Up: User's Guide to Macromolecular Crystallography Experiments
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The main purpose of the exposure control/dose mode is to correct for the electron current decay over time, by recalculating the exposure factor before collecting each image. With SPEAR3 operating in frequent fill mode, and typical shorter exposure times there is no real need to collect data in Exposure Control mode.
If the SPEAR3 frequent fill mode is temporarily disabled (e.g., because of an injector fault) staff can reenable the exposure control mode.
If exposure control is used, it is critical to determine the exposure time at the correct beam size, energy and attenuation before initiating data collection. To be safe, we recommend this procedure:
If exposure control is selected before effecting changes in attenuation and energy, the estimated exposure time per image will be displayed and highlighted in red.
In Blu-Ice, click on the units next to the energy input box to toggle between wavelength (Å) or energy (eV or keV).
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.
The white box in the sample camera video displayed on Blu-Ice represents the approximate FWHM beam size at the sample position at all the zoom levels (unlike the box displayed in the monitors at the beamline, which does not change size with the camera zoom. With the on-axis camera, the box shows what parts of the sample to beam hits. With the orthogonal sample camera, the vertical dimension of the box shows the size of the beam at 90 degrees.
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.
You can look at the image header with the Web-Ice Image Viewer; The program ADXV also displays the header.
The default beam position, should be at the nominal centers of the detectors (within 1 pixel) listed below (in mm):
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. Note: The convention used at SSRL may not be the same as on other sites. If the detector is offset and you cannot process the data, try flipping the Cy coordinate as described below.
To get the best data, we recommend 0.1 or 0.2 degrees oscillations and short exposures 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 parameters in Blu-Ice; we also advice to use Web-Ice to calculate the data collection strategy.
Important: Thin 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. Even if you do not exceed the maximum allowed dose with an increased exposure time, consider increasing the data multiplicity instead.
Small oscillation data can be processed at SSRL in a very straightforward manner either with XDS (see also the SSRL script autoxds) HKL2000 (from July 2010) and the most recent version of MOSFLM can be also used, although we have found that XDS tend to give the best results for oscillations much smaller than the crystal mosaicity.
Because the PILATUS has a very short readout time compared with the typical exposure time, it is not necessary to stop the phi rotation or close the shutter while the detector reads out the image; this results in faster data collection and, for very short exposures, may reduce systematic errors. The default setting for data collection mode is to collect the entire data set without closing the shutter or stopping phi.
For MAD and SAD data collections the shutter will close and phi will stop at the end of each wedge to change the energy or the crystal orientation. For native data sets, entering a wedge value different than the default of 180 will also cause the shutter to close, dividing the data collection run into several shutterless intervals. This can be advisable to be able to pause the data collection at some point - for instance, in order to check the preliminary results of data processing (see following section)
Choosing a wedge equal to the oscillation range effectively turns off shutterless data collection. This is not recommended, with the possible exception of MAD or SAD experiments on crystals that suffer significant radiation damage after a single shot.
The ''Pause'' button in the Blu-Ice data collection tab is designed to wait until the shutter closes (so that the last image is not bad). This means that after pressing this button, data collection will continue until the end of the current wedge.
Similarly, if there is a SPEAR3 current dump in the middle of the data collection, the collection will not pause, but proceed to the end of the wedge. Multiple images will be blank. For this reason, it is important to always monitor data collection with the PILATUS - or use wedges small enough that can be recollected without increasing dramatically the total dose absorbed by the crystal.
The Abort button can be used to stop the data collection, although a few images will be collected before the command takes effect. If you wish to resume the data collection after the pause, remember to recollect the last image written to disk.
The detector is programmed to change the gain at an energy of 9000 eV. Setting the new gain for all the pixels can take about two minutes and this will happen every time you start a data collection run at an energy that crosses that threshold in either direction: eg. if you collect at 12000 eV on one run and 8000 eV in the next run or vice-versa. If you want to do a MAD experiment on an absorption edge below 9000eV and the remote energy is above 9000eV consider manually selecting a different remote energy. Ask the support person for advice.
Despite the PILATUS 6M relatively large pixel size compared to CCD detectors, in our experience it is possible to resolve closely spaced diffraction spots (see for example, the PDB structure 3M8C); note that, unlike the CCD, this type of detector has a zero point spread function, which contributes to limit the spot size.
The Pilatus 6M has a large dynamic range: 1,048,576 counts. When the dynamic range is exceeded, the counter starts from zero again. The only indication for this is that holes may be observed in peaks. Blu-Ice marks in yellow pixels above 64,000 counts. These are not overloaded, but the coloring makes it easier to spot true overloads.
Unlike an integrating detector the PILATUS pixels have a count-rate limit (the counts per unit of time rather than the total number of counts over the entire exposure). Pixels that exceed the maxium count rate (recorded in the image header) are flagged in red.
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.
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).
The beamline status and current are displayed in the Blu-Ice status bar.
Additional information about SPEAR3 (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 SPEAR3 Web page. See also ''Monitoring the SPEAR3 status remotely''
SPEAR3 operates in frequent fill mode. The beam is topped up every 5 minutes. Data collection can continue normally during the injection in frequent fill mode. The normal variation of the beam intensity between fills is less than 1%. In this mode, the displayed current will always be close to the maximum injected current.
Under normal data collection conditions, we cannot detect any substantial differences between the quality of the data collected in frequent fill mode and with constant stored beam. It is also impossible to determine whether an injection took place during the collection of a diffraction image by examining the reflections in that image.
If a manual injection takes place, the data collection software will stop and restart when the temperature of the beamline optics has stabilized. If an image was being collected during manual injection, it will be recollected.
A message is displayed in the Blu-Ice status box while the beam is stabilizing following a manual injection.
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.
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 this is the problem, on-site users should repeat the hutch search. Remote users must call staff or, outside normal working hours, the duty operator (650-926-4040).
If the stoppers are open, try reoptimizing the beam. If the beam optimization does not solve the problem, call support staff.
To determine the cause of blank diffraction images, follow these steps:
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:
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.
It is advised to optimize:
Automatic optimization is also performed at regular intervals during data collection and therefore, manual optimization should not be necessary.
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)
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.
Check the relative load of the data processing server as described in ''data processing environment''.
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, the program LABELIT, and the mosflm and autoxds scripts provided at the SSRL can use the image header coordinates directly for autoindexing. However, if you use HKL2000, iMosflm or have your own scripts to run XDS you must specifically provide the offset center as follows:
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 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:
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.
You may also use the program display (from a Linux or Unix shell).
On-line Space Group diagrams are available at
The full International Tables are available at
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.
The beamline status and current SPEAR3 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 SPEAR3 status and fill history can also be accessed via the web.
For updates or inquiries about SPEAR3 you can call the duty operator 24/7 at 650 926-4040, or the beam information line at 650 926-BEAM (2326).
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)'', enlarge the NX client window as described in the remote desktop configuration (see section "Tuning the configuration" near the end of the page).
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.
Please consult the remote desktop documentation.
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).
|Technical questions: Webmaster
questions: Ana Gonzalez
|Last modified:Friday, 16-Dec-2016 11:16:08 PST.|