0.803     366
    1.80        5054    2451      3171       77.3%      30.9%     31.1%     4092    4.20    39.0%    43.1%    -3%   0.788     354
   total       73426   35268     43013       82.0%       6.5%      6.7%    60130   11.57     8.2%    11.7%    20%   0.963    5279


One hint towards the contents of the "crystal" is that the information about the simulated data contained the strings "1g1c". This structure (spacegroup 19, cell axes 38.3, 78.6, 79.6) is available from the PDB; it contains 2 chains of 99 residues, and a chain has 2 Cys and 2 Met. Thus we assume that the simulated data may represent SeMet-SAD. Using hkl2map, we can easily find four sites with good CCall/CCweak:

Simulated-1g1c-ccall-ccweak2.png Simulated-1g1c-hist2.png Simulated-1g1c-occ2.png Simulated-1g1c-contrast-vs-cycle2.png

I also tried the poly-Ala tracing feature of shelxe:

shelxe.beta -m40 -a -q -h -s0.54 -b -i -e -n 1g1c 1g1c_fa

but it traces only about 62 residues. The density looks somewhat reasonable, though.

The files xds-simulated-1g1c-I.mtz and xds-simulated-1g1c-F.mtz are available.

I refined against 1g1c.pdb:

phenix.refine xds-simulated-1g1c-F.mtz 1g1c.pdb refinement.input.xray_data.r_free_flags.generate=True

The result was

Start R-work = 0.3453, R-free = 0.3501
Final R-work = 0.2170, R-free = 0.2596

which appears reasonable.

Notes

Towards better completeness: using the first two frames instead of only the first

We might want better (anomalous) completeness than what is given by only the very first frame of each dataset. To this end, we change in the XDS.INP part of our script :

DATA_RANGE=1 2

then run the script which reduces the 100 datasets. When this has finished, we insert in XSCALE.INP

NBATCH=2 

after each INPUT_FILE line (this can be easily done using

 awk '{print $0;print "NBATCH=2"}' XSCALE.INP > x 

). The reason for this is that by default, XSCALE establishes scalefactors every 5 degrees, but here we want scalefactors for every frame, because the radiation damage is so strong. This gives:

      NOTE:      Friedel pairs are treated as different reflections.

SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
RESOLUTION     NUMBER OF REFLECTIONS    COMPLETENESS R-FACTOR  R-FACTOR COMPARED I/SIGMA   R-meas  Rmrgd-F  Anomal  SigAno   Nano
  LIMIT     OBSERVED  UNIQUE  POSSIBLE     OF DATA   observed  expected                                      Corr

    8.05        1922     467       476       98.1%       4.2%      6.6%     1888   20.04     4.8%     2.8%    84%   1.887     142
    5.69        3494     864       882       98.0%       4.5%      6.8%     3429   18.67     5.2%     3.1%    83%   1.635     297
    4.65        4480    1111      1136       97.8%       5.3%      6.7%     4395   18.89     6.1%     3.5%    66%   1.347     406
    4.03        5197    1325      1357       97.6%       6.2%      6.8%     5101   18.37     7.1%     4.3%    43%   1.156     499
    3.60        5916    1500      1533       97.8%       6.9%      7.1%     5804   17.83     8.0%     4.7%    36%   1.083     572
    3.29        6601    1657      1694       97.8%       7.6%      7.3%     6476   17.26     8.7%     4.9%    24%   1.029     634
    3.04        7081    1789      1830       97.8%       9.1%      8.0%     6949   15.50    10.4%     6.4%    17%   1.011     693
    2.85        7684    1946      1979       98.3%      10.9%      9.9%     7530   12.95    12.5%     8.1%    16%   0.950     751
    2.68        8101    2062      2100       98.2%      13.1%     12.1%     7935   11.18    15.0%    10.5%    10%   0.888     795
    2.55        8355    2156      2201       98.0%      15.2%     14.9%     8182    9.69    17.5%    12.3%     6%   0.867     837
    2.43        9195    2327      2376       97.9%      18.2%     18.6%     9003    8.20    20.8%    15.4%     6%   0.837     904
    2.32        9495    2377      2428       97.9%      21.3%     21.9%     9304    7.42    24.4%    18.4%     6%   0.800     934
    2.23        9939    2499      2551       98.0%      23.0%     23.3%     9753    7.13    26.4%    19.0%     4%   0.818     987
    2.15       10219    2577      2622       98.3%      25.4%     25.9%     9992    6.63    29.1%    20.6%     1%   0.797     998
    2.08       10712    2704      2766       97.8%      29.4%     30.8%    10508    5.80    33.8%    25.1%     4%   0.793    1071
    2.01       10900    2778      2839       97.9%      30.8%     31.2%    10649    5.50    35.3%    26.2%     4%   0.828    1060
    1.95       11361    2878      2937       98.0%      36.7%     38.2%    11134    4.71    42.1%    31.5%     1%   0.768    1136
    1.90       11641    2943      3000       98.1%      42.7%     45.1%    11405    4.12    49.1%    38.7%    -1%   0.775    1165
    1.85       12028    3069      3123       98.3%      54.0%     60.4%    11760    3.19    62.1%    47.5%     5%   0.735    1196
    1.80       11506    3003      3173       94.6%      62.1%     70.6%    11229    2.72    71.6%    60.6%    -2%   0.709    1148
   total      165827   42032     43003       97.7%      12.8%     13.3%   162426    8.79    14.7%    15.7%    15%   0.881   16225

showing that the anomalous completeness, and even the quality of the anomalous signal, can indeed be increased. I doubt, however, that going to three or more frames would improve things even more.

The MTZ files are at [2] and [3], respectively. They were of course obtained with XDSCONV.INP:

INPUT_FILE=temp.ahkl
OUTPUT_FILE=temp.hkl CCP4_I

for the intensities, and

INPUT_FILE=temp.ahkl
OUTPUT_FILE=temp.hkl CCP4

for the amplitudes. In both cases, after xdsconv we have to run

f2mtz HKLOUT temp.mtz<F2MTZ.INP
cad HKLIN1 temp.mtz HKLOUT output_file_name.mtz<<EOF
LABIN FILE 1 ALL
END
EOF

Using the default (see above) phenix.refine job, I obtain against the MTZ file with amplitudes:

Start R-work = 0.3434, R-free = 0.3540
Final R-work = 0.2209, R-free = 0.2479

and against the MTZ file with intensities

Start R-work = 0.3492, R-free = 0.3606
Final R-work = 0.2244, R-free = 0.2504

so: better R-free is obtained from better data.

The statistics from SHELXD and SHELXE don't look better - they were already quite good with a single frame per dataset. The statistics printed by SHELXE (for the correct hand) are:

...
<wt> = 0.300, Contrast = 0.591, Connect. = 0.740 for dens.mod. cycle 50

Estimated mean FOM and mapCC as a function of resolution
d    inf - 3.98 - 3.13 - 2.72 - 2.47 - 2.29 - 2.15 - 2.04 - 1.95 - 1.87 - 1.81
<FOM>   0.601  0.606  0.590  0.570  0.538  0.542  0.521  0.509  0.529  0.498
<mapCC> 0.841  0.813  0.811  0.786  0.763  0.744  0.727  0.740  0.761  0.722
N        2289   2303   2334   2245   2289   2330   2299   2297   2429   2046

Estimated mean FOM = 0.551   Pseudo-free CC = 59.42 %

...

Site    x       y       z  h(sig) near old  near new
  1  0.7375  0.6996  0.1537  20.4  1/0.06  2/15.05 6/21.38 3/21.54 5/22.03
  2  0.7676  0.7231  0.3419  18.8  3/0.13  5/12.15 1/15.05 3/21.34 4/22.43
  3  0.5967  0.4904 -0.0067  17.2  4/0.10  4/4.90 6/4.94 2/21.34 1/21.54
  4  0.5269  0.5194 -0.0498  17.1  2/0.05  3/4.90 6/7.85 2/22.43 1/22.96
  5  0.4857  0.6896  0.4039  -4.8  3/12.04  2/12.15 1/22.03 3/22.55 2/22.85
  6  0.5158  0.4788  0.0406   4.7  5/1.45  3/4.94 4/7.85 1/21.38 5/23.30

Why this is difficult to solve with SAD phasing

In the original publication ("Structural evidence for a possible role of reversible disulphide bridge formation in the elasticity of the muscle protein titin" Mayans, O., Wuerges, J., Canela, S., Gautel, M., Wilmanns, M. (2001) Structure 9: 331-340 ) we read:

"This crystal form contains two molecules in the asymmetric unit. They are related by a noncrystallographic two-fold axis, parallel to the crystallographic b axis, located at X = 0.25 and Z = 0.23. This arrangement results in a peak in the native Patterson map at U = 0.5, V = 0, W = 0.47 of peak height 26 σ (42% of the origin peak)."

Unfortunately, the arrangement of substructure sites has (pseudo-)translational symmetry, and may be related to a centrosymmetric arrangement. Indeed, the original structure was solved using molecular replacement.

Using the four sites as given by SHELXE (and default parameters otherwise), I obtained from the cctbx - Phase-O-Phrenia server the following

Plot of relative peak heights:

   |*
   |*
   |*
   |*
   |**
   |**
   |***
   |****
   |******
   |************
   |********************
   |*****************************
   |*********************************
   |***************************************
   |************************************************
   |************************************************************
   |************************************************************
   |************************************************************
   |************************************************************
   |************************************************************
   -------------------------------------------------------------

Peak list:
 Relative
  height   Fractional coordinates
    97.8   0.01982  0.49860 -0.00250
    80.2   0.17362  0.71758  0.83714
    71.5   0.02405  0.53538  0.48365
    63.9  -0.00511  0.07044  0.50289
    62.1   0.02410  0.94827  0.48807
    61.3   0.16922  0.28605  0.15985
    56.3   0.12047  0.50910  0.43665
    55.9   0.21871  0.26331  0.30008
    55.7   0.10931  0.47245  0.53659
    53.0   0.22211  0.23746  0.39503
    52.9   0.03449 -0.00661  0.98264   <------ this peak is close to the origin
    52.5   0.06905  0.02372  0.05632   <------ this one, too
    ...

so the strongest peak corresponds to the translation of molecules (0,0.5,0) but the origin peak is at 1/2 of that size, which appears significant.


Finally solving the structure

After thinking about the most likely way that James Holton used to produce the simulated data, I hypothesized that within each frame, the radiation damage is most likely constant, and that there is a jump in radiation damage from frame 1 to 2. Unfortunately for this scenario, the scaling algorithm in CORRECT and XSCALE was changed for the version of Dec-2010, such that it produces best results when the changes are smooth. Therefore, I tried the penultimate version (May-2010) of XSCALE - and indeed that gives significantly better results:

      NOTE:      Friedel pairs are treated as different reflections.

SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
RESOLUTION     NUMBER OF REFLECTIONS    COMPLETENESS R-FACTOR  R-FACTOR COMPARED I/SIGMA   R-meas  Rmrgd-F  Anomal  SigAno   Nano
  LIMIT     OBSERVED  UNIQUE  POSSIBLE     OF DATA   observed  expected                                      Corr

    8.05        1922     467       476       98.1%       4.0%      5.8%     1888   22.37     4.5%     2.5%    84%   1.952     142
    5.69        3494     864       882       98.0%       4.7%      6.0%     3429   20.85     5.4%     3.2%    77%   1.707     297
    4.65        4480    1111      1136       97.8%       5.1%      5.9%     4395   21.13     5.8%     3.3%    68%   1.518     406
    4.03        5197    1325      1357       97.6%       5.3%      6.0%     5101   20.57     6.1%     3.8%    48%   1.280     499
    3.60        5915    1500      1533       97.8%       6.0%      6.3%     5803   19.99     6.9%     4.1%    41%   1.169     572
    3.29        6601    1657      1694       97.8%       6.5%      6.5%     6476   19.42     7.5%     4.6%    27%   1.066     634
    3.04        7080    1789      1830       97.8%       7.6%      7.2%     6948   17.50     8.7%     5.4%    23%   1.037     693
    2.85        7682    1945      1979       98.3%       8.8%      9.0%     7528   14.75    10.1%     7.0%    15%   0.935     750
    2.68        8099    2062      2100       98.2%      11.0%     11.1%     7933   12.81    12.7%     9.1%    13%   0.881     795
    2.55        8351    2155      2201       97.9%      13.3%     13.7%     8178   11.16    15.4%    11.0%    12%   0.872     836
    2.43        9195    2327      2376       97.9%      16.5%     17.2%     9003    9.49    19.0%    15.1%     8%   0.838     904
    2.32        9495    2377      2428       97.9%      19.8%     20.3%     9304    8.62    22.7%    17.3%     4%   0.818     934
    2.23        9936    2498      2551       97.9%      20.8%     21.7%     9751    8.30    23.9%    17.5%     4%   0.830     987
    2.15       10217    2577      2622       98.3%      23.3%     24.0%     9990    7.74    26.7%    19.2%     4%   0.814     998
    2.08       10710    2704      2766       97.8%      27.1%     28.6%    10506    6.82    31.1%    23.5%     5%   0.812    1071
    2.01       10899    2777      2839       97.8%      28.1%     29.2%    10648    6.46    32.3%    25.0%     6%   0.813    1059
    1.95       11361    2878      2937       98.0%      34.4%     35.5%    11134    5.55    39.5%    30.3%     3%   0.780    1136
    1.90       11639    2941      3000       98.0%      40.5%     41.5%    11403    4.88    46.6%    35.9%     0%   0.787    1163
    1.85       12020    3068      3123       98.2%      52.2%     55.1%    11752    3.79    60.0%    47.4%     6%   0.775    1195
    1.80       11506    3003      3173       94.6%      60.8%     64.8%    11229    3.23    70.1%    58.8%     0%   0.765    1148
   total      165799   42025     43003       97.7%      11.7%     12.3%   162399   10.07    13.5%    14.8%    17%   0.908   16219

Using these data (stored in [4]), I was finally able to solve the structure (see screenshot below) - SHELXE traced 160 out of 198 residues. All files produced by SHELXE are in [5].

1g1c-shelxe.png

It is worth mentioning that James Holton confirmed that my hypothesis is true; he also says that this approach is a good approximation for a multi-pass data collection.

However, generally (i.e. for real data) the smooth scaling (which also applies to absorption correction and detector modulation) gives better results than the previous method of assigning the same scale factor to all reflections of a frame; in particular, it correctly treats those reflections near the border of two frames.

Phenix.refine against these data gives:

Start R-work = 0.3449, R-free = 0.3560
Final R-work = 0.2194, R-free = 0.2469

which is only 0.15%/0.10% better in R-work/R-free than the previous best result (see above).

This example shows that it is important to

  • have the best data available if a structure is difficult to solve
  • know the options (programs, algorithms)
  • know as much as possible about the experiment