Beamline Operating Software (BOS or B3)

Introduction

BOS, B3, or B4 is a software program used to control and operate Protein Crystallography beamlines. It is currently used at beamlines 5.0.1, 5.0.2, 5.0.3, 8.2.1, and 8.2.2 at the Advanced Light Source at Lawrence Berkeley National Laboratory. It was originally based on the SSRL Blu-ice software, so that users from both facilities could move between the two software packages with ease. BOS/B3 can be operated remotely, enabling users to ship samples to the ALS and collect data from their own lab or living room.


Logging in

If you are logging in remotely, you must first connect to the beamline computer using the NX client, as described here. Once you are connected to the beamline computer, open a terminal window on the desktop and type b3 or b4. A pop-up will appear asking for a login. Type in your login name and password (as supplied by the beamline staff). The data generated during your BOS session will be owned by the user name you login with.

Note: To completely restart the software (both the server and gui) type stopb3 and press return. Wait a moment, and then type startb3 and press return. Wait for the message “server online” then type b3.csh, b3, or b4 to open login window. Note that generally you do not need to use this option. 

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Mounting a Crystal

On the Center tab in b3 you will see a gui on the left side of the screen to control the robot.  Select the puck letter and the pin number from the drop-down menu, and then click on Mount. If there is already a pin on the goniometer, you can select Exchange, and this function will first remove the current pin, and then mount the pin you have selected.

The mount will take ~20 seconds if the gripper is already cold, and will take ~1 minute if the gripper needs to be cooled first (this happens automatically). For automatic mounting using the TaskQueue, see below.

In b3:

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In b4:


Centering a crystal

Click on the CENTER tab within B3 (picture below).

Click on the sample using the LEFT mouse button. This moves the area clicked beneath the cross hairs and circle, which is the center of rotation. You may have to click a few times to bring the loop into the center of the screen.

You can fine tune your centering using the arrow buttons to the right of the image.

Click on +90 (or -90) to rotate the sample by 90 degrees, and center again.

Click high mag. This will move the collimator stage in and switch the view to the high-mag camera. Note: the hutch door must be closed in order to move the collimator.

Turn up the microscope backlight if necessary (LED slider to right of view).

Click on the sample to center it. Rotate the sample 90 degrees. Center again.

Check the alignment by rotating the sample by 180 degrees. The center of rotation of the loop sample should stay beneath the crosshairs. Please contact staff if you are having trouble centering as an accurate center of rotation is very important.

 In b3:

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In b4:


Getting ready for data collection

 Click on the HUTCH tab in B3.

To change the detector distance, either type number into the box (in the picture above,the distance is 350) or click on the slide tab beneath the detector image. The distance will not change until you click on APPLY.

The resolution of the image corresponding to the detector distance you have chosen is shown by the red rings overlaying the detector image. In the picture above, the resolution at the edge of the detector is 2.4Å, and in the corner of the detector it is 1.8Å.

To move the detector to a 2-theta angle, either type a number into the box (in the picture above, the 2-theta angle is 0) or click on the slide tab to the left of the detector image. The detector angle will not change until you click on APPLY.

Change the wavelength by typing a number into the Wavelength box and clicking on the Apply button. Alternatively, you can type in an energy, such as 9500 eV.

The horizontal convergence is normally 2 mrad, as shown above in the box to the right of the wavelength. You can change this to a smaller value — the result will be a reduction in flux hitting the crystal, and also a reduction in thesize of the diffraction spots on the image.

Note that changing the energy (eV), the wavelength (Å), the beam divergence (mrad), or the detector position (mm) or angle (degrees) will update the resolution rings on the image.

Set the desired collimator size using the collimator x and y settings on the right hand side of the centering window.

When setting are selected press “Apply”.

 

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Taking a snapshot

Make sure your crystal is centered.

Click on the COLLECT tab in b3.

Click on the 0 in the upper right hand corner. The zero indicates snapshot mode.

Set prefix and directory for the data files. The directory will be created if it does not already exist.

The default MODE is binned (2048×2048), 18 Mb per image (Q315 detector).

Set the detector to desired distance from sample. Standard distances are 150-500 mm.

Set collection time (in seconds), frame number, phi (angle of crystal in degrees), oscillation angle, and energy (in eV).

If you click on UPDATE button in the upper right hand corner the current values (detector distance, energy, angle) are read in. Do not hit UPDATE if you want to enter new values for the snapshot.

Make sure the hutch door is closed and the safety shutter is open.

Click on START to take an image. The dark current is taken if the New Darks box is checked.

After the snapshot is taken, the image will appear in the workspace.

If the adxv viewer is not open, select Adxv Autoload or Restart adxv from the CCD pulldown menu.

Click on the image in the adxv viewer and hit H to autoscale. The scale and contrast can be set manually from the Adxv Control panel.

 

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Starting a run

Make sure your crystal is centered.

Click on the COLLECT tab within B3.

Click on the asterix to set up a run. Run 1 (indicated in the upper right corner) means you are in data-collection mode. Each time you click on the asterix the run number is updated. This way, you can set up several runs at once.

Set prefix and directory for the data files. Once you hit ENTER, the directory will be created if it does not already exist.

The default MODE is binned which is 8 Mb per image (Q210 detector) or 18 Mb per image (Q315 detector).

Set the detector to desired distance from sample. Standard distances are 150-350 mm.

Set the energy to collect data at. The maxium flux is at 1 A (12398 eV).

Set collection time (in seconds), oscillation angle, frame number, starting and ending phi (angle of crystal in degrees).

The resulting files will be listed in the middle panel. Adjusting the frame number and phi angles will update the list automatically.

If you click on UPDATE (upper right hand corner) the current values (detector distance, energy, angle) are read in. Do not hit UPDATE if you want to enter new values for the run.

If you enter more than one energy or if you click Inverse Beam, you can chance the number of frames per degree by adjusting the Wedge Size.

Click start. A dark current is taken if the New Darks box is checked. Note that if the exposure time is very different than the previous exposure, the software will take a new dark shot whether or not the box is checked.

As the images are taken, they will appear in the window. If the adxv viewer is not open, select Adxv Autoload from the CCD pulldown menu.

Click on the image in the adxv viewer and hit H to autoscale. The scale and contrast can be set manually from the Adxv Control panel.

 


Viewing images

Loading images

You can open the program ADXV one of several ways: (1) click on the ADXV icon on the desktop, (2) open a terminal window and type adxv at the prompt, or (3) within B3, click on the CCD menu and select either ADXV or ADXV-Autoload.

If you select ADXV-Autoload, the images will appear directly after they are collected.

If you select ADXV, then you can choose which image to load.

 

Scaling images

Move the cursor into the image space and hit H to autoscale. The scale and contrast can be set manually from the Adxv Control panel.

Right-click within the image to open up a magnified view. You can move the magnified window around by holding down the right mouse buttom while moving the mouse.

Left-click on the image for a line profile.

Middle-click to drag the image around within the screen.

Move the cursor into the image space and hit H to autoscale. The scale and contrast can be set manually from the Adxv Control panel.

 


Taking a fluorescence scan

Make sure your sample is centered.

On the scan page in BOS, select the edge you are trying to scan by clicking on the red K or L under the element. (When trying to decide which L-edge to use, keep in mind that the L3 gives the largest fluorescence signal.) A list of x-ray absorption edges can be found here.

Enter your filename and directory in the appropriate fields.

There are two ways to do the scan: (1)Click the “Use Optimized Convergence” button and type in a value of 0.1. Make sure the “Optimize Fluorescence” button is not selected. This will not optimize the convergence setting;instead, it will start the scan using the convergence value that you typed in (0.1). In most cases, the convergence setting of 0.1 works well. (2) Click the “Optimize Fluorescence” button. Make sure the “Use Optimized Convergence” button is not selected. This will optimize the convergence setting before starting the scan.

Once the scan starts, the curve will be displayed.

Once the scan is done, you can analyze the curve using either Kramers-Kronig program or the Chooch program, as described below.

 

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Kramers-Konig and Chooch

Kramers-Konig

If you have just run a scan, then click on the KK button to activate the Kramers-Konig calculation.

If you are loading a previous scan, then click on the load button. The directories are listed in the left column. Double-click on each subdirectory until you reach the directory containing your scan file. Double-click on the filename to load it.

On the right side of the screen, select the absorbing element and click on AutoFit Curve.

To zoom in on the plot, click with the left mouse button, drag a box around the area of interest,and click again with the left mouse button. To zoom out, click once with the right mouse button.

Click with the middle mouse button and the energy corresponding to the position of the cursor will be displayed at the bottom of the screen, along with the f’ and f” values at that point.

Find the maximum of the fluorescence scan (the f” curve) and the minimum from the f’ curve (which should roughly correspond to the inflection point of the f” curve). These energies (plus a remote point ~300 eV above the edge) are the values to use when collecting MAD data.

 

 

Chooch

For Chooch, click on the Chooch button in the Scan window.

Note that if you have loaded a scan, make sure you have selected the correct element and edge from the periodic table before running Chooch.

The calculated f’ and f” values will appear in the table below the button.

Click on the collect button within that table, and those values will be written into the Collect page for data collection.

A one-minute video tutorial on using Chooch can be found here.

 


The MAD experiment

From the fluorescence scan, you should know the energies for data collection: the minimum of the f’ curve (which corresponds to near the inflection point of the fluorescence scan), and the peak of the f” (fluorescence) curve. For example, for Se usually f’~12.662 KeVand f” ~12.658 keV. The remote energy is generally chosen to be ~50-300 eV above the peak.

There are several possible data collection strategies:

1) Collect one wavelenth at a time, with inverse beam, in 20-30 wedges. Collect the f”; (peak) first, then f’ (inflection), and finally the remote wavelength.

2) Collect the inflection and remote wavelengths at the same time, interleaving the inflection and remote energies (no inverse beam). Then, collect the peak.

3) Collect all wavelengths at once, interleaving the energies, with a wedge size of one.The staff will be happy to discuss the pros and cons of the different data collection strategies above.

 


Other Features

Saving a jpeg of your crystal

It may be valuable to use a copy of the centering view image in a report. To save this image, when viewing the Center tab click on the Camera pull down menu and select “Save as”.

 

Washing a crystals

From the Tasks pull-down menu in B3, select “Wash Crystal”. On sector 8, this will command the robot to pour a small amount of LN over your crystal. On sector 5, this will command the robot to take the pin from the goniometer and dunk it several times in LN, then return it to the goniometer.

 

Annealing a crystal

From the Tasks pull-down menu in B3, select “Annealer”. You will get a pop-up asking how long to anneal. Enter the time, and click on enter. A card will flip in front of the cryostream for the designated amount of time.

 

Tuning the beamline

From the Tasks pull-down menu in B3, select “Tuneup beamline”. This will atuomatically tune the monochromator and/or mirrors. The tuneup can take up to 5 minutes.

Thank you for reading!