MOSFLM 6.11 User Guide

2.2 Autoindexing

The following three queries ONLY affect REFIX autoindexing, if using the "new" DPS indexing, simply enter return to all three queries. The program asks if you want to change the spacegroup to 0 (ie spacegroup not known). For REFIX autoindexing if a spacegroup has been specified earlier then ONLY solutions for that spacegroup will be found, and if the spacegroup is wrong, the autoindexing may fail. The program asks if you want to fix the cell. This only has an effect if a cell and spacegroup have been defined. The default is not to fix the cell. The program then asks if the detector distance is to be fixed. The default is "yes", UNLESS the user has asked to fix the cell, in which case the default is to refine the distance.

Next, the user will be prompted to supply a filename for the output orientation matrix. Finally, for DPS indexing only, the user will be asked to enter a value for the maximum expected cell edge; the program makes an estimate of this. If the value given here is significantly (>25%) less than the true largest cell edge, the indexing will fail. Equally, if the true cell is VERY much smaller than the default value, it may also select an incorrect cell, with one or more parameters a multiple of the true cell. The default value will work in the great majority of cases.

All queries have a default, which can be selected by simply entering carriage return. The image will then be autoindexed.

When using the new DPS indexing, if a spacegroup and cell have been given, the cell parameters determined by the autoindexing will be permuted to best match the input values, but the user must still select the solution from the list provided. If using REFIX, the same information will force the image to be be autoindexed with this cell and no alternatives will be listed. If no spacegroup information has been given, for both algorithms the user will be presented with a list of choices, sorted on a "PENALTY" parameter (the lower the PENALTY the better). The user must select a spacegroup, and the cell is refined imposing that symmetry.

The success of the autoindexing can be checked by predicting the spots for the current image using the menu option "Predict". If not successful, try adjusting the intensity threshold "Min I/sig(I)" or the maximum cell length (for the FFT based algorithm) or read in another image (Read image menu option), find spots on it (Find spots) and repeat autoindexing (Autoindex). Spots from a satellite crystal can be removed using the Edit spots option.

2.3 Estimate mosaic spread

2.4 Run Strategy option

MOSFLM => STRATEGY
MOSFLM => GO

If you then want to reduce the total rotation range (to save time) and still get a maximally complete dataset type the following at the STRATEGY => prompt:

STRATEGY => ROTATE 60 SEGMENTS 2
STRATEGY => GO

For orthorhombic space groups, you should also try STRATEGY ALTERNATE if the predicted completeness in not as high as expected.

2.5 Determine oscillation angles with the TESTGEN option

Having determined what rotation range needs to be collected, you can check what the (maximum) rotation angle is to avoid getting (too many) spatial overlaps on the images. Remember you must have realistic estimates of the mosaic spread and minimum spot separation for this to be meaningful.

At the STRATEGY => prompt type:

STRATEGY => TESTGEN

STRATEGY => TESTGEN START -15 END 15
STRATEGY => GO

then type:

STRATEGY => TESTGEN START 45 END 75
STRATEGY => GO

To test for overlaps using a particular oscillation angle (eg 1.5 degrees) type:

STRATEGY => TESTGEN START 45 END 75 ANGLE 1.5
STRATEGY => GO

2.6 Integrate the first image to determine if the exposure time is OK

To get back to the Menu, type EXIT at the STRATEGY prompt:

STRATEGY => EXIT

An alternative way of dealing with backstop shadows (particularly for an extended backstop) is to use the NULLPIX keyword. This is used to define a minimum pixel value, and any spot that has a pixel within its measurement box with a value lower than (or equal to) this minimum will be rejected. Be sure that the value given is BIGGER than the background at the edge of the image, or all the high resolution data will be rejected !

Then select the "Integrate" menu option, and answer the questions (most can be answered by entering carriage return).

The program will put up the predicted pattern and then wait for input (unless the timeout mode is set). If the pattern is a good fit, choose menu option "Continue". If the pattern is not aligned with the spots, choose the option "Adjust" and follow the instructions to align the pattern with the spots. (The usual reason for poor alignment is an error in the direct beam coordinates, but as these are refined as part of the autoindexing this should not normally be a problem).

The image will then be integrated. Check the <I>/sigma<I> values (either in the terminal window or in the "mosflm.lp" file), and the Rsym if you have symmetry related fully recorded reflections on this image. The value of SDRATIO is a better guide than the actual Rsym, as the latter will depend on the intensity of the reflections. The SDRATIO should lie in the range 1 to 3.

2.7 Interpreting those "WARNING" messages

After the integration, the program will usually print a list of "WARNING" messages to the terminal window (or mosflm.lp). Don't worry about messages about the standard profiles at this stage, or large positional residuals (because the cell has not yet been accurately determined). However if the " OVERALL BACKGROUND RATIO (BGRATIO)" message is present, this suggests the detector GAIN may be wrong, and the input value (or default value if not input) need to be multiplied by the square of the value of the BGRATIO (get the default value from the mosflm.lp file, where all parameters are printed prior to the integration step). Beware, however, that images showing diffuse scatter will give a high BGRATIO even when the GAIN is correct (eg up to 1.5).

2.8 Getting accurate cell parameters

Accurate cell parameters are essential to obtain the best data quality. MOSFLM uses a post-refinement procedure to determine accurate cell parameters. For trigonal or higher symmetry, an accurate cell can usually be determined from a single "wedge" of data (typically 3-5 degrees), unless the unique axis is approximately along the X-ray beam direction in which case either a different phi value should be used or two segments of data. For orthorhombic or lower symmetry two "wedges" of data widely separated in phi will give the best results. In the latter case, one can either wait until a large rotation range has been collected before refining the cell, or one can start by collecting a few degrees at (say) phi 85 to 90, then start collecting from phi = 0.

When the appropriate data (images) are available, select the "Refine cell" menu option. Answer the queries. Although the default number of images to use is 2 (in each wedge), this is in fact the minimum number and better results will often be obtained by using 3 or 4 images in each wedge.

It is important to have a realistic estimate of the mosaic spread before refining the cell.

Post-refinement yields very accurate cell parameters, but has a relatively small radius of convergence. If the shift in cell parameters is more than 2.5 times the estimated error, the integration of the images and the actual refinement will be repeated. This will happen up to a maximum of 5 times. It is not unusual for 2 or 3 complete rounds to be required if the initial cell parameter estimates came from auto-indexing a single image.

2.9 Integrating a block of images

The next step is normally to integrate a block of between 5 and 10 images. Use the "Integrate" menu option as before. In this case, pay particular attention to the list of warning messages to see if any parameters or options need to be reset. It is also a good idea to check the appearance of the standard profiles (these are output to the terminal window but also to the file "mosflm.lp"). Make sure that adjacent spots are being adequately resolved, and that the peak is not spilling into those pixels marked as background. The PROFILE TOLERANCE parameters are crucial in determining the appearance of the standard profiles. Also try to ensure that NO profiles are being averaged. If necessary, change the minimum rms variation in the background (PROFILE RMSBG) or the number of different profiles (by defining PROFILE XLINES and PROFILE YLINES) to avoid profile averaging. Check that there are not too many reflections being rejected as "BAD SPOTS". If a significant number of strong reflections are being rejected for "Poor profile fit", if the cell has been accurately determined, the mosaic spread appears correct and the GAIN is correct, consider increasing the rejection value (REJECTION PKRATIO) from its default of 3.5 to 4.0. It should NOT be necessary to increase this above 4.0, but rejection of the strongest reflections may have a serious effect on structure determination.

2.10 Integrating the dataset

Once a block of images has been successfully integrated, the complete dataset can be integrated. If data processing is started before data collection is complete, use the WAIT keyword to make the program wait for an image to be completed before it tries to integrate it.

eg WAIT 15 for 15 minute exposures.

You may also wish to specify multiple MTZ files (one for each "block" of images) so that some data can be scaled and merged in SCALA before data collection/processing has completely finished.

Set the "Timeout mode" toggle (at the bottom of the "Processing parameters" window) so that the program will automatically continue 1 second after displaying each new image (this delay is set with keyword TIMEOUT).

Set the "Prompts" toggle to Off.

3: Determination of crystal orientation, cell parameters and spacegroup

3.1     Autoindexing Interactively
3.2     Autoindexing when running the program in background
    

3.1 Autoindexing Interactively

3.1.1 Finding Spots

3.1.1.1 Parameters used in Finding Spots

Threshold
Rmin
Rmax
X offset
Y offset
Min X size
Max X size
Min Y size
Max Y size
Min no of pix
X splitting
Y splitting

3.1.1.2 Displaying found spots

3.1.1.3 Editing found spots

3.1.2 Finding spots on other images

IMPORTANT All found spots are stored and will be used in autoindexing. If changing to a new crystal, spots found previously MUST be deleted using the "Clear spots" menu option.

3.1.3 Selecting images for autoindexing

3.1.4 Running the autoindexing

Autoindexing uses either the FFT based algorithm from DPS (Stellar, Bolotovsky and Rossmann, (1998) J. Appl. Cryst. 30, 1036-1040) or Wolfgang Kabsch's REFIX program (Kabsch, 1988, 1993) both of which have been incorporated into the MOSFLM program.

  18 150  cI   103.13   103.36   103.01    62.8  62.8  62.9  I23,I213,I432,
                                                             I4132
  17  64  tP    74.44    74.54    74.56    92.6  92.2  92.4  P4,P41,P42,P43,
                                                             P422,P4212,P4122,
                                                             P41212,P4222,
                                                             P42212,P4322,P43212
  16  63  oP    74.44    74.54    74.56    92.6  92.2  92.4  P222,P2221,P21212,
                                                             P212121
  15  63  tP    74.54    74.56    74.44    92.2  92.4  92.6  P4,P41,P42,P43,
                                                             P422,P4212,P4122,
                                                             P41212,P4222,
                                                             P42212,P4322,P43212
  14  62  hR   107.32   103.13   131.17    92.0  89.8 121.4  R3,R32
  13  41  oC   103.01   107.80    74.44    90.2  93.3  90.0  C222,C2221
  12  41  mP    74.54    74.44    74.56    92.2  92.6  92.4  P2,P21
  11  41  mP    74.56    74.44    74.54    92.4  92.6  92.2  P2,P21
  10  40  oC   103.01   107.80    74.44    89.8  93.3  90.0  C222,C2221
   9  40  mP    74.54    74.44    74.56    92.2  92.6  92.4  P2,P21
   8  26  cP    74.54    74.56    74.44    92.2  92.4  92.6  P23,P213,P432,
                                                             P4232,P4332,P4132
   7  24  mC   103.36   107.32    74.54    89.8  93.6  90.1  C2
   6  22  mC   103.36   107.32    74.54    89.8  93.6  90.1  C2
   5  19  aP    74.44    74.54    74.56    87.4  92.2  87.6  P1
   4   6  hR   107.32   107.52   123.58    89.9  90.0 119.8  R3,R32
   3   4  mC   103.36   107.32    74.54    90.2  93.6  89.9  C2
   2   2  mC   103.01   107.80    74.44    89.8  93.3  90.0  C2
   1   0  aP    74.44    74.54    74.56    92.6  92.2  92.4  P1

cell 150 50 100

3.1.4.1 How do you know if it has worked correctly ?

3.1.4.2 Errors reported by the autoindexing

3.2 Autoindexing when running the program in background

3.2.1 REFIX and general notes

Autoindexing is invoked by including the keyword AUTOINDEX. If no images are specified, the first image to be integrated (specified on the PROCESS keyword) will be used for autoindexing.

CELL 107 107 123 90 90 120
SYMM R32
AUTOINDEX
PROCESS 1 TO 30 [ ANGLE 1.0 START 0.0 ]

CELL KEEP 107.73 107.73 123.59 90 90 120

AUTOINDEX IMAGES 1 2 3

AUTOINDEX  IMAGE 1 PHI 0 1 IMAGE 20 PHI 50 51

AUTOINDEX IMAGE 1 PHI 0 1 IMAGE 20 PHI 50 51 IDENT test_2

AUTOINDEX IMAGE 1 PHI 0 1 IMAGE 20 21 IDENT test_2  is NOT allowed

AUTOINDEX THRESHOLD 30

FINDSPOTS THRESHOLD 10 RMIN 25 RMAX 75 SPLIT 0.5 0.5 
FINDSPOTS MINX 0.5 MAXX 1.5 MINY 0.5 MAXY 1.5 XOFFSET 25

3.2.2 DPS Indexing in background

The DPS autoindexing can be used to autoindex in a background job whether or not the spacegroup and/or cell are known. The algorithm used, however, will choose the highest symmetry characteristic lattice consistent with the solution unless instructed otherwise. Thus:

      AUTOINDEX DPS 
      PROCESS 1 TO 30 [ ANGLE 1.0 START 0.0 ]

      SYMM P212121

The program can be forced to choose a solution from the list of 44 generated thus;

      AUTOINDEX DPS SOLU n

The maximum cell length (in Angstroms) used in the search can be set manually;

      AUTOINDEX DPS MAXCELL xxxx

      AUTOINDEX DPS REFINE

4: Running the STRATEGY option

4.1     Overview of the STRATEGY option
4.2     Some Examples of the STRATEGY options
4.3     Determining the oscillation angle for each
        image (TESTGEN option)

4.1 Overview of the STRATEGY option

4.2 Some Examples of the STRATEGY option

4.2.1 No previous data has been collected

STRATEGY
GO

The program will determine the phi angle PHIZONE (see above), and generate a reflection list starting at that phi angle, for a total rotation determined by the Laue group. It will then generate a list of all unique reflections and merge the two lists. Finally it will give the completeness of the data for the rotation range generated:

 Optimum rotation gives  98.0% of unique data
 This corresponds to the following rotation ranges for the final run
 From   20.0 to 110.0 degrees
 Type "STATS" for full statistics
 ....
 STRATEGY =>

 STRATEGY => ROTATE 50 SEGMENTS 2
 STRATEGY => GO

 Optimum rotation gives  96.1% of unique data
 This corresponds to the following rotation ranges for the final run
 From   20.0 to  45.0 degrees
 From   65.0 to  90.0 degrees

 STRATEGY => rotate 50 segments 3 
 STRATEGY => go

 Optimum rotation gives  98.0% of unique data
 This corresponds to the following rotation ranges for the final run
 From   20.0 to  37.0 degrees
 From   52.0 to  69.0 degrees
 From   74.0 to  90.0 degrees

START 0 END 20
START 65 END 90
GO

START 0 END 30
GO

4.2.2 When some data have already been collected

STRATEGY START -10 END 20 PARTS 2 
GO
STRATEGY ROTATE 40 SEGMENTS 2
GO

MATRIX xtal_1.mat
STRATEGY  start -20 end 19 PARTS 2
GO
MATRIX xtal_2.mat
STRATEGY AUTO
GO

STRATEGY => PART 1                    ! Include all data from first crystal
STRATEGY => AUTO ROTATE 40 SEGMENTS 2 ! Use 2 segments (each 20 degrees for
				      ! second crystal

4.2.3 Optimising anomalous data collection

STRATEGY ROTATE 60 SEGMENTS 2 ANOMALOUS

4.2.4 A complete list of the STRATEGY subkeywords

4.3 Determining the oscillation angle for each image (TESTGEN option)

TESTGEN START 0 END 90 OVERLAP 3 MINOSC 0.5

STRATEGY AUTO
DISTANCE 80
DETECTOR SMALLMAR
MATRIX lyso_1.mat
SYMM 19
BEAM  90 90
DIVERGENCE 0.35 0.3
MOSAIC 0.2
SEPARATION 1.5 1.5
POLARISATION MONOCHROMATOR
WAVELENGTH  1.5418
RUN

5: Determining Accurate Cell parameters

5.1     Using Post-refinement to refine the cell
5.1.1   Doing post-refinement interactively 
5.1.2   Doing post refinement in background 
5.1.3   What the program actually does
5.1.4   Using several segments or different crystals 
5.1.5   Tips on post-refinement 
5.1.5.1 Processing images showing strong diffuse scatter

The unit cell parameters are refined as part of the autoindexing, but in general not all the parameters will be well defined (in particular, the cell parameter along the X-ray beam direction is ill-determined). Improved values can be obtained by using two or more images widely separated in phi for the autoindexing. However, accurate cell parameters are best determined by post-refinement, for which it is necessary to have a number (at least two) of abutting oscillation images. To obtain accurate cell parameters for orthorhombic or lower symmetry spacegroups, it is essential to have data from two orientations widely separated in phi, but for trigonal or higher symmetry only one "block" of data is normally required.

A pragmatic procedure is as follows:

If more than about 15 degrees of data are available from a single crystal, or several crystals in approximately the same orientation (within 20 degrees) use the "Refine cell" menu option (or the POSTREF SEGMENT option if running in background) to get an accurate cell and then do NOT refine it during integration.

If less than 15 degrees is available, use the refined cell from the autoindexing in processing and try post-refinement using an angular wedge of data, but if this is unstable (large sd's or shifts from cycle to cycle) then fix the cell parameters, as the values from the autoindexing, while they may be in error, will be sufficiently accurate to process a "local region" in reciprocal space, ie up to 10-15 degrees from the starting phi value.

5.1 Using Post-refinement to refine the cell

Post-refinement uses the distribution of the intensity of partially recorded reflections over the two images on which the partial is recorded to refine cell parameters, orientation and mosaic spread. It has the distinct advantage that the derived cell parameters are entirely independent of all detector parameters (crystal to detector distance and detector orientation) and distortions (ROFF and TOFF) which, if inaccurate, can lead to significant errors in the cell parameters derived from autoindexing.

**** IMPORTANT ****

The default post-refinement can ONLY use partially recorded reflections which extend over two images. Those which extend over three or more images CANNOT be used. Thus if the mosaic spread (plus beam divergence) is more than twice the oscillation angle, you MUST use the POSTREF MULTI option. If refining the cell interactively, use the "Keyword input" menu item to give these keywords.

5.1.1 Doing post-refinement interactively

Having obtained the crystal orientation by autoindexing, choose the "Refine cell" menu option. You can then select the number of "segments" of data to use in the refinement, the first image and the number of images to be used in each segment. Note that there must be at least two images in each segment, but there is generally little to be gained from using a total of more than 5-10 images in ALL segments (unless there are only a few partials on each image).

Note that when using data from two segments widely separated in phi, it is possible that the crystal orientation will have changed sufficiently that the orientation matrix for the first segment of data does not accurately predict the first image of the second segment. This can be quickly checked by reading in this image ("Read image" menu option) and then predicting the pattern ("Predict"). If the prediction is poor, there are two things that can be done. Either find spots on this second image and use them (together with the spots from the first image of the first segment) to repeat the autoindexing. This may give a matrix that predicts both images successfully. This should work unless the crystal orientation has genuinely changed between the two images (or the rotation axis is not normal to the X-ray beam). If this doe not work, you should derive a new orientation matrix for image from the second segment image. Remember to change the name of the file that the orientation matrix will be written to. REMEMBER to delete all the spots used to autoindex the first image if you have not already done so, or use the "Select images" menu option to choose only spots from the second image. Then use the "Autoindex" option to get an orientation matrix for this image. Under these circumstances, it is best to FIX the cell parameters for the autoindexing to those determined for the first segment of data (not yet possible for DPS indexing). This is because only a single set of cell parameters is allowed (for all segments) when doing the post-refinement. The "Refine cell" procedure allows you do define a separate orientation matrix for each segment of images.

Because the post-refinement uses partially recorded reflections, it is important to have a realistic estimate of the mosaic spread BEFORE starting post-refinement. In particular, if no value has been supplied (ie the mosaic spread is zero) the program will issue a warning message because it is unlikely that the post-refinement will work. Use the "Estimate mosaicity" menu item to obtain an initial estimate of the mosaic spread. The postrefinement will give a refined estimate of the mosaic spread, but this is not very reliable for mosaic spreads greater than about 0.7 degrees.

5.1.2 Doing post refinement in background

To use this option, the keyword :

POSTREFINEMENT SEGMENT <number of segments>

should be used, followed by PROCESS keywords defining the images to be included in each segment, with each PROCESS (see 5.3.1) keyword followed by a RUN keyword.

Example:

NEWMAT postref_3seg.mat      ! Defines the filename for the new matrix
POSTREF SEGMENT 3 
PROCESS 1 3 [ ANGLE 1.0 START 0.0 ] 
RUN 
PROCESS 43 45 [ ANGLE 1.0 START 42.0 ] 
RUN MATRIX test_88.mat 
PROCESS 86 88 [ ANGLE 1.0 START 85.0 ] 
RUN

Would use 3 segments of data (with phi values 0-3,42-45,85-88.) Note that a new MATRIX keyword has been given for the last segment, which could be necessary if the crystal has slipped during data collection. See section 5.1.1 for the best procedure to use when deriving an orientation matrix for the second or subsequent segments.

Note that the procedure uses only partially recorded reflections, and so in this case it would use partials then span images 1 and 2, 2 and 3, for the first segment etc. For this reason the PROCESS keyword MUST specify at LEAST 2 images, e.g.

PROCESS 1 1 ANGLE 1.0 START 0.0

would provide NO data for post-refinement.

If the sum of the beam divergence and mosaic spread is more than twice the oscillation angle, use the POSTREF MULTI keywords.

5.1.3 What the program actually does

During postrefinement, the images are not fully integrated (only the intensities of partially recorded reflections are measured, and by summation integration rather than profile fitting) so there is no output generate file or MTZ file. The crystal orientation will be refined for every image independently, but the cell parameters will only be refined once the final segment of data has been processed

(Note that the very last image (88 in the example above) is apparently (from the logfile) not measured at all...this is NOT an error, since the intensities of the partials at the start of image 88 are obtained while processing image 87.)

If the cell parameters change by more than 2.5 standard deviations from the input values, all images will be remeasured using the updated cell and another round of cell parameter post-refinement will be carried out. This will happen up to a maximum of 4 repeats. It is quite common that two or even three complete rounds of integration are required for convergence. For this reason it is not a good idea to include too many images in the refinement. A target of between 500 and 2000 reflection in the refinement is perfectly adequate.

It is recommended that the final cell parameters are then used to integrate all the images in the dataset, fixing the cell parameters in the post-refinement:

POSTREF FIX ALL

5.1.4 Using several segments or different crystals

Note that if the crystal has been slipping during data collection, it is possible to provide different MATRIX keywords for each segment of data, and supply a new orientation (eg derived by autoindexing the first image of the segment). When doing this, the orientation matrices for all segments (including the first) SHOULD BE OBTAINED FROM THE SAME INTERACTIVE RUN OF MOSFLM. This ensures that the matrices for the second and subsequent segments are all referred relative to the orientation matrix for the first segment. It is also a good idea to FIX the cell parameters when autoindexing the images from the second and subsequent segments, as only one set of cell parameters is allowed when refining the cell by post-refinement. It is also possible to provide new crystal identifiers for each segment (eg if the crystal has been translated and the images given a different identifier). It is also possible to use data from different crystals, but in this case there is the restriction that the orientation of the crystals must be the same (to within 20 degrees) and the relative phi values must be correct. Providing the different crystals are all indexed in the same run of MOSFLM, the relative phi values are taken care of automatically.

A possible complete example is then:

TITLE  Refine cell with 3 segments
DIVERGENCE 0.35 0.2
SYMMETRY 96
[DISTANCE 124.1]
[WAVELENGTH 1.542}
DIRECTORY /scr0/andrew/
BEAM 89.33 90.10
GAIN 1.2
NEWMAT postref_3seg.mat
POSTREF SEGMENT 3
IDENT oval1
MATRIX oval1.mat
PROCESS 1 3 [ANGLE 1.0 start 0.0]
RUN
IDENT oval2
MATRIX oval43.mat
PROCESS 43 45 [ANGLE 1.0 START 42.0]
RUN
IDENT oval3
MATRIX oval86.mat
PROCESS 86 88 [ANGLE 1.0 START 85.0]
RUN

When doing post-refinement, the crystal orientation around the X-ray beam direction (the X axis) is not defined (the refinement is based solely on the observed degree of partiality and not on the positions of the spots) and this parameter is therefore not refined, but missetting angles around Y and Z axes are refined (see Appendix IV for a definition of coordinate frames). The refinement of the detector parameter CCOMEGA allows for crystal slippage around the X-ray beam direction.

If only a narrow angular wedge of data is available for a low symmetry spacegroup (orthorhombic or lower) it is possible to FIX cell parameters that are not well defined (those closest to the direction of the X-ray beam)

eg POSTREF FIX A

5.1.5 Tips on post-refinement

In the great majority of cases the post-refinement will provide accurate cell parameters without any user intervention (providing the mosaic spread estimate is realistic). There are, however, some special cases where additional input is required to get the best results.

5.1.5.1 Processing images showing strong diffuse scatter.

It is not uncommon to observe diffuse scatter on the images, particularly for data collected at a synchrotron source. Sometimes this takes the appearance of a "halo" around the Bragg spot, because the intensity of some types of diffuse scatter peak at the positions of the Bragg reflections. This can cause difficulties in post-refinement, because it has the same effect as a crystal with a very large mosaic spread. Under these circumstances, it is best to refine the cell parameters using spots that are close to half-recorded, as the refinement is then less sensitive to the model for the "rocking curve". The minimum and maximum fraction recorded can be specified as shown below:

POSTREF FRMIN 0.4 FRMAX 0.6

will only use reflections that are between 0.4 and 0.6 recorded. (Default is 0.1 to 0.9).

6: Collecting data and processing the images

6.1     Overview
6.2     Special MOSFLM features
6.2.1   Accumulating profiles over several images
6.2.2   Addition of partials (ADDPART)
6.2.3   Post-refinement of orientation and cell
        parameters
6.2.4   Optimisation of measurement box parameters
6.3     Running a processing job
6.3.1   Running MOSFLM interactively
6.3.2   Processing the first block of data)
        (Non-interactively)
6.3.3   Finally, Processing the dataset

6.1 Overview

Before starting the serious data collection, integration of one or more images should be carried out to determine:

a) Is the crystal single?
b) Is the exposure time correct?
c) Is the crystal to detector distance correct (ie the whole of the detector is being used)?
d) Can the images be processed...are the spots separated and is the number of spatial overlaps small?

6.2 Special MOSFLM features

There are 4 features of MOSFLM which are unusual and require explanation. These are:

1. Accumulation of standard profiles over several images.
2. Addition of partially recorded reflections over adjacent images.
3. Post-refinement of cell parameters and crystal orientation.
4. Optimisation of the measurement box parameters.

6.2.1 Accumulation of profiles

In order to form well defined standard profiles (which are then used to evaluate the profile fitted intensities) fully recorded (or partially recorded) reflections over several images are added together. This improves the signal to noise and results in a better determined profile. The number of images used to form the profiles (usually between 5 and 10) is determined automatically by the program (in a way that avoids having just a few images in the final block). It can also be set manually by the BLOCK subkeyword on the PROCESS keyword line.

The positional refinement for all images in a block is carried out prior to forming the standard profiles and integrating the images. Thus each image is processed in two passes, the first pass for the positional refinement and writing all the "measurement boxes" for the spots to the SPOTOD file, and the second for actually evaluating the reflection intensities.

6.2.2 Addition of partials (ADDPART option)

The program has the option to add together the measurement boxes of the two halves of partially recorded reflections on adjacent images, thus giving the equivalent fully recorded reflection which can then be used to form standard profiles or for positional refinement of the detector parameters.

To make use of this option, the keyword:

ADDPARTials

should be given. (The default is now NOT to add partials).

Note that this procedure, which involves adding pixel values on two adjacent images, involves two assumptions:

Assumption 1

That the images have the same effective exposure time (ie total incident flux). If the rate of rotation of the spindle axis is determined by the ionisation chamber reading (as it may be on the MAR detector) then this assumption should be met. If not, then there may be an error introduced by this procedure especially if the incident beam is rapidly decaying (eg on an unstable synchrotron source). If post-refinement is being used (and by default it is used) then the program will print a warning message (to the summary file and the end of the logfile) if the exposure varies by more than 5% from one image to the next (as judged by the X-ray background).

Assumption 2

That the detector origin, orientation etc is identical for successive images, and that the images are exactly abutting (ie no overlap in rotation angle). These conditions will normally be met by the Mar, R-axis and Mac Science scanners, but mechanical wear can lead to the scanner not locking into the correct "home" position after a scan (it does one more or one too few rotations). This will show up as a variation in the ROFF distortion parameters in units of one pixel (0.15mm). The program keeps track of variations in ROFF,TOFF and CCOMEGA and will give a warning message if undue variation is detected.

**** IMPORTANT *****

If either of these assumptions is not met (this will be indicated by warning messages) then the ADDPART option should not be used.

With ADDPART, what are actually partially recorded reflections over 2 images are reclassified as fully recorded when stored in the MTZ file and they will therefore be used in scaling (SCALA). However, summed partials do carry a special flag, so that they are still classified as partials in the statistical analysis in SCALA. Thus information on partial bias, for example, is still available.

Because of the ability of SCALA to scale data when there are no fully recorded reflections, the use of this option less important than it once was. Because its use depends on the assumptions listed above, which may not always be met, the DEFAULT is now NOT to add partials.

6.2.3 Post-refinement of cell parameters and crystal orientation

By default the program will refine both cell parameters and crystal orientation using post-refinement during integration of the images. However, it is in fact preferable to determine accurate cell parameters prior to integration using the Refine cell menu option for interactive work or the POSTREF SEGMENT option in a background job. The resulting cell parameters are then input using a CELL keyword and the cell is NOT refined during integration (by using keywords POSTREF FIX ALL). This will refine the crystal orientation (and mosaic spread) but not cell parameters.

If cell parameters are refined in a processing job, the way in which the refinement is carried out depends on the crystal spacegroup. For crystals of trigonal or higher symmetry data from each pair of images in turn is used in the refinement. (This is equivalent to the POSTREF SINGLE mode.) Thus cell parameters, crystal orientation and mosaic spread are refined after every image using intensities on that image and the next one in the series. (For off-line scanners, reflections on the current image and the preceeding image are used).

For lower symmetry this is not recommended, because not all the cell parameters will be well defined using data from only one pair of images. Thus for orthorhombic and lower symmetries data is accumulated from a number of images and only then will cell parameter refinement be carried out (the crystal orientation is still refined after every image as this is well defined). By default, the number of images required for the cell parameter refinement (NADD) is set to correspond to a rotation of 10 degrees. However, this can be changed using the WIDTH subkeyword. Thus:

POSTREF WIDTH 15

specifies that 15 degrees of data must be accumulated for post-refinement of cell parameters. The actual WIDTH of data required for a satisfactory refinement will depend on the resolution (the higher the resolution, the fewer images are required) and the strength of the data (the weaker the data, the more images are required). Some experimentation may be required to find a WIDTH that gives a stable refinement. If the refinement appears unstable (ie large shifts in cell parameters) the WIDTH should be increased. If this is not possible (eg only a limited number of images have been obtained from the crystal before radiation damage set in) then the refinement of some or all cell parameters should be turned off. Thus

POSTREF FIX ALL

will fix all cell parameters.

POSTREF FIX C

will fix the "c" cell parameter etc. Normally one would fix the cell parameter that is closest to being parallel to the X-ray beam as this will be the least well defined. Alternatively, look at the standard deviations of the cell parameters to see which one(s) are least well defined. Normally the cell parameters obtained from autoindexing are quite adequate to measure 10-20 degrees of data from the image on which the autoindexing was run.

Once the appropriate number of images (NADD) have been processed, and the cell parameters have been refined for the first time, if there is a large shift in any cell parameter the program will start processing from the first image again, using the updated cell parameters. (The maximum shift allowed is determined by the subkeyword SHIFTFAC..thus

POSTREF SHIFTFAC 5

sets the maximum shift to 5 standard deviations...if larger than this the images will be reprocessed. The default value is 2.5.

From this point on, the cell parameters will be re-refined after every image, using data from the previous NADD images. For example, with 1 degree oscillation images and a width of 10 degrees, the first cell refinement will be carried out after processing image 10, using data from images 1 to 10. After processing image 11, cell parameters will be refined using data from images 2-11 etc. etc.

The missetting angles should ALWAYS be refined by post-refinement, but it may be necessary in some cases to suppress or limit refinement of cell parameters if the refinement is not stable.

The crystal mosaic spread is also refined by default, but the refined value IS NOT USED BY DEFAULT. This is because if the refinement is unstable, this can have rather drastic effects on the processing. If the refinement is stable, and there is evidence for a change in mosaic spread during the run (this often results from radiation damage), the refined values should be used by including the subkeyword USEBEAM:

POSTREF USEBEAM

If you wish to refine the horizontal and vertical beam divergence independently (good data is required to do this) use BEAM 2 :

POSTREF BEAM 2

Again, you need to include USEBEAM to actually make use of the refined values.

6.2.4 Optimisation of measurement box parameters

By default the program will automatically determine the best measurement box parameters. It will first determine the spot size from spots in the centre of the image (parameters for this search are set by keyword SPOT). This information is used to set initial sizes for the overall dimesnions (NXS,NYS in figure below) and the corner and rims parameters (NC,NRX,NRY). Following detector parameter refinement using spots from the centre of the first image, the program will then optimise the rim and corner parameters NRX,NRY and NC.

            <------------------ NXS = 23 --------------->

       ^    - - - - - - - - - - - - - - - - - - - - - - -  ^
       !    - - - - - - - - - - - - - - - - - - - - - - -    NRY =2
       !    - - - - - -                       - - - - - -  ^
       !    - - - - -                           - - - - -
       !    - - - -                               - - - -
       !    - - -                                   - - -
       !    - - -                                   - - -
       !    - - -                                   - - -
    NYS =17 - - -                                   - - -
       !    - - -                                   - - -  ^
       !    - - -                                   - - -  !
       !    - - -                                   - - -  !
       !    - - - -                               - - - -  !
       !    - - - - -                           - - - - -  NC =8
       !    - - - - - -                       - - - - - -  !
       !    - - - - - - - - - - - - - - - - - - - - - - -  !
       ^    - - - - - - - - - - - - - - - - - - - - - - -  ^
            <NRX> = 3

Figure 1. The measurement box used in MOSFLM. NXS and NYS (odd integers) define the overall size of the measurement box in pixels. NRX and NRY define the widths of the background rim and NC defines the corner cutoff. In the figure a "-" denotes a background pixel, all other pixels belong to the peak. There is no "safety rim" between peak and background.

The algorithm employed is as follows. The parameters NRX, NRY, NC are varied in turn and the value giving the highest ratio of the integrated intensity I to the standard deviation in the intensity sigma(I) is found. This is the notional optimum value for that parameter. The total intensity for this value is checked, and if it is less than the maximum intensity (for any value of the parameter) by more than a factor 0.01 (TOLERANCE), then that parameter is decreased by up to IBOUND pixels.

For example, the max I/sigma(i) might be found for a X rim value of 4, but the intensity might be only 97% of the maximum intensity found for any value of NRX (eg 1). NRX will therefore be decreased, one pixel at a time, and for each value the integrated intensity tested against the maximum value. If for NRX=2, the intensity is within 0.01 (ie 1%) of the maximum value, then 2 will be taken as the optimal value for NRX.

Thus it can be seen that the higher the TOLERANCE parameter, the SMALLER the optimised peak area will be. It is difficult to define a "correct" value for TOLERANCE, because this will depend on the degree of diffuse scatter associated with the Bragg peaks and how well adjacent spots are resolved. Values between 0.01 and 0.04 are typical, it should NOT be necessary to use values above 0.04. Note that two values may be supplied to the TOLERANCE keyword. In this case the first value is used for profiles in the centre of the image (closest to the direct beam) and the second value for the outermost profiles. An interpolated value is used for other profiles. The default value for the innermost profile is 0.01. For very close spots this can be increased to 0.02 (rarely larger).

For the first round of this iteration, when optimising NRX and NRY, NC is set to zero. NRX and NRY are varied from 1 to a maximum value which would give a peak dimension of 5 pixels. NC is varied from the smaller of NRX and NRY up to the smaller of (NX-2) and (NY-2).

Two rounds of optimisation are performed, the second round using the results of the first.

The optimisation is first carried out on the average spot profile for the central region of the detector. The optimisation of the overall dimensions (NXS,NYS) is only carried out at this stage. The background rim parameters are optimised for ALL the standard profiles.

The background rim parameters are reoptimised for each new BLOCK of images.

If the optimisation of the standard profiles causes problems (because of unusual spot shapes with long tails or other features) it can be suppressed using keywords:

PROFILE NOOPTIMISE

In this case the measurement box parameters will still be optimised for the average spot profile using spots from the centre of the detector (this profile is NOT used for integration). This box will then be expanded automatically to allow for the increase in spot size due to obliquity of incidence on the detector, but the measurement box parameters will NOT be optimised for the standard profiles (one for each area of the detector) that are used for integration. To suppress the optimisation of the measurement box parameters altogether (so that the program will use the parameters supplied on the RASTER keyword), give the keywords:

PROFILE NOOPTIMISE ATALL FIXBOX

To just suppress the optimisation of the overall size of the measurement box (parameters NXS,NYS) include keywords:

PROFILE FIXBOX

6.3 Running a processing job

Normally at this stage one would go straight on to process the first BLOCK of images (See "6.3.2 Processing the first block of data" below).

6.3.1 Running MOSFLM interactively

The following keywords might be used for an interactive run. Before starting MOSFLM, it is convenient to edit them into a file (eg comm) to save typing them several times. Then at the mosflm prompt simply type:

@comm

TITLE Processing test data
! Crystal parameters 
MATRIX oval_2_3.mat 
SYMMETRY 96 
MOSAIC 0.1

! image parameters 
IDENT oval 
PROCESS 1 TO 1 [ ANGLE 1.0 START 0.0 ]
DIRECTORY /scr0/andrew/ 
EXTENSION image

! beam parameters 
DIVERGENCE 0.35 0.2 
POLARISATION MIRRORS

! detector parameters 
BEAM 89.33 90.10 
BACKSTOP CENTRE 88.5 91 RADIUS 14 
GAIN 1.2

! The following are read from the image header if not supplied on 
! keywords for Mar, ADSC, RaxisIV and Mac Science. The phi values (on 
! PROCESS keyword) will also be read from the header if not supplied.

DISTANCE 124.1 
WAVELENGTH 1.542

!HKLOUT oval.mtz
!SEPARATION 1.3 1.3
!RASTER 17 17 9 3 3
!GENFILE oval_1to1.gen
!RESOLUTION 3.0

PLOT 
RUN

*** NOTE WELL ***

If the data has been collected at a synchrotron source, the polarisation of the beam and the horizontal and vertical divergences (horizontal here means in the plane of the X-ray beam and the rotation axis) should be given. Values default to those for the SRS at Daresbury, UK.

The TITLE is written to the mtz and generate files.

MATRIX is the filename for the orientation matrix derived from a previous autoindexing or cell parameter post-refinement run. If you want to override the cell parameters or missetting angles in this matrix use the CELL and MISSETTS keywords respectively.

SYMMETRY gives the space group of the crystal, either as the name or the number in International Tables. Note that axial systematic absences (eg 0k0 with k odd in spacegroup P21) are measured by MOSFLM, so that the symmetry can be checked. Lattice absences however (due to face or body centring) will NOT be measured.

The IDENTifier (oval) is used as a template for the image file names, which have the form:

oval_003.image

for image number 3 for example. There is an absolute limit of 40 characters for IDENT. The extension (image in this case) is set using the EXTENSION keyword.

The template keyword is an alternative to the IDENT keyword. In this case:

TEMPLATE oval_###.image

would work identically.

PROCESS gives the images to be generated, in this case from image 1 to image 1 (ie only one image is going to be examined), with a rotation angle of 1.0 degrees, starting at phi=0 (relative to the phi values given in autoindexing). Note that for Mar Research, ADSC, RaxisIV and Mac Science scanners, the phi values need not be given, they will be taken from the image header.

DIRECTORY gives the directory name where the images are stored (up to 10 different directories can be given on one or more DIRECTORY keywords).

EXTENSION defines the extension of the image filenames (default is "image" for Mar Research scanners, "osc" for R-axis, "ipf" for Mac Science, "cor" for ESRF CCD, and "image" for unrecognised detectors.

DIVERGENCE... if two values are given, they are the horizontal and vertical beam divergences (which will differ if a monochromator is used). Only a single number need be given for isotropic divergence. "Horizontal" in this context actually means in the plane containing the rotation axis and the X-ray beam, which is horizontal in the case of the Enraf Nonius oscillation camera and the Mar Research IP scanners, but vertical for standard Raxis machines.

POLARISATION specifies the polarisation of the X-ray beam, and can be given as PINHOLE or MIRRORS (both specify an unpolarised beam), MONOCHROMATOR (for a GRAPHITE monochromator) or SYNCHROTRON followed by the degre of polarisation (0.86 for SRS).

BEAM defines the direct beam position, in mm, relative to the position of the first pixel in the image. This can be determined by taking a wax image (or plasticine) and measuring the centre of the circles using the X-windows display.

Special note for Raxis II scanners: The definition of the detector coordinates in the R-axis software is different to that adopted in MOSFLM. Thus if the direct beam coordinates have been obtained from the R-axis software, then the X and Y coordinates must be interchanged.

BACKSTOP Defines the centre and radius of the backstop shadow. Reflections lying within this circle will not be integrated. The position and size of the shadow are best determined using the X-windows display.

GAIN defines the gain (adc units per X-ray photon) of the detector. This should be constant for a given detector (and a fixed wavelength). It is CRUCIAL to have a reasonable estimate of the gain as many aspects of the program use counting statistics derived standard deviations to determine acceptable spots for refinement, profiles etc. The correct value of the gain should give a BGRATIO of unity (the BGRATIO is printed as part of the MOSFLM output, as a function of intensity). The actual value of BGRATIO obtained can be used to get an improved estimate of the gain using the relationship:

true gain = estimated gain * (bgratio)**2

The gain is typically between 1 and 2, but can be as high as 5 for Raxis II detectors. See section 8.1 for a way of estimating the GAIN of your detector.

IMPORTANT. The GAIN should not be interpreted too literally. The way it is derived (section 8.1) is only strictly correct if all pixels are independent, which in practice they are not. For this reason, this parameter should NOT be used to assess the relative performance of different detectors.

DISTANCE is the crystal to detector distance in mm.

WAVELENGTH is the radiation wavelength (defaults to 1.5418).

HKLOUT gives the name of the output mtz file. This contains the Lp corrected intensities and standard deviations, with the reflection indices reduced to the asymmetric unit. This file can be used (after sorting) as input to the CCP4 programs SCALA. HKLOUT can also be given on the command line, but if both are given that specified by the keyword takes precedence. If not given, the filename is made up from the image identifier and the first image number, so in this case would be "oval_001.mtz".

SEPARATION (in mm in detector directions X and Y, ie horizontal and vertical for the Mar IP scanner) gives the minimum allowed separation of two spots before they are flagged as spatially overlapped (spots are treated as being ellipsoidal in shape, the numbers given being the full axial lengths in the X and Y directions). No attempt is made to integrate spatially overlapped reflections. If not given, the program will work out suitable values based on the spot size in the centre of the image. It will also determine if the "CLOSE" option needs to be used (see SEPARATION in the helpfile for more details). For spots that are not, in fact, completely resolved, the values determined by the program may be too conservative and lead to a very large number of spots being rejected as overlapped. In such cases, the SEPARATION should be defined explicitly.

RASTER gives the parameters of the measurement box (see Fig. 1 above). As explained in section 6.2.4 above, these values will be determined automatically if not supplied.

GENFILE specifies the generate filename. If not given, it will default to the MTZ filename, but with the suffix ".gen"

RESOLUTION: If not given, the resolution is set by the physical size of the detector. Both high and low resolution limits can be given. A "dynamic" high resolution limit, which depends on the mean I/sig(I), can be set using the CUTOFF keyword. A specific resolution range can also be excluded (eg to eliminate an ice ring) with the EXCLUDE keyword.

PLOT invokes the X-window interactive graphics option

RUN will start the processing

The image will be displayed with the predicted pattern overlaid. Fully recorded reflections are displayed as blue boxes, partials as yellow boxes, spatial overlaps as red boxes and reflections rejected as being too wide in phi (default 5 degrees, can be reset with keyword MAXWIDTH) as green boxes. Clicking (left mouse button) on the centre of a box will result in the reflection indices, phi value and phi width being displayed in the "Output" window. If examining the image after integration, the profile fitted intensity and standard deviation will also be given. See below for more details on examining the image after integration.

The image should be examined carefully to ensure that the predicted pattern actually matches what is on the image. If it does not, then any of the relevant parameters (cell dimensions, missetting angles, beam divergence, etc etc) can be adjusted and the pattern re-predicted (use the predict menu item). A brief description of the various menu options is given below:

Predict If the cell dimensions or any other parameter in the "Display Parameters" window is changed, selecting this menu item will re-calculate the predicted reflections and display them.

Clear prediction This will delete the predicted pattern. It can be restored by choosing the "Predict" menu item.

Adjust If there is an error in the beam coordinates or camera constants the calculated pattern will be displaced relative to the observed pattern. The "Adjust" option allows this to be corrected. The mouse is used to input the calculated and the observed positions of two spots. From this the program calculates the shift, rotation and scale factor required to superimpose these two spots. The values of the shifts required are given and the user given the choice of accepting the transformation or not.

Auto-refine This will refine the crystal missetting angles using the AUTOMATCH option (see help library for more details), which essentially adjusts the missetting angles to try to optimise the fit of the predicted to the observed pattern. This can converge from initial errors of 1-2 degrees, but the final parameters are NOT as accurate as those obtained from post-refinement. With the use of REFIX, this option should not normally be necessary. Note that it will use the default parameters for the refinement (eg only using data to 6A). You may wish to modify these using the appropriate keywords...see help library documentation. After refinement it will go on to measure the image. **** THIS OPTION IS NO LONGER SUPPORTED ****

Integrate This will close down the display and measure the image in the normal way. You are given the option of displaying the image again after positional refinement has been carried out, or after the integration has been carried out. (See below)

Find hkl If this menu item is activated, the user is prompted for the hkl indices of the spot he wishes to find. A blue cross is drawn on the position of the spot. A warning is given if the spot does not lie in the displayed part of the image. This can be useful in identifying bad spots.

Pick This will display the actual pixel values in a box around the cursor position (the size of the box can be set in the "Display Parameters" window.

Measure Cell Allows measurement of cell parameters, but the distance and wavelength must be set correctly.

Circles Puts up resolution circles

Beam/ backstop Will determine the centre of a circle defined by clicking with the mouse at a series of points lying on the circle. After selecting "Fit circles" select a series of points with the mouse and then selet "Fit points". The rms fit, radius and centre of the circle will be given. The user has the option to update the direct beam coordinates to the circle centre. Used to determine the direct beam position from wax rings.

EXIT Closes down the display. If the image was being displayed with the IMAGE keyword, the mosflm prompt will return. If all keywords for processing are given, then MOSFLM will proceed to measure the image(s).

Examining the image after integration

There is the option to update the display after integration (At the bottom of the "Processing Parameters" window, this is one of a number of On/Off or Yes/No toggles). If this option is chosen, once the profiles have been determined and the image integrated, the image plus the predicted pattern will again be displayed again. (Note that there is an overhead associated with this, because the image will have to be read into memory again (unless only a single image is being processed)).

If there are any "bad spots" (ie poorly measured reflections) on the image a window will be displayed which gives the user has the option to examine and/or edit the badspots. If this option is selected, the image will be displayed with a new menu option "Bad spots". Bad spots can then be reclassified as acceptable and other (accepted) spots can be re-classified as rejected.

In addition to the predicted reflections, vectors will be drawn indicating the difference between the predicted and observed spot positions for FULLY RECORDED reflections (these vectors are in red....it may be necessary to use the "Clear Prediction" menu option to see them clearly. The vectors can be scaled using the "Vector scale" menu item in the Processing parameters window. Also a minimum intensity threshold for the display of these vectors can be set using the "Threshold" menu item.

The vectors will of course be longer for weak spots than strong ones, but for all reflections the direction of these vectors should be random. If this is not the case, it suggests errors in crystal cell parameters or orientation, or misclassification of fully recorded/partially recorded reflections, or the existence of spatial distortion which is not being correctly modelled.

"Badspots" will be indicated as red crosses, rejected reflections as blue croses. Rejected reflections will normally be those containing a zero pixel value (because the measurement box extends outside the scanned area of the image plate); these are NOT classified as "badspots". They may also arise if a very large number of background pixels have been rejected.

Overloaded reflections will be indicated as green crosses. Note that you MUST include keywords PROFILE OVERLOAD in order to estimate the intensities of overloaded reflections by profile fitting.

By clicking on a reflection, the LP corrected profile fitted intensity and standard deviation will be given in the output window.

6.3.2 Processing the first block of data (Non-interactively)

It is usually advisable to process a block of (say) 10 degrees of data prior to processing the complete dataset (as this is quite time consuming) just to check that the processing is satisfactory.

The commands for MOSFLM might now be:

TITLE Procesing test data

! Crystal parameters 
MATRIX oval_2_3.mat 
SYMMETRY 96 
MOSAIC 0.1

! image parameters IDENT oval 
PROCESS 1 TO 10 [ ANGLE 1.0 START 0.0 ] 
DIRECTORY /scr0/andrew/ 
EXTENSION image

! beam parameters 
DIVERGENCE 0.35 0.2 
POLARISATION MIRRORS

! detector parameters 
BEAM 89.33 90.10 
BACKSTOP CENTRE 88.5 91 RADIUS 14 
GAIN 1.2 
DISTORTION ROFF 0.3 TOFF 0.1

! The following are read from the image header if not supplied on 
! keywords for Mar, ADSC, RaxisIV and Mac Science. The phi values (on 
! PROCESS keyword) will also be read from the header if not supplied.

DISTANCE 124.1 WAVELENGTH 1.542

!The following are optional, if not given, the program will set 
!suitable defaults. 
!HKLOUT oval.mtz 
!SEPARATION 1.3 1.3 
!RASTER 17 17 9 3 3 
!GENFILE oval_1to1.gen 
!RESOLUTION 3.0

PLOT 
RUN

See above (6.3.1 Running MOSFLM interactively) for a description of each of the keywords.The only difference to the commands described above is that the PROCESS keyword has been set up to process the first 10 images. The ADD subkeyword on the PROCESS line specifies that the batch number on the output mtz file should be (1000+image number) (ie 1001-1010 in this example).

DISTORTION specifies the ROFF and TOFF values for this scanner (See Appendix IV). It is not normally necessary to specify these values unless thay are large (greater than 0.3).

The program will then form the standard profiles by summing reflections over the first block of images 1 to 5 and print the resulting profiles. The number of images in a block is set by the program, but may be set explicitly by the PROFILE BLOCK keywords).

6.3.2.1 Formation of the Standard Profiles

The standard profiles are determined in a number of areas across the detector. By default the detector is divided into 9 regions for data to a resolution lower than 2.5A and 25 regions of which only 22 lie within the active area of a circular detector for resolution higher than 2.5A. Alternatively the user can define the set of lines parallel to the detector X and Y axes which define the standard areas. This is done with keyword PROFILE XLINES... YLINES...

EXAMPLE:

PROFILE XLINES 0 45 90 135 180 YLINES 0 45 90 135 180

will divide the a detector 180x180mm into 16 areas. See MOSFLM Help file for more details.

A separate standard profile is evaluated for each of these areas.

The program prints out some statistics on the standard profiles, followed by statistics on profiles that it has averaged (if any) and followed by a representation of each of the standard profiles using a single character (0- 9, then A-Z) to represent the value at each pixel (A [ denotes a negative value). In these representations, a minus sign denotes the background region, and a * denotes rejected pixels. Background pixels which are overlapped by the peak regions of neighbouring spots are automatically rejected by the program. It will also warn you if the peak regions of neighbouring spots overlap.

6.3.2.2 Inadequate profiles

Each standard profile has to satisfy two criteria before it is considered acceptable. There must be at least ten contributing reflections, and the rms variation in the background plane (after rejecting outliers) must be less than 10 (after scaling the profile to a maximum value of 255). If a profile fails to pass either test, then it is averaged using the profiles from neighbouring areas on the detector. Profile averaging should be avoided if at all possible. The averaging inevitably produces a profile that is less broad than the original profile because it is dominated by the stronger, lower resolution data. Look at the printed profiles before and after averaging to confirm this.

Accumulating the profiles over a BLOCK of say 10 images (see below) should help provide a sufficient number of reflections, but is unwise to accumulate over too wide a phi range because this will average out any genuine variation in the profiles with phi (eg due to a change in effective diffracting volume). Both rejection criteria can be changed using subkeywords (NREF for number of reflections, RMSBG for rms variation in background, on the PROFILE keyword line) and it is usually better to avoid averaging by changing these criteria if necessary:

PROFILE RMSBG 20 NREF 5

6.3.2.3 Spots running into each other

The program will give a warning if it detects that the peak areas of adjacent spots overlap. There are two possible ways around this:

1) Increase the SEPARATION parameters. Making the separation significantly smaller than the actual spot size in the centre of the image can lead to serious problems and is NOT recommended.

2) The actual spot size that the program works with when testing for peak overlaps (after rejecting those that are too close as determined by the SEPARATION parameters) is determined by the "profile optimisation"...that is when the program works out the best values of the measurement box parameters NC, NRX, NRY, which is done independently for each of the standard profiles. If there is significant diffuse scatter on the image, the "optimised" raster parameters may well produce a peak area that is actually slightly broader than the true Bragg peak and includes part of the "diffuse" peak. This can be checked by examination of the standard profiles...if the peak area contains many pixels with values of 0 or 1 then it suggests the peak is too broad. This effect can be overcome by increasing the TOLERANCE parameter (See the help library for more details on how the optimum parameters are derived and the effect of the TOLERANCE parameter). The default value for this parameter is 1% ie 0.01. Increasing it (try steps of 0.005, but it should not be necessary to go above 0.04) will result in a reduction of the optimised peak size. It is up to you to decide what the optimimum value is on the basis of the appearance of individual spots in the image.

6.3.2.4 Output Statistics

The other statistics produced are for general information, and are described in the MOSFLM help library under "Output". Probably the most useful is the breakdown of I/sig(I) as a function of resolution. This will give an immediate idea of the quality of the data...particularly at the high resolution end. For guidance, a mean I/sig(I) of 3.0 will give an R-merge of between 20% and 30% in SCALA. If there are symmetry related fully recorded (or summed partial) reflections on a single image, statistics are also provided on the agreement between their intensities.

Check the following in this job:

1) Check the standard profiles look OK (ie the peak is within the peak region).

2) Check the weighted residual is about 1.0.

3) CHECK FOR WARNING MESSAGES. These are given at the end of the logfile and in the summary file. They will point out possible problems and suggest a way around them.

6.3.3 Finally, Processing the dataset

The whole philosophy of MOSFLM is to allow the entire dataset (or all images obtained from a single crystal) to be processed in a single job. To make this possible, the crystal orientation can be refined continuously for every image, to take account of possible crystal slippage, and the cell parameters can also be refined if the initial estimates are not accurate. An accuracy of 1 part in 1000 or better is required for optimal processing of high resolution data.

There are a large number of adjustable parameters within MOSFLM, but considerable effort has gone into making the program select an appropriate value for these parameters. The program defaults should therefore ALWAYS be used unless there is a very specific reason for changing parameters (eg if it is suggested in the warning messages in the summary or logfile)

See section 9.3 for a complete example command file.

7: Interpreting the output

7.1     The log files
7.2     The summary file
7.3     Checking the quality of the data

7.1 The log files

7.2 The summary file

7.2.1 Statistics on Processing

7.2.2 Results of the post-refinement

7.3 Checking the quality of the data

8: General tips

8.1     Estimating the GAIN of a detector
8.2     Processing images with no (or very few) fully
        recorded reflections
8.3     Processing images when the spots are not fully resolved
8.4     Processing data from other detectors, or standard
        detectors with different rotation axis orientation. 

8.1 Estimating the gain of the detector.

IMPORTANT. The GAIN should not be interpreted too literally. The way it is derived is only strictly correct if all pixels are independent, which in practice they are not. For this reason, this parameter should NOT be used to assess the relative performance of different detectors.

8.2 Processing images with no (or very few) fully recorded reflections

8.3 Processing images when the spots are not fully resolved

8.4 Processing data from other detectors

9: example command files

9.1     Autoindexing an initial image (interactively)
9.2     Determining an accurate cell
9.3     Integrating a series of images

There follow a number of example command files. In general, it is best to let MOSFLM choose appropriate default values for processing, and to only specify additional parameters if you get warning messages suggesting changes (these warning messages are given in the Summary file and at the end of the logfile).

It is usually NOT a good idea to start with someone elses command file, as they have have set some parameters which are not appropriate for your data.

9.1 Autoindexing an initial image

This will normally be done interactively. The commands can be put in a file (eg "runit") and executed by typing @runit at the MOSFLM prompt.

=> TITLE My lysozyme data               ! This title is transferred to the
                                        ! MTZ file

=> IMAGE lyso_001.image [PHI 0 TO 1]    ! Filename of first image. For Mar,
                                        ! ADSC, Mac Science and RaxisIV images
                                        ! the phi values will be taken from
                                        ! the image header if not given here.

=> BEAM 150.0 149.0                     ! Direct beam coordinates

If not a Mar Research IP scanner:
=> DETECTOR RAXISIV                      ! or RAXIS (for RAXIS II) or DIP2000 etc

If not processing Mar Research, R-axis or Mac Science images:
=> WAVE 0.91                            ! For Mar, ADSC,RaxisIV and Mac Science this
=> DISTANCE 300                 ! information is taken from the header
                                        ! but can be overwritten using the 
                                        ! keywords.

=> SYMM p43212                  ! If known, give cell and symmetry
=> CELL 79 79 38                        ! otherwise omit completely.

Not essential for first stages, but needed for integration
=> DIVERGENCE 0.1 0.03          ! If isotropic, the beam divergence
                                        ! can be included in the mosaic spread.
=> SYNCHROTRON POLARIZATION 0.9 ! Defaults to 0.86 (SRS, Daresbury UK)
=> GAIN 1.7                             ! See section 8.1 for a way to
                                        ! estimate the gain if not known.
=> GO

Further options are then usually invoked via the menu of the X-window interface.

9.2 Determining an accurate cell

This example assumes that an orientation matrix has been obtained for the first image, and that accurate cell parameters are to be determined using two (one can use one or more) segments of data. Note that this can be done interactively via the X-windows menu, the example given below is for a background job.

 ipmosflm spotod /scr0/andrew/f1adpx3.spotod \ 
<< eof-ipmosflm
TITLE Postrefining the cell with two segments 

! Source parameters
WAVE 0.91                       ! For Mar, ADSC, RaxisIV and Mac Science
                                ! this information is taken from the
                                ! header but can be overwritten using
                                ! the keywords.

SYNCHROTRON POLARISATION 0.86   ! Default polarisation is 0.86 (SRS)
DIVERGENCE 0.10 0.02            ! Horizontal and vertical divergence
DISPER 0.00020                  ! Wavelength dispersion

! Detector parameters
DETECTOR RAXISIV                 ! If not a Mar Research IP scanner:
                                ! can be  RAXIS (for RAXIS II), DIP2000 etc

BEAM 150.0 149.0                ! Direct beam coordinates
GAIN 1.2                        ! Detector gain
DISTANCE 300                    ! For Mar, ADSC, RaxisIV and Mac Science
                                ! thisinformation is taken from the
                                ! header but can be overwritten using
                                ! the keywords.
BACKSTOP CENTRE 148 151 RADIUS 12       ! Beamstop shadow

! Crystal parameters
SYMMETRY P212121
MATRIX image_001.mat            ! orientation matrix for first image
MOSAIC 0.22                     ! Mosaic spread, should be a reasonable estimate

! Image parameters
IDENT f1adpx3                   ! Sets template for image filenames
DIRECTORY /scr1/andrew/images/  ! Directory where images are stored

! Processing parameters
POSTREF SEGMENT 2               ! postrefine using two segments
PROCESS 1 to 4 [START 0 ANGLE 1]  ! Images to be used in first segment.
                                ! For Mar, ADSC, RaxisIV and Mac Science
                                ! the phi values are taken from the
                                ! header but can be overwritten using the 
                                ! keywords.
NEWMATRIX f1adpx3_postref.mat   ! Filename for output orientation matrix
                                ! This will contain the refined cell
                                ! and the refined missetting angles for
                                ! the first image (1 in this case).
GO
PROCESS 86 to 89 [START 85 ANGLE 1.0]   ! Images to be used in second segment
! MATRIX image_086.mat          ! If necessary (ie crystal has slipped) can
                                ! specify an orientation matrix for the first
                                ! image of the second run.
GO
eof-ipmosflm

Possible additional keywords:

1) If there is significant diffuse scatter in the image, or if the mosaic spread is very large (greater than 0.5 degrees) it is usually best to limit the post-refinement to using reflections that are nearly half-recorded, using the FRMIN, FRMAX keywords. This will make the refinement less dependent on the model of the rocking curve:

eg POSTREF FRMIN 0.4 FRMAX 0.6

2) If there is an ice ring or spots on the image, all spots within a specified resolution limit can be rejected.

eg RESOLUTION EXCLUDE 3.66 3.72

9.3 Integrating a series of images

This example assumes that an accurate cell has already been obtained (using a POSTREF SEGMENT run) so no further refinement of cell parameters is required. Note that integration can be done interactively via the X-windows menu, the example given below is for a background job.

ipmosflm spotod /scr0/andrew/f1adpx3_1to90.spotod \
summary f1adpx3_1to90.sum \
coords /scr0/andrew/f1adpx3_1to90.coords \ 
<< eof-ipmosflm 
TITLE Postrefining the cell with two segments ! Source parameters 
WAVE 0.91               ! For Mar, ADSC, RaxisIV and Mac Science this
                        ! information is taken from the header 
                        ! but can be overwritten using the
                        ! keywords.
SYNCHROTRON POLARISATION 0.86 ! Default polarisation is 0.86 (SRS)
DIVERGENCE 0.10 0.02      ! Horizontal and vertical divergence DISPER 0.00020
                          ! Wavelength dispersion
                          ! Detector parameters 
DETECTOR RAXISIV           ! If not a Mar Research IP scanner:
                          ! can be RAXIS (for RAXIS II), DIP2000 etc
BEAM 150.0 149.0          ! Direct beam coordinates 
GAIN 1.2                  ! Detector gain
DISTANCE 300              ! For Mar, ADSC, RaxisIV and Mac Science this 
                          ! information is taken from the header 
                          ! but can be overwritten using the 
                          ! keywords. 
BACKSTOP CENTRE 148 151 RADIUS 12    ! Beamstop shadow
! Crystal parameters 
SYMMETRY P212121 
MATRIX f1adpx3_postref.mat ! orientation matrix previous job. 
MOSAIC 0.22                ! Mosaic spread, should be a reasonable estimate

! Image parameters 
IDENT f1adpx3                      ! Sets template for image filenames
DIRECTORY /scr1/andrew/images/     ! Directory where images are stored 


! Processing parameters 
POSTREF FIX ALL            ! do not refine cell, only crystal orientation

PROCESS 1 to 90 [START 0 ANGLE 1] ADD 1000 ! Images to be integrated.
                                           ! For Mar, ADSC, RaxisIV and Mac Science
                                           ! the phi values are taken from the header 
                                           ! but can be overwritten using the 
                                           ! keywords. The batch numbers in the output 
                                           ! MTZ file will be the image number plus 1000 
                                           ! (ADD keyword).

HKLOUT f1adpx3_1to90.mtz      ! Name of output MTZ file. 
GO 
END 
eof-ipmosflm
# Delete temporary files 
/bin/rm /scr0/andrew/f1adpx3_1to90.spotod 
/bin/rm /scr0/andrew/f1adpx3_1to90.coords 
/bin/rm f1adpx3_1to90.gen 

Possible additional keywords:

PROFILE TOLERANCE 0.02 0.03 
PROFILE XLINES 0 75 150 225 300 YLINES 0 75 150 225 300 
SEPARATION 0.75 0.75 CLOSE 
AUTOINDEX
CELL KEEP 283.09 107.60 139.65 90.000 90.000 90.000
REJECTION PKRATIO 4.0 
RESOLUTION EXCLUDE 3.66 3.72 
RESOLUTION ANISOTROPIC 3.5 2.4 2.3
TWOTHETA 15
BEAM SWUNG_OUT 99.96 124.5 
LIMITS RSCAN 147.0 

These are described below.

1) Optimising the Standard Profiles

Depending on the strength of the images, the degree of diffuse scatter, the spot separation on the images, the crystal mosaicity etc, it may be necessary to adjust the PROFILE TOLERANCE parameters to get well defined standard profiles. The appearance of the standard profiles should always be checked in the logfile, to ensure that adjacent spots are not included in the "peak" region, that long diffuse tails are not being included in the peak, and that not too many profiles are being averaged (see below).

eg PROFILE TOLERANCE 0.02 0.03

The first value is used for profiles near the centre of the image, the second value for profiles at the outside, and an interpolated value for profiles inbetween. Defaults are 0.01 0.01 for a lab source (wavelength 1.542) and 0.01 0.03 for a synchrotron.

It should not normally be necessary to use values above 0.04.

For weak images, you may find that some profiles are being averaged. This is to be avoided if possible. Consider if you are trying to integrate beyond the true resolution limit of the crystals. If not, try to avoid the averaging by one of the following:

i) If profiles are being averaged because there are very few reflections, but the reflections are reasonably strong so that the profiles look OK, reduce the minimum number of reflections required (default 10): eg PROFILE NREF 5

ii) If the profile is being averaged because the rms variation in the background (after scaling the peak to 255) is too large, but in fact this is because there is significant diffuse scatter which should not be included in the Bragg peak, then increase the allowed rms variation (default 10.0): eg PROFILE RMSBG 20.0

iii) Try setting up fewer standard profiles, by defining the regions on the detector where the profiles are to be set up using the XLINES,YLINES keywords:

eg PROFILE XLINES 0 75 150 225 300 YLINES 0 75 150 225 300

will give 16 areas over which a profile will be determined.

2) Specifying the minimum spot separation

The program will automatically determine the minimum allowed spot separation based on the size of spots in the centre of the first image to be processed. Spots closer than this will not be integrated. However, if the spots are very close together, the minimum spot separation determined by the program may be too conservative and result in many spots being rejected. To avoid this, set the minimum spot separation explicitly. Note that in such cases the "CLOSE" option for spot integration should be used (see the Help library under "SEPARATION" for further details of this option). The program also decides if the "CLOSE" option need to be invoked, again based on the very first image to be processed. It is a good idea to ensure that if the CLOSE option is used for one segment of data (eg the 90 images processed in the job above) then it is also used for all other data from this crystal, by specifying its use explicitly.

eg SEPARATION 0.75 0.75 CLOSE

or if just wanting to enfore the use of the CLOSE option:

SEPARATION CLOSE

3) Refining cell parameters during integration

In this case, replace the POSTREF FIX ALL keywords with

POSTREF WIDTH 10

where WIDTH specifies the width (in phi) of data to be used in the refinement. For trigonal or higher symmetry a few degrees of data is usually sufficient, for lower symmetries at least 10 degrees is usually necessary. THIS IS NOT RECOMMENDED: It is usually preferable to determine the cell initially using the POSTREF SEGMENT option.

4) Determining the orientation as part of the job

It is possible to invoke the autoindexing as the first part of a processing job. By default the indexing will be done on the first image to be processed, but the images to be used can be specified explicitly (See section 3.2). In these circumstances the unit cell derived from the autoindexing will be used in the integration, unless the KEEP subkeyword is given with the CELL. Usually an accurate cell (from post-refinement) will be available and should be used in the integration:

 AUTOINDEX [ IMAGE 1 2 3 ] 
CELL KEEP 283.09 107.60 139.65 90.000 90.000 90.000 

5) Specifying the size of the integration box

The overall dimensions of the integration or measurement box are determined automatically based on strong spots from the first image to be processed. If you want to make the box smaller or larger than this, specify its size explicitly and tell the program not to change it:

 RASTER 25 25 17 3 3 
PROFILE FIXBOX 

The corner and rim parameters (17 3 3 in above case) will still be optimised, but the overall dimensions will not (use PROFILE NOOPTIMISE (ATALL) to suppress optimisation).

6) Excessive numbers of "Bad spots"

Excessive numbers of "Bad spots" is usually a sign that the processing is not as good as it should be and there are errors in the cell parameters, crystal orientation, mosaic spread or in the detector itself. However, if the cause of the bad spots cannot be corrected, it may be preferable to change the rejection parameters to avoid too many reflections being rejected. This is particularly true if a large number of strong reflections are being rejected because of a poor profile fit, because this could have a significant effect on Patterson based methods used in Molecular Replacement, definition of solvent masks etc.

eg REJECTION PKRATIO 5.0

This should ONLY be done as a last resort, it is much better to find the cause of the rejections. See REJECTION keyword in Help library for more details.

7) Ice rings

If there is an ice ring or spots on the image, all spots within a specified resolution limit can be rejected.

eg RESOLUTION EXCLUDE 3.66 3.72

8) Anisotropic diffraction

If the crystal diffraction is very anisotropic, an anisotropic resolution limit can be applied.

eg RESOLUTION ANISOTROPIC 3.5 2.4 2.3

9) Inclusion of partially recorded reflections in refinement and profiles

The program will decide automatically if partials should be included in the refinement of the detector parameters and formation of the standard profiles. If however you want to explicitly force the program to do so use the following keywords: REFINEMENT INCLUDE PARTIALS for detector refinement PROFILE PARTIALS for profile formation. To explicitly EXCLUDE partials from profile formation use: PROFILE FULLS

10) Processing data from an offset detector (in two-theta)

To process data collected from an offset detector, the swing angle needs to be specified, and the direct beam coordinates should be those corresponding to a two-theta value of zero, unless the SWUNG_OUT subkeywords are given. The areas for the standard profiles MUST be specified explicitly using the PROFILE keyword.

TWOTHETA 15 BEAM SWUNG_OUT 99.96 124.5 ! Direct beam position on swung out ! detector PROFILE XLINES 0 50 100 150 200 YLINES 0 50 100 150 200

11) Excluding a "shadow" around the rim of the image

eg. LIMITS RSCAN 146.5

12) Excluding more general "shadows"

In addition, rectangular areas of the detector can be excluded from spot finding and integration by use of the LIMIT EXCLUDE keywords. See the helpfile for details.

13) Resetting the saturation point of the detector

If there is evidence that the detector is saturating before the default cutoff value, it can be reset with the OVERLOAD CUTOFF keywords:

eg OVERLOAD CUTOFF 75000

14) Limiting the resolution

Normally the resolution limit is set by the physical dimensions on the detector. This can be overridden by the RESOLUTION keyword:

eg RESOLUTION 3.5

Both inner and outer resolution limits can be set:

eg RESOLUTION 20.0 3.5

The resolution can also be determined by the quality of the data, so that the resolution limit is determined by the mean value of I/sig(I) within a resolution shell.

eg RESOLUTION CUTOFF 5.0 |

Appendix I - Changes in MOSFLM

Appendix II Setting the measurement box parameters manually

==>

Appendix III Overview of MOSFLM

1) Introduction

The MOSFLM program

a) Overview.

---------------------------------|
|                            Read in first two images
|                   (the first image is the 'current image')
|  ------------------------------|-------------------------------------------|
|  |             Generate reflection list for current image                  |
|  |          using the latest parameters (orientation cell, etc)            |
|  |                             |                                           |
|  |       Refine detector parameters, initially using reflections from      |
|  |        the central region, then over the entire detector.               |
|  |        Optimise the measurement box parameters for the average          |
|  |        spot profile of centre of image. This is done for the first      |
|  |                image of every new BLOCK of data.                        |
|  |                             |                                           |
|  |                             |                                           |
|  |     Simultaneously integrate the two halves of partially recorded       |
|  |        reflections for use in post-refinement                           |
|  |                             |                                           |
|  |         Refine crystal orientation etc. using post-refinement           |
|  |                             |                                           |
|  |                 Are shifts acceptably small ?                           |
|  |                             |                                           |
|  |                             |----------YES---------------               |
|  |                             |                           |               |
|  |                             NO                          |               |
|  |                             |                           |               |
|  |            Is post-refinement in single image mode ?    |               |
|  |                             |                           |               |
|  |------------- YES -----------|                           |               |
|                                NO                          |               |
|                                |                           |               |
|              Is this the first post-refinement run ?       |               |
|                                |                           |               |
|--------------------YES---------|                           |               |
                                 NO                          |               |
                                 |                           |               |
                       Print warning message                 |               |
                                 |----------------------------               |
             Is refinement residual acceptably small ?                       |
                                 |                                           |
        STOP------------NO-------|                                           |
                                YES                                          |
                                 |                                           |
 Have sufficient images been accumulated to form standard profiles ?         |
                                 |                                           |
                                 |-------------- NO --- Read next image -----
                                YES
                                 |
           Rewind scratch file. Form standard profiles.
       Optimise measurement box parameters for each profile.
       Reject all background pixels overlapped by neighbouring spots.
                Integrate all images in this block
             Write intensities back to generate file.
  Apply Lp corrections, reduce indices to asymmetric unit write to MTZ file.
          Calculate R-factor for symmetry related reflections.
             Write summary file information.
                                 |
                Process next block of images (if any)

c) Generation of the reflection list

Generation of the reflection list is performed using the Reeke algorithm.

d) Initial refinement of detector parameters.

The first step in the refinement of the detector parameters is the location of up to 60 suitable reflections. Generally, only fully recorded reflections are selected, as the position of the centroid of a partially recorded reflection will depend on its degree of partiality. For crystals with a high mosaic spread, there may be too few fully recorded reflections to allow a satisfactory refinement, so partially recorded reflections may have to be used. Similarly, overloaded reflections will have a poorly determined centre of gravity, although for very intense images it may be necessary to include some overloaded reflections.

e) Location of reflections in outer region of the detector and further refinement.

g) Extracting the measurement boxes from the digitised image.

A final list of all reflections to be integrated is prepared and sorted on the detector X coordinate. Using a circular buffer and working through the digitised image stripe by stripe, the pixel values corresponding to the measurement boxes of these reflections are accumulated and written to a scratch file. The size of the peak area of the measurement box is expanded automatically (by 2 pixels at a time to maintain an odd number of pixels) in order to allow for the increase in spot size due to obliquity of incidence of the diffracted beam on the detector. This is a function of the spot size (determined by collimation and mosaic spread of the crystal), the effective detector thickness and the Bragg angle. If post-refinement is to be carried out (image plate data only), then for each reflection that is partially recorded at the end of the oscillation range of the current image, the identical pixels on the next image in the series are also accumulated. This requires both images to be stored in memory. This is only practical if the detector has a fixed origin and orientation (eg the Mar Research IP scanner) so that the predicted position of the partially recorded reflection is identical in the digitised image on both images. It is unclear at present whether images scanned using an off-line image plate scanner will give sufficiently reproducible results for the method to be applicable. The measurement boxes for the two halves of the partial are then integrated (using summation integration) to give the intensity of the two components, which in turn gives an observed degree of partiality:

h) Post-refinement of crystal orientation, cell parameters and beam parameters.

The degree of partiality of a partially recorded reflection is a function of the crystal orientation, cell parameters and beam parameters (mosaic spread and horizontal and vertical divergence of the X-ray beam). These parameters can therefore be refined using the observations P(obs) described in section (g) if there is a model for the rocking curve, that is, the relationship between the distance of a reciprocal lattice point from the Ewald sphere at the end of the rotation and the resulting intensity of the reflection on that image expressed as a fraction of the total intensity. This rocking curve is therefore used to convert the observed partiality P(obs) to the position of the reciprocal lattice point at the end point of the rotation. It is this conversion which is a function of the mosaic spread and beam divergences. The position of the reciprocal lattice point is then a function of the crystal orientation and cell parameters. It should be noted that this is independent of the reflection coordinates on the detector or the crystal to detector distance.

i) Formation of the standard profiles.

j) Reflection integration.

Following formation of the standard profiles, the reflections on all images contributing to the profiles are integrated. The first step in this procedure is fitting the best background plane to the background pixels in the measurement mask. In order to deal with outliers (due to cosmic rays, spots from a satellite crystal etc) in the background region of the reflection, the initial background plane is determined using a fraction (between 0.5 and 1.0) of the total number of background points, selecting those pixels with the lowest values. The constant component of the background plane is then adjusted to correct for the systematic bias introduced by selecting the lowest pixel values (assuming a Gaussian distribution). This plane is then used to reject outliers, which are defined as pixels with values which deviate from the plane by more than a fixed number (usually 3) standard deviations, where the standard deviation is based on Poissonian counting statistics. The same procedure is applied to determining the centre of gravity of spots used in positional refinement (sections d and e). The profile fitted intensities and standard deviations are evaluated using weighted profile fitting and methods based on those originally described by Rossmann (1978). For every reflection, a new profile is evaluated by linear interpolation of the standard profiles of the regions surrounding that reflection. The interpolation is based on the distance of the reflection from each standard profile, where the coordinates of the profiles are calculated as the intensity weighted mean of all the reflections contributing to that profile. For most reflections, four different profiles will contribute to the variable profile, but for reflections near the periphery of the detector there may be only three or two contributing profiles, while no sensible interpolation can be done for the outermost reflections. This procedure provides a more accurate modelling of the way in which the profile varies across the face of the detector.

ii) Orientation refinement by pattern matching.

Appendix IV Coordinate systems used by MOSFLM

a)     Detector  Coordinates
b)     Camera Constants
c)     Coordinate Transformations
       References
    

a) Detector Coordinates

						  /!
		      Y-axis			 / !
			^			/  !
			!		       /   !
			!		      /    !
			!   / 		     /  Xd !
			!  /		    / * ^  !
			! /		    ! 3 !  !
			!/      X-ray beam  !   !  !
			/-----------------------/--!---->X-axis
		       /                    !  / *1!
		    <-/-		    ! /    !
		     /  \+ve phi	    ! Yd  /
		    /   /		    ! 2  /
		   /			    ! * /
		  Z-axis		 Ys ^ _/
		Rotation		    ! /| Xs
		 axis			    !/
                                            O                          
			     Figure. 3

b) Camera Constants

The coordinates of the direct beam position XCEN, YCEN in the scanner coordinate frame must be supplied by the user. Any deviation in the refined position of the centre of the diffraction pattern from these coordinates are denoted by the camera constants CCX, CCY. It should be noted that CCX, CCY are defined in the detector frame (Xd, Yd), NOT in the scanner coordinate frame. This a positive CCX represents a displacement along +Ys, while a positive CCY is a displacement along -Xs. Any deviation of the angle OMEGA from its expected value of 90 degrees is referred to by the camera constant CCOMEGA. The camera constants allow for errors in the user defined position of the direct beam (CCX and CCY) and in the alignment of the scan direction of the image plate relative to the camera (and detector) axes (CCOMEGA).

For film data the camera constants CCX, CCY denote the deviation of the refined position of the centre of the diffraction pattern from the midpoint of fiducial marks 1 and 3 (Fig. 3), as appropriate for an Enraf Nonius oscillation camera. They are expressed in the detector frame (Xd, Yd), so that they are independent of the orientation of the film on the scanner. The line joining fiducial marks 2 and 3 is assumed to be parallel to the detector axis Xd , and any deviation is denoted by the camera constant CCOMEGA. Note that for film data, the value of OMEGA will not necessarily be 90 degrees (normal orientation) or 0 degrees (rotated orientation) because it will depend on exactly how the film is placed on the scanner. The orientation of the film on the scanner does NOT affect the relative orientation of the line joining fiducials 2 and 3 and the detector axis Xd at the time of collecting the data. The camera constants allow for errors in the positioning of the cassette on the carousel and misalignment of the camera itself.

c) Coordinate Transformations

References