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Subsections
Data collection and processing
The following sections describe how to carry out an experiment at
from sample screening to full data collection making use of the
integrated data collection and analysis environment at SSRL.
For additional help setting up data collection, please consult
the Blu-Ice documentation.
Automated crystal screening
The high throughput screening system implemented at SSRL makes it
possible to automatically collect and analyze test images and fully
characterize the sample in a semi- or fully automated fashion.
To set up automated crystal screening follow the steps below or
watch the video tutorials
''Uploading the Sample
Spreadsheet''
and ''Automated screening from
Blu-Ice''.
- Upload a spreadsheet containing sample information to
http://smb.slac.stanford.edu/crystal-server
as described in the SAM guide . You can do this
any time before or during beamtime.
Note: If no spreadsheet is available, sample characterization must be
carried out for each sample manually or with the Web-Ice Autoindexing
interface (the latter will keep a permanent record of the results
which can be accessed by the user).
- Once you are enabled to use the beamline, assign the spreadsheet to the cassette or
Unipuck position in the beamline dewar (if you are a remote user,
the beamline support staff will tell you the correct position or
assign it for you).
- Start Blu-Ice and select the Screening Tab. Select
the samples you wish to screen, the input directory and image
collection parameters as described in the Blu-Ice
manual. Some
tips:
- Collect two images at different phi
orientations in order to trigger automated autoindexing (check the
first two ''Collect Image''
entries
in the Action Sequence list of the Blu-Ice screening tab). If you
collect only one image, the spots will be analyzed and assigned a
score, but autoindex will not be carried out.
Important: The images must be collected to your area in the /data
disk. Please do not try to use a specific data disk (eg /data1 ,
/data2 , etc.) in the directory path. Use /data/''your-id'', or else
the software may fail to write the image to the disk.
- You may pause the screening after the loop has been
centered. This allows you to adjust the sample centering using the
click to center
option.
This is only recommended if you use loops much larger than the
crystals (which is not a good idea). It is faster to
select a large beam
size
in the Blu-Ice hutch tab and screen all the samples without pauses.
- It is possible to pause the data collection after the test
images have been collected - this will trigger automated strategy
calculation for this sample following autoindex. This option is not
recommended, unless you are very sure that you want to collect data
from this particular sample (e.g., if the samples have already
been screened). It is more time efficient to screen all samples
automatically and then remount the best ones for data
collection.
Tip: It is possible to use automated sample characterization
with the Blu-Ice screening tab without using the robot (in manual
mode). The software will prompt you to change the sample in this case
A summary of the autoindexing results (symmetry, resolution and
mosaicity estimates and a score) will be written to the
sample list information displayed in Blu-Ice and Web-Ice shortly after the
images have been collected. This information can be used to help
select the best samples for collection of a complete data
set. Follow the instructions below or consult the
Video Tutorial
To see the screening results in the Blu-Ice screening tab, make
sure that you use the
''Results''view
to see these columns.
You can inspect the results in detail with Web-Ice:
- Use the Web
button
above the sample information list: Click on the arrow to open the
drop down menu, and select Web-Ice. Blu-Ice will open a new
browser window (if you do not see the browser window, look for
minimized or hidden browser windows). You will be directed to the
Web-Ice Screening
tab.
- In the Web-Ice Cassette Summary
page
sort the samples by score, mosaicity, resolution, or rmsd by clicking
on the title of the corresponding columns.
- Select the sample you want to inspect and click on Cassette
Details (in the gray navigation toolbar). This will let you see the analyzed image, spot
statistics, crystal JPEG and autoindexing results for that
particular sample.
Important: Do not relay blindly on the score or other image
statistics as a means of selecting the best crystal - always
inspect the results displayed in the Cassette
Details page.
Figure 35:
Cassette details navigation in Web-Ice Screening tab: Header
displays the image header; Spot Statistics displays the results of
image analysis before autoindexing; Crystal image shows a camera
shot of the crystal; Autoindex shows the autoindex results and
image score; Details is a directory browser and image display tool
to inspect log and output files.
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Once the optimal sample for data collection has been selected,
monochromatic (non-anomalous), and simple MAD and SAD experiments
from a single crystal can be set up in a very easy way using
Web-Ice. Follow the steps below or look at the Video Tutorial
- Mount the sample (either manually or
using the robot. If you use the robot, select the sample in the
sample information list in the Blu-Ice Screening tab and select
the Stop immediately following Loop Alignment in the
Action sequence widget; then
click the Start button.
- Move the sample camera zoom to High, and adjust the sample centering if necessary. If the box
defining the beam is much larger than the crystal, adjust
the beam
size
- Go back to the Blu-Ice screening tab and start
Web-Ice - or go to the URL
http://smb.slac.stanford.edu/webice/
and log in using you Unix user ID and password.
- Once in Web-Ice select the Autoindex Tab.
- Select New run from the gray toolbar menu in the Autoindex
tab. Enter a unique run name (the software will ask you to try
again if the run name has already been used under your user name)
Figure 36:
How to generate a new run in Web-Ice.
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- Select the option to collect 2 images and autoindex. If required, by
the software, select the beamline from the drop down menu on the
right of the gray navigation toolbar.
Figure 37:
Selecting a beamline in Web-Ice.
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- The program will take you through all the steps to set up the
test image
collection
and (for MAD and SAD) fluorescence scan. Some tips:
- Use the screening results to set up the
optimal parameters. If you want to increase the resolution ,
remember to increase the exposure time too; however, it is
important you do not overload spots, this can cause a problem with
the exposure time estimate.
- If you know that your crystals consistently index in a higher
symmetry than the correct one (e.g., a monoclinic crystal with the
angle close to 90 degrees), supply the correct Laue group
and cell.
- Once you have started the autoindex/strategy run, you can monitor progress by
looking at the Beamline log and Autoindex Log in the
Log page in Web-Ice. The Setup page also prints
messages as the results become available. Inspect the autoindexing
results in the Autoindex Summary page, image integration
results in Solutions page, and (very important!) look at the
images in the Predictions page. For MAD and optimized SAD
experiments, examine the Scan. If everything looks fine, look
at the Strategy page.
Important: Never collect data without inspecting the test
diffraction images and the predicted pattern
- If the crystal space group is well known, make sure that the
strategy for the correct Laue symmetry is selected (remember that if
the Laue symmetry is not declared when setting up the run, the
strategy selected by default corresponds to the lowest symmetry for
the best autoindexing solution).
- If the predicted resolution is lower than the target
resolution, try recollecting the test images with a longer exposure
time. Do this by clicking on the Recollect button; the
program will display a page where you can edit the data collection
parameters. Note: Increasing the exposure time per image will
increase the radiation dose by the same amount. E.g., if the
estimated dose is 1.5e7 Gy, you will reach the limit by doubling
the exposure time. Always verify that the dose limit is not
exceeded unless you have additional good quality crystals.
- If the predicted resolution is higher than the target,
recollect the test images by clicking on the Recollect
button. This will not only test if the diffraction limit estimated
by the software is correct, but will also recalculate the correct
oscillation angle per image and optimal exposure time, both of
which are dependent on the resolution. Use the initial exposure
time (used to collect the first test images) and the new sample to
detector distance recommended by the software.
Figure 38:
Results menu for a Selected Run in the Web-Ice autoindex tab.
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- Once you have obtained a satisfactory strategy (see the notes
below), start data
collection from by clicking on the Collect button. This will
simultaneously export the data collection to Blu-Ice AND start
the collection. If you wish to make additional adjustments
(e.g. enabling dose
mode), you can Export the strategy
to Blu-Ice. Like the Collect button, this will create a run
in Blu-Ice, but will not initiate collection. This will provide you
the chance to edit the experimental parameters in Blu-Ice; once
this is done, start the data collection as described in the
Blu-Ice documentation
- Pausing and interrupting the data collection can be done at any
time from Blu-Ice.
For monochromatic experiments the priority is to maximize
unique completeness. If the estimated dose for the experiment is low
enough, consider collecting additional data beyond the starting or
ending phi to increase data redundancy.
Completeness for the low resolution shells is
important, so decrease the exposure time or use additional
attenuation if the strategy page displays a warning about overloaded
spots in one or both of the images.
If ultra-high resolution data are required and you are on a
beamline equipped with a CCD detector (e.g. BL14-1) it may not be
possible to collect to the resolution limit without overloading the low
resolution reflections. In this case, collect an additional
low-resolution pass:
- Run Web-Ice to calculate the strategy for the high resolution
pass. Once this is done, export the strategy to
Blu-Ice. This will create a data collection run in Blu-Ice but
will not initiate data collection.
- Examine the high resolution test images to determine the
resolution
at which no overloaded reflections are present.
- Run Web-Ice again to determine the strategy for the low
resolution pass. The target resolution for this pass should be 1/2
Å above the resolution
mentioned in the above step. This
will allow the data sets to be scaled together. Export the low
pass strategy to Blu-Ice.
- Start data collection from Blu-Ice. To ensure
proper measurement of the critical low resolution reflections, it is recommended
to collect the low resolution pass first (if the high resolution
data are collected first and the crystal degrades, proper scaling
of the two passes may be difficult).
A low pass may not be necessary when collecting thin phi sliced
data on the high dynamic range Pilatus detector, but check the
reflections at low resolution.
For MAD and SAD experiments is it very important to limit the dose
received by the crystal during the experiment. The Web-Ice strategy
already incorporates some mitigation procedures (e.g., data collection
in wedges, use of two wavelengths for MAD experiments). If the Web-Ice strategy
still results in a dose exceeding or at the limit given by the software,
consider decreasing the exposure time. This can be done manually
either after exporting the data collection parameters to Blu-Ice, or
in the edit window Web-Ice displays prior to initiating data
collection. The dose is proportional to exposure time, so reducing the
exposure time by half will reduce the dose by half at the only expense of a
slightly lower data resolution.
For certain space groups and crystal orientations, it is also possible
to reduce the absorbed dose by selecting the phi range to maximize
unique data set completeness (maximizing Bijvoet pair completeness is
the default strategy for MAD and SAD). This usually works for MAD data
with a medium to strong anomalous signal.
In unfavorable cases (very small, weakly diffracting crystals) it
may be impossible to collect a data set without inflicting serious
radiation damage to the crystal. In this case, several crystal will
most likely be required for successful structure solution.
Low signal experiments
If the expected anomalous signal is very low (less that 1 % of
the average reflection intensity), collection of additional redundancy
may be required for structure solution. This can be done by manually
adjusting the ending phi before proceeding with data collection in
Blu-Ice or Web-Ice. As mentioned above, the exposure time may also
require adjusting to avoid excessive irradiation of the crystal.
On the most intense beamlines, particularly BL12-2, it is
possible to collect data from small crystals of dimensions less than
20 microns. However, because diffracted intensity decreases with the
crystal volume faster than the deposited dose, use of such small
crystals often prevents being able to collect a full data set from a
single crystal before radiation damage severely affects the data. This
is also often the case for data collection at above cryo and room
temperatures, were often crystals last about a factor of 100 less than
at cryo. In this cases, it is necessary to stitch a data set from data
collected from different crystals. For high symmetry space groups it
is often possible to obtain good completeness by starting data
collection on a random orientation for each crystal. For low symmetry
or scarce samples, it is useful to determine a data collection
strategy for each crystal that maximizes the total completeness. This
can be achieved with the collection strategy tool available
in
Web-ice.
Some tips for multicrystal experiments:
- It is a good idea to estimate the dose before starting data
collection so that you have an idea of how long the crystal will
last. Collecting a low dose low resolution data set from a single
crystal may also give you some idea of the total lifetime of the
crystals in the beam and help you plan the experiment.
- Do not overexpose the crystal: When aiming for high resolution,
it is tempting to use very long exposure times to measure very faint
spots at 2 /AA/ in test shots...only to find that not only have those spots
vanished after collecting the first image, but you cannot even merge
those single shots together. In general, slightly lower resolution
data are better than no data.
- While collecting data, monitor the quality of incoming diffraction images using the Collect Tab strip chart. This will tell you when your crystal begins to deteriorate, so that you can determine when to stop data collection or which images to exclude from processing.
- To maximize the lifetime of the crystal, always expose as much
volume as possible: Fully bathing large crystals in the beam at room
temperature will result in a longer lifetime (or give higher
resolution data for the same total exposure time) than making the beam
small and shooting several data sets on different parts of the
crystal.
- It is important to process the data on the fly. If there is a large
variation of the unit cell or other lack of isomorphism between
different crystals, you may need to collect data from more samples
than anticipated to achieve a quality data set. A program such as
``pointless''
or ``sortmtz''
can be used to combine the reflections in
a single file previous to scaling. When using
autoxds, use the
name_xds.mtz files as input to obtain a single file. Note that pointless can also be used to ensure that the indexing for the
different crystals is consistent in the space groups where this can be
an issue. The program
xia2 has a very effective algorithm for processing and merging multiple datasets; xia2.multiplex can also be used to refine the combined dataset and to detect non-isomorphism.
- Autoindexing only a few degrees of data may not always be
reliable. It is useful to provide the indexing software with the
symmetry and unit cell if known. When using the autoxds script or
xia2 this can
be done from the command line.
- Similarly, pointless can misidentify the space group in an
incomplete data set, as there may not be enough reflections to fully
characterize all symmetry elements presents in the crystal. If this
becomes a problem, the option ``-c'' will force the program to use the
input space group.
Data processing
The following
directories are automatically created the first time you log in to a
SSRL px computer (these directories are accessible from all computers):
Use the remote data processing servers (pxproc01 - pxproc24) to
process the data; from the beamline workstations, use the SSRL menu
option of the Xfce panel in the Linux beamline
computers and the remote Unix
desktop. You
can also right-click on the Linux Xfce desktop and select Data
Processing from the desktop menu. Clicking on Select Least
Loaded displays the
load. Avoid using a computer is the load is close
to the total number of CPUs (displayed next to the computer name).
Note that, since 2017, Web-Ice attempts automated data processing
after a data set has been collected. The purpose of this tool is to provide
quick feedback of the data quality. Please consult
the Web-Ice data processing help.
For
more information about the beamline machines, consult the web document
http://smb.slac.stanford.edu/facilities/computing/
Figure 39:
Logging to the data processing servers from the Linux Xfce panel.
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Figure 40:
System load window
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Commonly used software packages for data processing are
available at the macromolecular crystallography beamlines. If you are
unfamiliar with a particular application, consult the relevant documentation:
For a complete list of supported and unsupported software installed in the
SSRL computers, see
http://smb.slac.stanford.edu/facilities/software/xtal_software
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