Table of Contents

Data collection and processing


The following sections provide a complete description of an experiment at am SSRL macromolecular crystallography beamline, from shipping crystalline sample to SSRL to data collection to data processing and analysis.

For additional help setting up data collection, please consult the Blu-Ice documentation.

How do I get a computer account and control the beam lines remotely?

A computer account for SSRL Macromolecular Crystallography is necessary for all on-site and remote access experiments. This account will allow you to log in to a virtual desktop, enable you to use the Blu-Ice beamline control software, and give you access to our crystallographic computing cluster.

How do I prepare my samples for robotic sample exchange?

The Stanford Automatic Crystal Mounting System (SAM) is the centerpiece of macromolecular crystallography at SSRL. Enabling both on-site and remote access experiments, the SAM robot allows semi- or fully-automatic mounting and exchange of crystalline sample.

How do I ship my samles to SSRL?

  • IMPORTANT: Prior to shipping of samples, complete the appropriate shipping form! This will ensure that you will be notified when your samples arrive to SSRL.
  • The SAM robot manual has detailed instructions for loading and shipping both cassettes and UniPucks. IMPORTANT: If you are shipping cryogenically-preserved samples, please test your dewar!

How do I upload information about my samples to the beamline control software?

  • Uploading sample information is desirable if collecting X-ray diffraction data from a cassette or UniPuck, and required for automatic crystal screening and data collection. Sample information is entered into a Microsoft Excel spreadsheet (the template for which can be downloaded from the SSRL Sample Database. You can do this at any time before or during beamtime, but prior to data collection. NOTE: if no spreadsheet is uploaded, some characterization must be carried out fo reach sample manually; this option is only available if collecting data on site.
  • Once enabled to use the beamline, assign the spreadsheet to the appropriate cassette or UniPuck position in the beamline dewar. If using the beamline remotely, beamline support staff will do this for you.
  • NOTE: an uploaded sample spreadsheet can be edited via the SSRL-PX computing system.
  • How do I mount a sample using the SAM robot?

    Once the cassette or UniPuck is in place and the sample information spreadsheet has been uploaded, the Screening Tab in Blu-Ice can be used to mount and inspect the samples. This can be done automatically for the entire cassette or UniPuck, for a subset of samples, or for each sample individually, as preferred. The Screening Tab allows you to:

    Samples can also be mounted and inspected individually via the SAM robot interface which is found in the Sample Tab. This system is independent of the spreadsheet-based Screening Tab, and allows more extensive viewing and manipulation of the individual crystals and their crystallographic analysis, such as

    How do I evaluate crystals and diffraction quality?

    It is useful to evaluate the crystals in the cassette and UniPuck and select the best-diffracting prior to performing the full X-ray diffraction experiment. Blu-Ice contains a variety of tools that can be used for this purpose.

    How do I find the best-diffracting crystal(s)?

    The automated crystal screening procedure includes automated indexing of the collected test images, with the results (symmetry, resolution, mosaicity estimate, and an overall image score) displayed in the Screening Tab in Blu-Ice. These results can be used to determine which of the crystals yields the best diffraction.

    You can evaluate the results of the screening in several additional ways:

    • The sample spreadsheet would be updated with screening results and can be downloaded after screening concludes.
    • A visual image of each mounted crystal is saved and can be inspected to ensure that, in cases where no diffraction was seen in the test images, the crystal was correctly centered or present in the loop.

    How do I collect test images?

    While the screening tab will display some basic information about diffraction quality for each sample, it is nevertheless advisable to visually inspect test images collected from the most promising crystals. Test image-sets conventionally consist of two images (collected at PHI angles of 0 and 90 degrees), and are collected automatically during screening, or could be acquired manually in the Run 0 subtab of the Collect Tab.

    When collecting images manually, it is useful to use "coarse slicing" (i.e. 1-degree oscillation) for test snapshots, switching to "fine slicing" (e.g. 0.1-degree oscillation) for full datasets. The extent of per-image oscillation can be set via the "delta" parameter in the Collect Tab.

    What should I look for when examining my diffraction images?

    You can view diffraction images in two ways:

    • X-ray diffraction images will show up in the Collect Tab's image display window. This display is fairly rudimentary, with a few basic image manipulation options, which include zoom, brightness control, and display of resolution estimate at the mouse pointer position. There is also an option to open this image in ADXV for a more detailed inspection.
    • Alternatively, image files can be located in the user directory into which they were written and displayed via the user's favorite image viewing program, most of which are available on SSRL-PX's computing cluster.

    When inspecting diffraction images, it's useful to look for a few specific features.

    • Look for a series of concentric rings, which can be contiguous or broken-up. These are most likely caused by ice crystals. Most modern data processing software can bypass this, but a really icy crystal can be problematic.
    • Look for obvious artifacts, such as shadows cast by equipment that somehow is positioned in front of the beam. This is unlikely to happen in standard setup, but might crop up in some custom configurations.
    • Raise the brightness of diffraction to a high level (paradoxically, this will make the image darker) and try to locate the faintest high-resolution spots. Mousing over a spot will reveal the resolution at which it is observedin most viewers; as a rule of thumb, that resolution would likely be ~0.5 Ångstroms less than the cutoff established by processing software.
    • Zoom in and inspect individual spots at various resolutions (you might have to play with brightness to see them properly); look for signs of splitting (i.e. bimodal distribution of dark pixels in a spot), smearing, or extreme elongation that would signal high mosaicity. These might explain poor merging statistics of the processed dataset, should one be collected from this crystal.
    • Look closely at the lines of spots forming the lattice pattern; if some of the lines are at unusual angles (e.g. very acute) to others, this may indicate multiple lattices. Most data processing software can handle this eventuality, though sometimes it can be problematic.

    How do I find the best-diffracting volume of my crystal?

    The rastering technique allows you to find the best-diffracting volumes of your crystal using low-level diffraction. In the Raster tab, you can use a user-friendly GUI to define a three-dimensional grid encompassing your sample; the software will scan the grid with a highly-attenuated beam, collecting low-level diffraction images and analyzing them for diffraction quality. A heat map representing diffraction quality by a certain metric (resolution, number of spots, or a combined "score") will be overlayed on top of the sample after rastering is concluded, indicating where the best diffracting volume(s) can be found.

    NOTE: this technique can also be used to locate crystals that are hard to see because they are small and/or obscured by ice, cryoprotectant, or precipitate. Rastering can also be used during autonomous screening or data collection to ensure that each crystal is correctly centered.

    How do I set up the best data collection strategy for my crystal?

    Choosing optimal collection parameters is an extremely important part of the structure determination process, as everything subsequent involves manipulation of the data and the model in silico. By collecting the highest quality data possible the subsequent steps will be easier and more successful. The following tutorial will help you determine the best values to use for the parameters listed below:

    What rotation range and oscillation angle should I use for data collection?

    Fast Strategy: With the use of Pixel Array Detectors (PADs) like the Pilatus and the Eiger detectors on the SSRL beamlines, one fast and easy collection strategy is to measure a full 360° rotation of the crystal using a very small rotation (oscillation) angle, called fine-phi-slicing. To get the best data, we recommend 0.1 or 0.2 degree oscillations and short exposures (~0.2 s) per image. However, a larger oscillation (0.5 -1 degree) should be used for crystal screening or test shots. If in doubt, use the default collection parameters in Blu-Ice; we recommend examining the test images and using the resolution calculator to set the detector distance and the dose calculator to set the appropriate amount of X-ray attenuation (typically over 90% at BL12). For collecting many datasets in a hurry, the screening tab can be used to collect 360° datasets from all crystals in a puck or cassette in an autonomous mode; select the rastering option to have each crystal optimally centered during this process.

    Optimized Strategy: However, sometimes simply collecting 360° of data can be problematic, particularly with crystals prone to radiation damage or crystals with long unit cell edges, so a better strategy is to calculate starting and ending angle and check the rotation angle to make sure there are minimal overlaps:

    • To determine the best rotation range, collect some test images which can be used with iMosflm to calculate a data collection strategy following our online tutorial.

      The goal of this step is to maximize the completeness of your dataset while collecting the least amount of data, to reduce radiation damage. This means that you have been able to measure every possible reflection emanating from your crystal. Sometimes the geometry of the crystal relative to the X-ray beam, coupled with the space group of your crystal and the underlying symmetry means that it may not be possible to collect 100% of the reflections but you certainly want a completeness in the high ninety percent range, and this is generally practicable from most crystals.

    • iMosflm strategy can also be used to verify if your chosen rotation step size (oscillation angle) will prevent problems from overlapping reflections, something that arises in cases where you have long unit cell which makes reflections closer together.

      Overlapping reflections may be difficult to separate during data processing and a lot of them may be discarded, giving you incomplete data which can lead to anomalies in your electron density maps. If your crystal has a long unit cell and diffracts to high resolution, you may need to decrease the oscillation angle. Sometimes you may also need to sacrifice resolution for completeness by moving the detector to a longer distance so that the spots are separated. Alternatively, the detector can be offset horizontally and/or vertically to allow for the capture of the high angle reflections. You should consult your User Support person if you encounter a case like this.

    What crystal to detector distance should I use for data collection?

    This parameter will determine the resolution limit of your data on the detector surface and ultimately the resolution and completeness of your final data set. The resolution is directly related to the angular spread of the diffracted X-rays (relative to the direct X-ray beam) from your crystal. Measuring higher angle diffraction spots (reflections equate to high resolution) requires either a closer detector distance or higher X-ray energy. A resolution predictor in Blu-Ice gives a visual representation of the relationship between the detector distance, energy and the resolution.

    The detector should be just close enough to the crystal to make sure all high angle reflections are measured. If you have the detector too far, the high angle reflections will be off the edge of the detector surface and will not be measured. Keep in mind, using a longer crystal to detector distance will actually increase the signal-to-noise of your collected data because the background (air) scatter decreases faster as detector distance is increased (by the square of the crystal to detector distance). If you have a very large unit cell, you may also need to increase your detector distance (as described in the previous section).

    What beamsize should I use for data collection?

    • In general, we recommend matching the X-ray beam size to your crystal size. The beamsize is variable at all the SSRL macromolecular crystallography beamlines and may be adjusted using the beamsize widget.
    • When your crystal is significantly larger than the largest beamsize, you may want to try helical data collection, which will both rotate and translate the beam during collection. Helical collection at BL12-1 and BL12-2 is now done in a shutterless mode which makes this option a very fast way to reduce radiation damage effects on your dataset.
    • In some cases, it is difficult to see your crystal which can make it difficult to choose the correct beam size. In other cases, the crystal may not diffract well in all areas. For these scenarios, we recommend using a low-dose X-ray beam raster/grid search to both locate the best diffracting area of your crystal and to help estimate the optimum beam size.

    What wavelength should I use for data collection?

    This can depend upon several factors. If you are collecting data from a native crystal (one containing only light atoms (C, O, N, H and S) then we recommend using the wavelength at which each beamline has been optimized. Generally this is the selenium absorption edge (12658 eV, 0.9795 Å) for beamlines BL9-2, BL12-1 and BL12-2, and just above the gallium K-edge (10375 eV, 1.1950 Å) for beamline BL14-1 (as synchrotron scientists, we have a tendency to describe the X-ray wavelength in electron-volts rather than Angstroms, and the energy widget on the Hutch Tab in Blu-Ice allows you to change between the two units). At these wavelengths, beam intensity is maximized and air absorption is minimal. Like the crystal to detector distance, changing the wavelength will change the resolution at the edge of the detector, and you can use the resolution predictor on the Hutch Tab in Blu-Ice to test the relationship between the X-ray wavelength (or energy), the distance and the resolution.

    If your protein crystals contain a heavy atom and you wish to utilize these atoms to help solve your structure using MAD or SAD phasing, you should consult the directions on how to measure a MAD scan, and also talk to your User Support person if you are unsure how to do these specialized data collection experiments.

    What exposure time and attenuation should I use?

    To get the best data, we recommend you keep a consistent exposure time and rotation range of 0.1 degrees oscillations and 0.2 s exposures per image. Then you should increase the attenuation (reduce the X-ray transmission) to optimize the total X-ray dose on your crystal.

    Once you have determined the start, end and oscillation angles for your dataset (such as using iMosflm strategy), use the Run Dose Estimate window in the Collect Tab in Blu-Ice to determine the optimal X-ray transmission settings (the amount of X-ray attenuation to use). This calculator will compare the X-ray dose from your input collection strategy with a suggested dose limit, which is defined as the dose which reduces the sample to half its diffracting power or the loss of useful anomalous signal. You should select a dose limit based on your experiment, such as under 30 MGy for experiments performed at ~100 K, or 3 MGy for sulfur SAD experiments at ~100 K, or only 0.38 MGy for room temperature experiments.

    NOTE: Fine sliced images with the correct exposure time look very weak - this is to be expected, as all the reflections are partials, and you should refrain from increasing the exposure time until the pattern looks strong, since this may result in rapid radiation damage. Rather than increasing the exposure time, if your data collection strategy does not exceed the maximum suggested X-ray dose, we suggest to instead collect a larger rotation range to increase the multiplicity of your dataset.

    Trade-offs: As you reduce the x-ray transmission, the highest angle diffraction may become weaker and eventually fall off – reducing the resolution of your dataset. However, if you do not lower the x-ray transmission, your crystal may be damaged during data collection, which will cause fall-off of diffraction resolution during data collection lowering the completeness of the dataset, visible in the data collection strip chart. Therefore, in some cases, you may need to combine data from multiple crystals to optimize the resolution and completeness of your dataset. Here is a tutorial for combining multiple datasets using xia2.

    How do I collect an X-ray diffraction dataset?

    Once a promising crystal has been identified, X-ray diffraction data can be collected via the Collect Tab in Blu-Ice. The general procedure consists of several relatively simple steps:

    • Initiate a data collection run
    • Edit the run parameters. There are quite a few of these, though the most important ones are:
      • prefix - determines a filename template that is unique to the dataset to be collected.
      • directory - designates the directory where the images will be written; make sure it's a directory in your designated space, otherwise the images won't be collected! (It is also advisable to create separate folders for separate datasets, in order to make them easy to find.)
      • distance - determines crystal-to-detector distance; it is useful to adjust it depending on the resolution of your diffraction.
      • delta - determines the rotation along the PHI axis during a single exposure.
      • time - determines the time of exposure (and thus the amount of X-ray radiation delivered to the crystal).
      • start and end - determine the ranking numbers of the first and last frames of the dataset.
    • Check the radiation dose estimate for the run, which is calculated automatically; if the predicted dose exceeds the maximum X-ray dose that can be delivered to a macromolecule before altering its structure, also known a the "Garman limit", it is advisable to make the run shorter, either by reducing the number of frames, the exposure time per frame, or the percent of X-ray photons that are transmitted to the sample.
    • Once satisfied with the data collection parameters, press the Collect button.

    To get more strategy advise on the optimal parameters to use in the Collect tab, we suggest reviewing the data collection strategy portion of this User Guide.

    How do I set up screening or data collection to run autonomously for multiple crystals?

    The spreadsheet display window of the Screening Tab includes a checkbox next to each listed sample. Any subset of these can be selected prior to screening, ranging from a single sample, to several, to all of them. After that, automatic screening is as simple as pressing the "Start" button, although the specific screening action sequence can also be customized.

    Instead of collecting test images, the automated screening procedure can be set up to collect a larger (up to 360°) dataset for each crystal.

    • This the fastest way to obtain complete datasets from all crystals in a cassette or UniPuck.
    • NOTE: if you use this options, we recommend checking the "Rastering" option in the screening action sequence to ensure that each crystal is automatically centered prior to data collection.

    How do I monitor diffraction quality and radiation damage?

    The Collect Tab includes a diffraction quality strip chart, which enables real-time monitoring of the experiment. The chart displays basic statistics for each frame collected, allowing you to see trends in crystal behavior as it's exposed to the X-rays. For example, a clear deterioration of estimated resolution would indicate possible accumulation of radiation damage. A drop or gain in diffraction resolution during data collection can also occur if the crystal is not well centered in the X-ray beam, or if the crystal volume exposed or crystalline order varies with rotation direction.

    The results displayed in the strip chart can also be cross-checked against the overall run dose estimate; taken together, this information can suggest a change of strategy for the next run.

    Furthermore, beamline 9-2 is equipped with an inline UV-vis microspectrophotometer. While useful for a variety of applications, the microspectrophotometer can be utilized to monitor the X-ray exposure of a crystal during data collection.

    How can I tell if my protein contains a metal in the active site?

    Determining whether your protein of interest contains one or more coordinated metal ions, as well as identifying their types, is useful for a variety of applications in structural biology. The Excitation Scan feature found in the Scan Tab can be used for this purpose.

    Additionally, the MAD Scan feature can be used if a specific metal or heavy atom is present in the structure, for the purposes of determining the optimal energies for single- or multi-wavelength anomalous dispersion (SAD/MAD) data collection.

    How can I get the most out of an extremely large or long crystal?

    In cases where a crystal is much larger than the available beam size, and especially if the crystal is very long, helical data collection is an appealing option. In a helical approach, oscillation images are collected while the sample is translated along a pre-determined path. This technique

    • Optimizes diffraction resolution while minimizing the effects of radiation damage by exposing the largest possible volume of the crystal.
    • Takes advantage of crystal size and ensures that you get the most out of your sample.
    • Can have other potential uses, e.g. a multi-crystal strategy where a number of microcrystals in a fixed target can be exposed sequentially.

    How do I process the data I have collected?

    All datasets collected at the SSRL macromolecular crystallography beamlines are processed automatically after the run is concluded. If you wish to re-process the dataset on your own, SSRL-PX computing offers a powerful data processing environment with the latest stable versions of all major data processing packages installed, configured, and tested.

    How do I view the results of automated data processing and what programs are used for this?

    The automated data processing pipeline writes its results to a folder with a path that looks like /data/account_name/data_collection_directory/auto-processing-file_name. Scripts for each step of the autoprocessing are located in the appropriate sub-folders and can be edited and re-run manually. Please consult the auto-processing webpage or the README file located in the auto-processing folder for more information.

    How do I access the SSRL-PX data processing environment?

    The following directories are accessible from all SSRL-PX computers and remotely when you log into your user account:

    • /data/username - this folder is used to store all of your raw and processed data. (Here, "username" refers to the username you use to access your account.) IMPORTANT: the files on /data drive are NOT backed up!
    • /home/username - this is a much smaller disk space which you can use to store any text files, notes, README's, etc. or install your own local versions of specific software. This drive is backed up.

    To access the data processing software (as well as all of the crystallographic software that we host), log onto one of the data processing machines (pxproc01 - pxproc24) as follows:

    • Log in to the remote desktop.
    • On the toolbar at the bottom of the screen, click the "SSRL logo" button to display the list of pxproc servers. Clicking the up-arrow button will display a pop-up menu of the servers (see figure, left); cicking on one of the server names will open a terminal window, where you will be logged into that server.
    • Alternatively, clicking the larger "SSRL logo" button will launch an app showing a list of the servers with information of how much each one has been used recently (see figure, right). Double-clicking on a specific server name will open the terminal window, where you will be logged into that server.
    Image toolbar Image least-load

    What data processing software is available for my use?

    Commonly used software packages for data processing are available at the macromolecular crystallography beamlines. SSRL-PX currently offers tutorials for the following software:

    For a complete list of supported and unsupported software installed on the SSRL-PX computing cluster, see our software webpage.

    What structure solution and refinement software is available for my use?

    For a complete list of supported and unsupported software installed on the SSRL-PX computing cluster, see our software webpage.