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Up: User's Guide to Macromolecular Crystallography Experiments
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Subsections


Frequently Answered Questions

If you have questions that are not covered below, please consult the on-line documentation or use our local Google search.

Contact support staff for additional questions. During your beamtime, use the beamline cell phones.

Data collection

When do I use Exposure Control mode (former dose mode)?

Exposure Control mode2 is used to equalize the exposure for images in long experiments by multiplying the exposure time by a exposure factor based on the readings from the I2 ion chamber. This corrects for the SPEAR electron current decay over time. The exposure factor is recalculated before collecting each image.

As a rule of thumb, collecting data in Exposure Control mode is advisable when the exposure time per frame exceeds 20 seconds or the total data collection time exceeds 1-2 hours. It can also be used for MAD experiments to achieve a similar level of exposure at all wavelengths (but see below)

It is critical to determine the exposure time at the correct beam size, energy and attenuation before initiating data collection using Exposure Control. To be safe, we recommend this procedure:

  1. Move to the correct values of energy, attenuation and beam size from the Blu-Ice hutch tab BEFORE starting the data collection
  2. Optimize the beam
  3. Enable the dose mode and normalize the dose
  4. Start data collection from the Blu-Ice collect Tab

It is not recommended to use very high beam attenuation in conjunction with Exposure Control, as the ion chamber readings may become too low to calculate a consistent exposure factor (in this case, a No beam error will be given by the software).

For more information about Exposure Control mode, consult the Blu-Ice documentation.

When should I normalize the exposure factor

The exposure factor should be normalized every time a data collection from a new sample is started. This sets the factor to 1. Note that the exposure factor may change significantly after a table optimization or change of energy following normalization; the factor should not be renormalized in this case unless you really intend for subsequent images to receive a different exposure.

How can I convert from wavelength to energy?

In Blu-Ice, click on the units next to the energy input box to toggle between wavelength (Å) or energy (eV or keV).

What is the optimal beamstop to sample distance?

The backstop should be placed at a position where it allows collection of reflections in the 30-40 Å resolution range. At most wavelengths, it will be possible to collect even lower resolution, however, this will be at the expense of additional air scatter that may obscure weak reflections and reduce the diffraction signal over noise.

Using Web-Ice to calculate the data collection strategy will automatically calculate a reasonable beamstop to sample distance. In addition, the Blu-Ice resolution predictor shows the low resolution limit at the given beamstop and energy values.

Sample mounting

What is the size of the white box on the beamline monitor?

At maximum zoom, the box is 0.1 mm x 0.1 mm. At medium zoom, 0.25 mm x 0.25 mm and at low zoom it is 1 mm x 1 mm.

Note: The white box in the sample camera video displayed on Blu-Ice represents the approximate beam size at the sample position and is therefore more reliable to determine if the crystal is in the beam than the monitor box. Additionally, the size of the Blu-Ice box also changes to reflect the scale at different camera zoom levels.

The automated loop centering leaves the camera near the medium zoom level. Is there anything wrong?

No, this is normal operation. The system uses the medium zoom level to do the alignment, in case large loops are used.

Also, during the crystal screening mode, maximum zoom is used for recording JPEG images of the crystal.

Detectors

What are the normal, binning and dezingering detector options?

  • Binning combines the readout of 4 pixels into one pixel, resulting in a larger dynamic range and better signal-to-noise characteristics. Although binning degrades somewhat the spatial resolution, this is not an issue (the source and sample are the main contributors to the spot size). Because non-binning produces large files ( 70 MB), only binning mode is so far allowed for the Quantum-315 detector. There is no binning/unbinning option for the MAR CCD detector.

  • Dezingering rejects spurious spots on the detector (e.g. from cosmic rays) by splitting the total exposure between two images; pixels with similar intensity values for the two images are averaged; pixels with very different values are set to the smallest value. Dezingering should be used for exposure times that are longer than 20 seconds.

Can I display the image header?

You can look at the image header with the Web-Ice Image Viewer; The program ADXV also displays the header.

How do I determine the direct beam position?

The default beam position, should be at the nominal centers of the detectors (within 1 pixel) listed below (in mm):

  • MAR325 CCD: 162.5, 162.5
  • Q315 CCD: 157.5, 157.5

When a detector offset is selected in the Blu-Ice hutch tab, the data collection software writes the true beam center coordinates Cx,Cy to the image header.

Determining data collection strategy

What is a reasonable exposure time for beamline X?

If in doubt, use the default values: in the Blu-Ice Collect tab, click the Default button; in the Screening Tab, click Reset defaults. If your crystals diffract very poorly or you are collecting the images at extra long or short wavelengths you may have to increase the time.

We recommend to use Web-Ice to determine the optimal exposure time from the initial test shots of the crystal.

How long do I need to expose a crystal at SSRL for RIP phasing?

For radiation damage sensitive samples, the best strategy is to do a two-wavelength MAD or SAD experiment without exceeding the maximum recommended dose (use Web-Ice to obtain an estimate of the absorbed dose). Overdosing the crystal results in a unit cell expansion which most often prevents accurate measurement of any kind of phasing signal in the data. Radiation induced intensity difference are no easier to measure than anomalous or dispersive differences in this case.

For some derivatives (e.g., brominated DNA), the heavy atom may become cleaved at very low doses. The program SHARP has been reported to deal well with this particular case, using the loss of occupancy of the anomalous scatterer to enhance MAD or SAD phases, as long as the total dose is kept to a reasonable value (i.e., you should not exceed the dose limit in Web-Ice).


Problems during data collection

How can I monitor the SPEAR beam?

The beamline status and current are displayed in the Blu-Ice status bar.

Additional information about SPEAR (including status of all beamlines and the 24-hour fill history) is displayed on a monitor at the beamline (top left corner of the console) and on the SSRL SPEAR Web page. See also ''Monitoring the SPEAR status remotely''

How often is the beam refilled?

Currently SPEAR is topped up every 8 hours, at 6:00, 14:00 and 22:00. The top up lasts only a few minutes. Occasionally the stored beam is lost during injection and a longer refill is required.

After the refill it takes a couple of additional minutes for the temperature of the beamline optics to stabilize.

Will data collection pause during beam refills?

During data collection, the beam status is monitored by the beamline control software. Data collection will stop when SPEAR is down and restart when the temperature of the beamline optics has stabilized after the beamline is reopened.

A message is displayed in the Blu-Ice status box while the beam is stabilizing following an injection.

Additionally, the beam in the hutch is monitored before each image is collected.

How does the search and reset procedure work?

Completely search the hutch for persons before activating the search reset. The hutch door must be closed and locked before the search alarm stops ringing, otherwise the procedure must be repeated.

Is there beam in the hutch?

If the beamline is open and Blu-Ice repeatedly displays the message ''waiting for beam'', check that the beamline stoppers switch is open on the key panel in the control rack (all green LEDs should be lit). Remote users can see the stoppers LEDs by selecting the appropriate panel video preset in Blu-Ice or Web-Ice.

If the stoppers are open, check the beam attenuation; The attenuation level depends on the beam energy, so a filter combination appropriate for data collection at a high energy can fully block the beam at a lower energy. Also, note that a combination of high attenuation and small beam size can prevent data collection using Exposure Control (old ''dose'' mode), because there are not enough counts to calculate the exposure factor precisely enough. In this case, you should collect data in time mode.

If beam attenuation is not the problem, try reoptimizing the beam. If the beam optimization does not solve the problem, call support staff.

Why do I get blank diffraction images?

To determine the cause of blank diffraction images, follow these steps:

  1. Verify that there is beam in your sample (see the previous question).

  2. If there is no image displayed, try opening the image with a different program (e.g, ADXV, Mosflm) If the image looks only blank on Blu-Ice or Web-Ice, the image server may have crashed. Contact support staff (please, send an e-mail during non-working hours, as this problem does not affect data collection).

  3. Check the contrast in the image display: Images with no diffraction spots displayed at a high contrast level can hide diffraction features.

  4. If you see very weak scatter on the image, verify that the exposure time is not too short - this can happen if Exposure Control is on, and the exposure factor is very small.

  5. If the image is truly ''flat'' (no beamstop shadow nor variation in the background level of the image at any contrast level), verify that there is not a cover on the detector (remote users can look at the Hutch camera video from Blu-Ice or Web-Ice).

  6. If nothing is blocking the detector look at the shutter controller to determine if the shutter is opening. The switch on the controller should be on "auto" and a red LED light should light up when the shutter is open, as shown in shutter-controller. Remote users can use the Panel camera to view the shutter controller. In addition, when the shutter opens you should be able to see an increased reading for the I_beamstop beam monitor. The I_beamstop reading will be low at long wavelengths (low energies). Verify that there is beam on the beamstop by going to a shorter wavelength (higher energy).

    Figure 42: Shutter controller showing open shutter status
    \includegraphics[width=0.5\textwidth]{images/shuttercontrol.ps}

  7. If there is diffuse diffraction but no spots, check the centering of the crystal. Open up the slits to increase the beam size and collect images at different crystal orientations. If you get diffraction with a large beam size or at some phi positions but not others, the phi axis may be misaligned. Contact support staff.

  8. If there is no difference with large slits or at different phi positions, the crystal may not diffract (even if it was diffracting previously). Check the cryojet temperature (Blu-Ice will display an error in the status display if the temperature raises above 120 K); dismount the sample and try another sample. If you do not observe any diffraction or scatter from any sample, contact support staff.

Why can't I open Blu-Ice?

The first time you log in to Blu-Ice it will request your password. Make sure that it is typed correctly. If you cannot log in, contact the user support staff and let them know what Unix account you are using.

If you cannot start Blu-Ice from the icon in the XFCE menu:

  1. Open a terminal on the local workstation or NX client.

  2. Log in to a different beamline workstation. For example, if you are trying to open Blu-Ice from bl92a, log in to bl92b or bl92c. Use the command:

    >ssh bl92a

    >go

    Note: The NX client tries to open Blu-Ice on the ''c'' workstation; if Blu-Ice fails to open, make sure that you use the ''a'' or ''b'' computer.

Why is Blu-Ice not responding?

  • Verify that the Blu-Ice window is active.

  • If you lose permission to connect to Blu-Ice, contact support staff.

  • If some buttons are inactive (grayed out) move the mouse over the button. Blu-Ice will display a message explaining why the button is inactive. If the message tells that the control software for a hardware component is off-line (''DHS off-line'') contact support staff.

  • If the Blu-Ice status window displays the messages ''MOTOR STOP BUTTON LATCHED'', an emergency stop button may have been depressed accidentally. To reset the motors, press the green motor reset button. If collecting data remotely call support staff, or, after working hours, try contacting the duty operator at 650 926 4040.

  • If the entire Blu-Ice interface fails to respond, try exiting the Blu-Ice client and starting a new one. If the fault persists, call support staff.

  • Sometimes, a computer crash or a network problem can hang the system. In this case, other programs and processes will also be affected. If the window manager program is hanging, log on to the host computer from another terminal and kill the processes. To list processes:

    >ps -u "your_id"

    and kill them with

    >kill -9 "process_id"

  • If all the computers at the beamline are hanging or data collection will not proceed, the file system may have crashed. Contact support staff.

What does the message ''Detector error'' mean?

A detector error message in Blu-Ice can have many different causes; although often staff intervention is required in order to continue data collection, it is a good idea to retry the image collection before calling staff, as some errors (e.g., a transient network glitch) do not disable the detector permanently.

Important: Note that the ''Detector Error'' message will still be displayed after a problem with the detector has been fixed; the message will only disappear once an image has been collected without errors.

When should I optimize the beam?

It is advised to optimize:

  • After changing the wavelength from the Hutch tab. Automatic optimization is performed after wavelength changes during MAD data collection.

  • After changing the beam size.

Automatic optimization is also performed at regular intervals during data collection and therefore, manual optimization should not be necessary.

Data processing and graphics software

Why can't I run graphics software?

Graphics programs can be run on the local beamline Linux machines (eg. bl15a, bl15b and bl15c on BL1-5). The program coot runs on the NX client and Linux data processing servers, but not on the older Alpha blcpu computers. Note that graphics applications are generally very slow on the NX client and it may be more time effective to transfer the maps or reflection files to your local computer!

I get an HTTP error when trying to display or refresh a page in Web-Ice

Often this will be a transient server glitch. Try refreshing the page again. If the problem persists, contact support staff (use e-mail outside working hours)

Why doesn't program X run?

Most crystallographic software packages are only installed on the data processing servers and not on the local beamline computers. Graphics programs are installed only on the local beamline workstations.

Why is Mosflm/HKL2000/other running slow?

Check the relative load of the data processing server as described in ''data processing environment''.

Why can't I autoindex my images?

The detector may have been offset from Blu-Ice: Check the detector positioner vertical and horizontal values in the Blu-Ice hutch tab. If they are not 0, look at the center coordinates in the image header (the Web based software Web-Ice can be used to display the image header).

  • Web-Ice knows about the detector coordinate system and can use the image header coordinates directly for auto-indexing.

  • The Mosflm and HKL2000 coordinate systems are related to the beam center coordinates (Cx,Cy) by the following equations:

    x = detector-width - Cy

    y = Cx

    The mosflm script used at the SSRL automatically transforms the beam coordinates. For HKL2000 and XDS you must provide the offset center as explained in the documentation.

  • XDS uses the same coordinate system as the detector header, but it requires the coordinates in pixels. In this case, the XDS center (x,y) is related to the image center coordinates by:

    x = Cx/pixel-size

    y = Cy/pixel-size

    The pixel size is also given in the image header.

Indexing may also fail if the diffraction is weak, if there are many ice rings or if there is a double lattice. Editing the spots manually in Mosflm or HKL2000 often circumvents these problems. Web-Ice uses a different spot-finding algorithm and it is worth trying if other software fails.

If the diffraction pattern is misindexed (this should be very rare, but is a possibility if the spots are very close and the r-merges after scaling are above 20 or 30%) try using Web-Ice (Web-Ice does not assume that the input center is correct, but searches for the optimal coordinates over a small area).

Local users can determine the accurate center position by following these steps:

  1. Move the detector to the distance used for data collection.

  2. If the resolution at the edge of the detector is 3Å or higher, use the Si sample, otherwise use the polyethylene sample. Both samples are located in the same compartment on the beamline tool board.

  3. Collect a diffraction image. For the Si sample, use a delta phi of 15 degrees and 5 s exposure time. For the polyethylene sample, use a delta phi of 0.02 degrees and a 1 s exposure time. If the detector saturates, attenuate the beam.

  4. Run the program center on one of the blcpu servers to calculate the direct beam position from the image.

Important: The beam center position in the image header should be accurate to within 0.1mm. In the extremely rare event that it is off my a larger amount, make sure to tell user support staff.

How do I display the JPEG snapshots of the crystal?

You can use the screening tab or the image tab in Web-Ice to display the crystal snapshots.

You may also use the program display (from a Linux or Unix shell).

Are the International Tables for Crystallography available?

On-line Space Group diagrams are available at
http://smb.slac.stanford.edu/facilities/software/spacegroups/. This site is for local access only; the NX Client can be used to view the tables remotely.

The full International Tables are available at
http://it.iucr.org/. The IUCr site is fully accessible from SSRL or the NX client (or off-site if your local institution has a license).

Is it OK to leave files on the /data disk?

Users are responsible for backing up by their data by the end of their beam time. Images stored on the /data disk can be deleted at any time. Special requests to keep files on /data should be made to support staff. The /home area can be used to store small files indefinitely.


Remote access


How can I monitor the SPEAR beam remotely?

The beamline status and current SPEAR intensity are displayed in Blu-Ice. The video tools in Blu-Ice and Web-Ice can also be used to look at the beamline SPEAR monitor; in addition, the SPEAR status and fill history can also be accessed via the web.

For updates or inquiries about SPEAR you can call the duty operator 24/7 at 650 926-4040, or the beam information line at 650 926-BEAM (2326).


HKL2000 displays a screen size error

In order to run HKL2000, the NX client window must be at least 1100 x 900 pixels. If HKL2000 gives the error ''HKL2000 requires screen width larger then 1100 and screen height larger then 900 (sic)'', stretch the NX client window by left-clicking on a corner and moving out the mouse. Old versions of the NX client do not support dynamic resizing of the window. In this case, it is possible to reconfigure the window size (but updating to the latest version of the client is strongly recommended instead):

  1. Exit your current NX session.

  2. Open the NX Client as described in the installation instructions and Click on the Configure button.

  3. Find the Display options under the General menu and select ''Available Area''. If you are already using this option, select ''Custom'' instead and use the W and H input boxes to select the appropriate size.

  4. Save the configuration and restart a NX session.

The Mosflm GUI does not fit in the NX client window

Make sure that the NX client display is set to ''Available area'' (see above), and use the command mosflm_SD to run Mosflm (SD stands for ''small display'').

I cannot open an ADXV window from Blu-Ice

There is a limit on the number of ADXV processes allowed to run on the NX server in order to stop it from running out of memory (currently the limit is 5). To inspect new image, close some old ADXV windows. You can also use Web-Ice to inspect the images.

Why can't I connect to the NX server?

Please consult the remote desktop documentation.

Why can't I see staff phone numbers on the web?

Most staff phone numbers are only visible from on-site only. They can be viewed remotely by opening a browser within the NX Client.

Computers

Can I change the default settings for my SSRL account?

Click on the Settings icon in the Xfce panel to access the Settings Manager GUI. Exception: Use only the Blank Screen as a screen saver (animated screen savers use a lot of CPU, which can affect remote access through the NX client).

The previous group left the workstations locked. How do I log in?

You can log out the previous user with the keys Ctrl-Alt Backspace. Please do not do this unless you are certain that the previous group or support staff have finished working at the beamline. If in doubt, call support staff.


Footnotes

... mode2
Dose mode is a misnomer since it implies that collecting in this mode will result in the same dose (deposited energy per mass unit) on the sample. This is not strictly true.