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Subsections
Autoindex and strategy calculation
The Autoindex Tab is used to autoindex test diffraction
images (with the option to collect the test images too) and to
calculate data collection strategies that optimize data
completeness. It can also be used to inspect a data collection
strategy generated during sample screening.
The autoindex and strategy results for each sample are grouped
under as a Run. Each run has got a unique name. The autoindex
Tab navigation toolbar contains links to display
a summary of all the user's runs (under user Runs), start an
interactive application to generate new runs (New Run) or
inspect or edit one of the runs (Selected Run)
Figure 4:
Navigation toolbar in the Autoindex tab
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The Runs page displays a table
summarizing all the autoindexing results generated by Web-Ice.
Figure 5:
Autoindex tab page showing a summary of all autoindex runs.
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For each run, the following are listed:
- The images used for autoindexing.
- The crystal score calculated as
, where is
the resolution limit in Å, rmsr (described above) is in mm, and
is the mosaicity in degrees.
- The number of spots in the image and
the number of reliable diffraction spots (found after rejecting the
weakest and strongest spots).
- The number of ice rings.
- The autoindex
software best guess of the crystal lattice.
- The estimated diffraction resolution limit based on
statistics.
The runs can be sorted by alphabetical order or by time of
creation. The Update reloads the page (this is useful to
display results generated by Web-Ice in the background since the last
visit to the page)
Clicking on a run name allows the user to edit and reuse the run
or inspect the results. Once a run has been selected, it will be
highlighted in the user Runs table. The user can revisit a
selected run by clicking on the name, or using the Selected Run
link in the navigation toolbar.
To clear the run and all its contents, click the link [Delete] in the Commands column of the user Runs table.
Generating a new run
Selecting New Run link in the Autoindex navigation toolbar opens a new run form
page. The user has three options: 1) autoindex existing
images stored in the MC computers, 2) import a strategy
calculated during screening of the currently mounted sample, or 3) to collect and autoindex new test images from a mounted sample. The
last two options require the user to connect to a
beamline and they can only be used during beamtime.
A name for the run must be chosen. The run name must be a single word of
alphanumeric characters.
Figure 6:
New run form page.
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Pressing the enter key or clicking the
Create button will create the run; clicking on the Cancel
button will return to the user Runs page.
The Run generation tool requires javascript enabled on the browser.
Autoindexing existing images
This option assumes that the user has already used Blu-Ice to collect the
diffraction images and, for anomalous dispersion experiments, the
fluorescence scans required to characterize the sample. In this case
Web-Ice can read these files and supply a data collection strategy to
the user.
The option is selected by clicking the ''Autoindex existing
images'' button in the new run form page and
clicking the Create button. This will take the user to the Setup page which guides the user through the following run set up steps (The Prev and
Next buttons can be used to navigate between the various steps):
- Choosing the images: The directory and
image names can be entered by the user or
selected using the Browse button.
Figure 7:
Selection of directory and images for autoindex.
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- Choosing the strategy: The user has the option to select
the strategy using beamline specific parameters; this option is
disabled unless the user has selected a
beamline; this option takes into consideration the detector
position and energy limits enabled at the beamline, and uses the
beam intensity to provide an estimate of the absorbed dose. The
option not to calculate the strategy is useful if the user does not
intend to collect data from the crystal, but only examine the
diffraction quality (e.g., during manual sample screening).
- Choosing the type of experiment: The types
available are Native, MAD (three wavelengths) and SAD (SAD is
also the recommended mode to collect data for Isomorphous Replacement phasing). Note
that two oscillation ranges maximizing the unique and anomalous
completeness will be always calculated regardless of the experiment
type. The range optimizing anomalous completeness will be selected by
default for MAD and SAD experiments.
Figure 8:
Selection of MAD experiments type for ''Autoindex Only'' option.
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- For MAD and SAD experiments, the energies may be input by the
user, or imported from an autochooch summary file; use the directory
browser tool to locate the summary file, select it by clicking on the
radio button next to the file name and then click on the Load
Scan File button. Important: The File filter in the Preferences tab
must contain the entry *summary for the directory browsing tool to
display this file type.
- To do a two-wavelength MAD experiment, change
the appropriate energy input box to 0 (it is best to leave out
the ''peak'' or the ''inflection'' energy - the remote energy should always
be collected on MAD experiments).
It is also possible to enter the file name
(specifying the full path name in the input text box next to the Load Scan File button. Note that only the ''peak'' energy needs to be
entered for a SAD experiment.
- The element and edge input boxes will be read in from the summary
file. Note: If a summary file is not given, it is recommended to
supply the heavy atom type. This makes it possible to give a more
accurate estimate of the radiation dose for the experiment.
- Other options: The user can select the Laue
group and cell if known. If given, the program will calculate the
strategy only for that space group. If not given, the strategy
will be calculated for the lowest space group belonging to the predicted
Bravais lattice.
For SAD and MAD experiments it is possible to enter the
number of residues and heavy atoms in the monomer. If given, and the
atom type is known, they will be used in the dose calculation for the
strategy and also the exposure time and total number of images (based
on the estimated signal).
The page also displays the program used to calculate the starting
and total rotation angle. BEST is used to calculate the
full oscillation range for data collection; for multicrystal
data collection, MOSFLM is used instead.
Figure 9:
Symmetry and strategy options.
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Collecting test images and autoindexing
This option allow the user to collect test images and a
fluorescence scan from a sample. In the current version, the sample
must be previously mounted by hand, or using the robot via
Blu-Ice, and centered by the user. The slit size should also be set to
an appropriate value before hand.
The option to collect images and autoindex can be selected from the New Run
form after choosing an accessible beamline from the
toolbar. Creating the run will take the user to the Setup
pages. The Prev and Next buttons can be used to navigate
between the following steps:
- Choose sample: In the current version of
Web-Ice, the user may only collect images from samples which have been
already mounted either by the robot (via Blu-Ice) or manually. In
later releases, it will be possible to use the robot to mount samples
from Web-Ice.
- Choose Directory and image root name: The directory where the
test images will be written to can be typed in or, if it already
exists, selected using the Browse tool. The image root name will
default to the run name.
- Choosing the strategy: The user has the option to select
the strategy using beamline specific parameters; this option takes into consideration the detector
position and energy limits enabled at the beamline, and uses the
beam intensity to provide an estimate of the absorbed dose. The
option not to calculate the strategy is useful if the user does not
intend to collect data from the crystal, but only examine the
diffraction quality (e.g., during manual sample screening).
- Choose the type of
experiment. For SAD and MAD
experiments, there is the option to collect a fluorescence scan from
the anomalous element of interest; to do this, select the Perform
Fluorescence scan option, and click on the absorption edge of
interest in the periodic table appearing at the bottom of the
page.
For SAD experiments, the periodic table will enable selection of
elements without an absorption edge in the beamline spectral
range. Clicking on the element name will select the lowest energy
attainable at the beamline for maximizing the anomalous signal
present in the data. Important: This choice of energy does not guarantee
that the anomalous signal can be detected. In some cases, collecting
high redundancy data will also be necessary; in the current version
this can only be done by manually extending the phi range supplied by Web-Ice.
Figure 10:
Selection of absorption edge to do a fluorescence scan
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If the option to collect the scan is not used, the
energies may be input by the user or imported from an autochooch
summary file; use the directory browser tool to locate the
summary file, select it by clicking on the radio button next to the
file name and then click on the Load Scan File button. You can
also enter the file name (specifying the full path name in the input
text box next to the Load Scan File button. Note: only the ''peak'' energy is
required for a SAD experiment.
To do a two-wavelength MAD experiment, change one of the energy input boxes to 0 (it is best to leave out
the ''peak'' or the ''inflection'' energy - the remote energy should always
be collected on MAD experiments).
Note: If a fluorescence scan is not collected and summary file is not given, it is recommended to
supply the heavy atom type. This makes it possible to give a more
accurate estimate of the radiation dose for the experiment.
- Choose other options: The user can choose the
exposure time, oscillation angle, beam attenuation and resolution for
the test images. The default exposure time is a reasonable value for
well diffracting crystals at the selected beamline. The default
resolution is based on the current detector sample and energy (or the
absorption edge energy for SAD or MAD) and the
detector type installed at the beamline. The default attenuation
corresponds to the current value at the beamline; usually, no
attenuation is necessary for the test images, except for BL12-2. Changing the resolution will make the
detector move at the start of the image collection. The Laue symmetry
and cell can also be given if
known; the multicrystal strategy mode
can also be selected here; this will change the strategy program used to
calculate the oscillation range for data collection from BEST or MOSFLM.
For SAD and MAD experiments: The number of heavy atoms and the number of
residues per monomer may be also specified here. If given, they will
be used in the dose calculation for the strategy.
Figure 11:
Options for test data collection and autoindexing.
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Multicrystal strategy
The multicrystal strategy mode allows to select the optimal
starting and final orientation phi in cases where a full data set
cannot be collected from a single crystal, often because of radiation
damage during data collection of microcrystals, metalloproteins or
samples at room temperature. This option can be selected by choosing
the multicrystal option in the Other Options setup page.
Upon selecting the multicrystal strategy option, the strategy
calculation program will change to MOSFLM and
a menu to select multicrystal strategy options will appear.
Figure 12:
Options for multicrystal strategy.
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If the crystal mounted is the first one in the multiple crystal
experiment, the Previous run in the multicrystal series input
box must be left empty. Once a strategy calculation run has been
calculated for the crystal, a file containing information about the
orientation of the crystal and the associated strategy is written
out in the webice directory for the run. When another crystal in the
multicrystal series is mounted, the immediately preceding run must be selected using the drop down
menu. This will automatically read in and use the file storing the
strategy parameters for all previous crystals in the series and
updated with the values determined from the current sample.
The user can specify the expected rotation range that can be
collected from the crystal before damage takes place in the
rotation angle input box, which defaults to 10 degrees. For the
first sample, accurate selection of the rotation range is not critical
and could be even left blank -in this case, the program will calculate
the standard range to maximize completeness for the crystal symmetry and
orientation. However, in general, a fuller completeness will be
achieved with a more accurate estimate of the rotation range, based
either on the dose calculated by the strategy or the actual decay
observed from previous samples.
When a previous run is selected, the program displays the
starting and end phi angle determined by the strategy for the
previous runs. If these values do not match the data collected from
the crystal they can be edited to reflect that data that is actually
available (for example, if the crystal was either damaged earlier or
lasted longer than the input rotation range)
Note that, for multicrystal MAD and SAD experiments the input
angle range will be used, unlike in single crystal mode, where twice as much data are collected
by default to improve the multiplicity
Importing the screening strategy
If the sample has been mounted and analyzed during a screening
experiment, a new run can be created to view the
optimal data collection strategy for that sample. To do this from the
autoindex tab, follow these steps:
- Make sure that the appropriate beamline is
selected. Click on New Run and then on the Import button in the New Run Form page. The software will display some
information pertaining to the sample. A default run name will be built
from the spreadsheet ID and the sample name is also supplied.
- Edit the default run name if desired, and click Import
Run.
Note: It is also possible to import the strategy from the Screening
Tab or by launching the Web-Ice application
from the screening tab in Blu-Ice.
Important: The import feature is only enabled if the sample
is still mounted on the goniometer. If the sample has been dismounted,
the autoindex must be repeated in order to obtain the optimal phi
range for data collection.
After the run setup has been completed, a summary of the
user's choices is displayed. The user can click the
Edit Setup button to change the choices or the Run button
to start collecting
or autoindexing the images.
The Run Status will be periodically updated in the Run
box. Messages will be displayed when autoindexing results
are ready for viewing. It is possible to use other Web-Ice tools while
the processing is running.
Clicking the Update button will reload the run contents.
The user may choose to abort the data collection or autoindex run
by clicking the Abort button displayed after starting
autoindexing.
Figure 13:
Running Autoindex
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Viewing the run results
The Selected Run displays a series of
links to facilitate viewing all the run analysis
results.
Figure 14:
Selected Run navigation links
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The [Scan] page only appears if the Perform
Fluorescence Scan option was selected for a MAD/SAD
experiment. This page will display a graph showing the raw
fluorescence counts as the scan is being carried out, as well as
messages from the scan operation. Once the scan is processed with
autochooch, a plot of f' and f'' will also be available. The
plots can be saved as PNG images by right-clicking on them and
selecting the Save Image As... option from the menu.
The [Log] link allows the user to inspect the detailed
autoindexing log (with information about each program and version that
is run and exit status) and data
collection status on the connected beamline. The latter log is shared with
Blu-Ice
The [Autoindex Summary] page displays the refined values for the direct
beam coordinates (Beam x and Beam y) and the distances; an
initial estimate for the mosaicity based on how many of the
observed spots are predicted by the best solution; and a list of
indexing solutions.
For each indexing solution, the programs displays the following:
- Metric fit: This is a measure of the distortion of the
unit cell required to fit the symmetry. This increases with the
symmetry but often remains below 0.1 for good
solutions.
- rmsd: Root mean square deviation between the observed and
the predicted pattern. Usually less than 0.5 for good cases.
- # spots: Number of spots predicted by the solution (up to
a maximum of 600).
- Crystal system, Unit Cell (in Å) , Volume
(cell volume).
A summary of the refinement and integration results for the
indexing solution determined by LABELIT is shown at
the top of the page. Note that for strong diffraction images the
estimated resolution limit may extend well beyond the limits of the
detector; in order to collect data to this resolution, you should then
move the detector closer to the sample; changing the exposure time by
a large amount may have an effect on this estimate.
Note: be
aware that the estimated mosaicity value can be underestimated for
well diffracting crystals. The estimated value treated as suspect
until several contiguous images are available for postrefinement.
The profile fitting for the central region of the
images and intensity integration statistics are displayed below the
''Integrated Solution'' table.
To access the entire MOSFLM log and summary file for the
integration, see the [Details] link.
Predictions
To view the predicted spots, click on the [Prediction] tab. This page displays the observed spots, and the
predicted spots after autoindexing and integration with MOSFLM for
either image used for the autoindexing. The image display parameters
can be modified as described in the Image Viewer
documentation.
The observed spots used for autoindexing are shadowed in
green.
The predicted spots surrounded by a blue circle are fully
recorded. The ones surrounded by a yellow circle are partially recorded.
Ice rings and the initial resolution estimate (before
autoindexing) are also marked.
Details
[Details] displays the directory tree containing all the
files generated by Web-Ice during the autoindex and integration,
including scripts, orientation matrix files, logs and output
reflection files. Access to these files is useful to validate the
image analysis results and to refine the strategy calculation by
making changes to the input files and
re-running them manually.
The contents of
non binary files can be displayed by clicking on the file name.
- The top directory in the tree /data/user-id/webice/autoindex/run-id contains
- The LABELIT subdirectory contains the autoindexing
results and the MOSFLM matrix, input scripts and output files for each
possible autoindex solution
- The solutionxx subdirectory contains the integration
results for the solution number xx (since 2015, the results for the triclinic
solution are not calculated). It
also contains the dose calculation results (in the RADDOSE directory)
and the strategy input scripts and log files for each possible
symmetry consistent with the autoindexing solution (in directories
named after the corresponding Laue group).
- The SCAN directory contains all the output files from the
fluorescence
scan
(if a MAD or SAD experiment has been selected). These files are also
written to the image directory.
- If MOSFLM is chosen as the strategy program, a BEST directory containing the input and log files from the
program BEST (used to calculate the exposure time) will also be
generated.
The strategy calculation results are shown under the [Strategy] tab.
The optimal oscillation range for the Laue group determined by
LABELIT and the
overall completeness are displayed in the ''Space Group''
table. An upper limit for the oscillation per image (delta phi) is
also shown in this table. Above this delta phi value, more than 3%
reflection in one or more images in the phi range will
overlap. Note: The maximum delta value is based on the mosaicity
estimation. If the mosaicity is underestimated, the delta phi value
will be overestimated.
Figure 15:
Space group summary table. Unless the space group was
preselected before autoindexing, the optimal collection range is
calculated for all the lowest symmetry group in the predicted lattice.
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The full recommended data collection strategy for the determined
space group is displayed at
the bottom of the page. The BEST strategy
calculation results can also be displayed by selecting the appropriate tabs
above the data collection strategy
table1. Clicking on the phi ranges displayed in the
table will toggle between the strategy to optimize anomalous data and
unique data completeness.
Figure 16:
Strategy navigation tabs to toggle between the strategy
summary table and the completeness and overlap
analysis for each Laue class in the autoindexing
solution.
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The Data Collection Strategy table shows all the required
input for a Blu-Ice data collection run based on the strategy
calculation results. Below is an explanation of how each quantity is
determined. If the results are not suitable for the users'
experiment it is possible to re-run the calculations as explained in
the troubleshooting section.
- Experiment type: This will also display a warning if a
non-default total oscillation range is selected in the Space
Group Table (e.g., if the user selected a SAD experiment, but clicked
on the phi range maximizing unique completeness).
- Mosaicity and Score: These values are shown for
information purposes only. If the mosaicity is flagged as poor, or
the score is low, it is strongly recommended to
inspect the images before proceeding to data collection.
- Oscillation start and oscillation end: For native data
collection, the default values optimize the unique data completeness.
For SAD and MAD experiments, the values maximizing the
anomalous pair completeness are selected instead, and the total
oscillation range is doubled (ie, the multiplicity of the data is
doubled for each wavelength), since increased multiplicity is better
for anomalous signal detection.
- Oscillation angle: The oscillation angle
calculated by BEST is used. This should not exceed the maximum
delta phi value displayed in the strategy summary table.
- Oscillation wedge: For conventional data collection, it is
suggested to collect the entire run in a single continuous patch. For
MAD and SAD experiments, data collection in wedges is preferable, to
minimize the amount of damage between Bijvoet or dispersive
pairs. When feasible, the wedge size is chosen so that the total dose
received by each wedge of data does not exceed 0.025 MGy. However, if the dose wedge-based size is smaller
than a minimum wedge size (based on the amount of time needed to
change energy on each individual beamline), the latter will be
selected instead.
- Resolution: This is the resolution for which the strategy
has been calculated. A warning is displayed if the
maximum resolution predicted from the image integration statistics
(shown under the [Autoindex] and [Solutions] links) is
higher than the value at the detector edge.
- Exposure time: BEST is used to estimate the optimal
exposure time to collect data with an
of 2 at the
resolution given above. For SAD and MAD experiments, the
target is chosen so that a default signal of 3% can in principle be measured
at low resolution. If the user provides the number of anomalous
scatterers in the molecule, the signal will be estimated from that
amount. Note: While this is a better criterion to determine the
strategy in phasing experiments, theory and reality may differ. If the
expected anomalous signal is small, increasing the data multiplicity
beyond what is recommended by the software may be a good course of action.
If the exposure per image is too short, it will be reset
to the minimum allowed value; in this case, a suitable beam
attenuation will be calculated to avoid image overexposure.
- Attenuation; Calculated to avoid too short exposure
times2.
- Detector to sample distance: Detector to sample distance
to obtain the resolution given above. If the predicted resolution is
shorter, a warning will be displayed with the required
sample-to-detector distance.
Note: If the detector distance to achieve the maximum resolution
in the warning differs noticeably from the other value, the values for
the exposure time and oscillation angle may not be valid any more. In
this case, it is recommended to recollect test images at the new
distance and repeat the strategy calculation.
- Beamstop Distance: Minimum beamstop to sample distance
required to collect data at a low resolution of 40 Å. To collect
data at a lower resolution (at the expense of a higher image
background) the beamstop must be moved further back. Note that the
optimal value is different at wavelengths other than the one used to
collect the test images.
- Energy: For native crystal experiments, the energy is read
from the image header; this is also the default for SAD experiments,
unless the user has selected the element from the periodic table with
the Perform Fluorescence Scan
option. In these cases, the software may
display a warning suggesting a higher energy for crystals diffracting
to very high resolution; if so, it is recommended to recollect
test images with appropriate values of energy and detector distance
and repeat the strategy calculation.
In all other cases, the optimal energies for MAD and SAD data collection will be
calculated based on a fluorescence scan or values supplied by the user.
- Detector type: This is read from the image header and
displayed for information purposes.
- Detector mode: Dezingering is suggested for CCD detectors
if the exposure time per image is longer than 30 seconds.
- Inverse beam: This option is selected for SAD experiments
(inverse beam = Yes). For the other experiment types this is set to No
(no inverse beam).
- Number of images: Obtained by dividing the total
oscillation range by the oscillation per image.
- Absorbed dose: Estimate of the absorbed dose assuming no
heavy atoms present in the sample, a solvent content of 50% and a
uniform crystal size of 100 microns. For MAD experiments, the dose at
each energy will be given. If the total dose exceeds the
Garman
limit, a
warning will be displayed; for MAD and SAD experiments, a more strict
limit (half the Garman limit) is used, as the accurate measurement of anomalous
differences is easily affected by radiation damage; in addition, for
MAD experiments, only data at
two
energies
will be collected. Other measures to reduce the absorbed dose are left
to the user's discretion: decreasing the exposure time per image is
the best option in most cases. However, if an abundant supply of
crystals is available, decreasing the number of images is also an
option.
Important: The dose calculated for MAD and SAD experiments can be
significantly underestimated if the protein buffer/cryoprotectant
contains a high concentration of ions such as Ca, Cl or SO4 or heavier
atoms). Please see the troubleshooting section to find out how to
calculate a more realistic dose in this case.
The values given in the Strategy
table can be used for subsequent data collection using
the Export Run Definition, Recollect Test Images and Collect Data set buttons in the [Strategy] page.
Figure 17:
Options after strategy calculation.
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- Export Run Definition: This button does not initiate data
collection automatically, but fills out a Blu-Ice run entry. The user
can then open a Blu-Ice client and start the data collection. This
option is useful if the user wishes to modify certain beamline
operation parameters which are not accessible from Web-Ice )(e.g.,
dose mode or beam size) or to queue the data collection run. Some of
the settings (oscillation start and end, oscillation angle, exposure
time, detector and beamstop-to-sample distance and energy) can be
edited after clicking the button and before the run is exported.
- Recollect Test Images: This is recommended if the user
wishes to repeat autoindexing and strategy calculation based on the
warnings given by the strategy. Clicking the button allows the user to
edit the oscillation angle, exposure time, detector and beamstop
distance and energy before recollecting two images and repeating the
analysis.
- Collect Data Set: This button starts data collection. The
user may edit the oscillation start and end, oscillation angle, exposure
time, detector and beamstop-to-sample distance and energy before
data collection is initiated.
Important: Web-Ice will recalculate the dose when the exposure
time, attenuation or oscillation range are changed in the
Export/Collect page. However, the resolution does not change. The
resolution corresponds to the diffraction limit of the crystal, and it
is difficult to predict the new resolution when the exposure is
changed without analyzing another pair of test images first.
Footnotes
- ...strategy-links1
- Before September 2015, the software
calculated the strategy for all the space groups; this took a long
time and has been discontinued.
- ...
times2
- Note that the actual attenuation may differ slightly from the
calculated attenuation. Also, on MAD experiments, the attenuation at
the remote wavelength will be slightly different to the attenuation at
the edge energies.
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