Scan Tab

• Overview
• MAD Scan
• Selecting an X-ray Absorption Edge
• Analyzing and examining the Fluorescence Scan
• Output files
• Saving, reading and printing fluorescence scans
• Excitation Scan
  • Pitfalls and caveats
• Appendix: Absorption edge and main emission line energies

Overview

The Scan Tab is used for energy and excitation scans. The energy (MAD) scans are used to select the appropriate wavelengths for anomalous dispersion experiments (optimized SAD and MAD). The excitation scan is useful to identify and verify the presence of anomalous scatterers in the sample

[Fig 1. The Scan Tab in Blu-Ice]

MAD Scan

• This mode is used to scan the x-ray energy against the fluorescence emitted by the sample and subsequently determine the optimal energies for MAD data collection.

[Fig 2. Scan Mode and Scan Parameter Menus]

Selecting an X-ray Absorption Edge

• The desired absorption edge can be selected from the Periodic Table tab.
• Select an edge by clicking on a particular edge (K, L1, L2, L3) and press the Scan button.

For some elements, such as those shown to the left, you can select different edges-- L1, L2, L3. As a rule of thumb always select L3 for heavy elements; however, if this edge is not accessible at the beamline, try scanning the L2 edge instead. The L1 edge, on the other hand is usually to small to provide a good dispersive signal, and a SAD (instead of MAD) experiment above this edge is recommended if neither the L2 nor the L3 edges are accessible. Consult the support staff for help with selecting the edge.

• The directory and prefix determine the location and name of the scan output files. The update button will set the directory to /data/yourid and prefix to test. If you used the screening tab to mount the crystal, the update button will set the directory and prefix to the directory and crystal name used in the screening tab.
• The Start button will start the scan operation. Before starting scanning the energy, the software will attenuate the beam to avoid saturating the fluorescence detector. Several beam filter combinations are tested. You can monitor this from the hardware tab or in the electronics rack filter control panel .
• Stop will interrupt the scan and move all the motor positions to their initial values. Abort will stop all motors inmediately, but the beam filters will be returned to their original configuration.
• Click on the Plot tab next to the Periodic Table tab to display the plot of the spectra.



[Fig 3. Plot of fluorescence scan showing raw counts (on an arbitrary scale)]

• The Log tab displays the fluorescence readings during the scan
• If the initial scan looks noisy (large oscillations in the fluorescence counts from point to point comparable to the edge height), the scan time can be increased to improve the signal.

Analyzing and examining the Fluorescence Scan

• The program autochooch automatically calculates the anomalous scattering factors from the fluorescence data. The f" and f' plots are displayed in the Plot tab, as well as the suggested peak (maximum f") , inflection (minimum f') and remote (high f" and f') energies for MAD data collection.

[Fig 4. Plot of f" and f' values calculated from the fluorescence scan]

• The selected energy values can be adjusted by moving the vertical cursors (the blue line in Figure 4). The cursors can be moved by clicking on them with the middle mouse button. Right clicking on the cursors will give options to change the cursor color, cursor thickness, and to delete the cursor. This will update the energy values selected for data collection
• Checking The Link to Run Definition via Update box allows exporting the energy values into the collect tab (this is the default after a successful scan). Pressing the Update button in the Collect Tab will then import the energies for MAD data collection.
• You can display the raw fluorescence counts by clicking the right mouse button on either side of the plot and selecting a new Y axis label.
• By clicking the right mouse button on the transform or any other lines in the plot, users can change various parameters like line color, line thickness, symbol shape and color, etc.
• Placing the mouse over the plot line will display the x and y coordinates for each point. This is useful to find out the f' and f" values for energies other than the ones written out by autochooch. The f' and f" for the autochooch energies can be obtained from the Log tab or the summary file. If you use a different remote wavelength to the one selected by the software, you can get the f' and f" values from these tables.
• To zoom in the plot, left click on a point of the plot window, hold down the mouse key and drag the mouse to define the zoom rectangle. The zoom level changes after releasing the mouse button. Right clicking on the plot window displays a menu with the option to zoom out.

Output files

The following files are written out after successful completion of a fluorescence scan:

1. name-scan: Contains the raw fluorescence readings
2. name-smooth_exp.bip and name-smooth_norm.bip: Intermediate files from autochooch
3. name-fp_fpp.bip: Anomalous scattering factors (f" and f') calculated by autochooch
4. name-summary: Summary of the scan, including the values for the peak, inflection and remote wavelengths and the f" and f' values for each of them

A summary of the scan is also displayed in the Log tab

Saving, reading and printing fluorescence scans

Right-clicking the mouse on the plot window opens a menu which allows saving, printing and opening fluorescence scan files.

• The print option will send the current scan plot to the default printer.
• The open option allows to read in previous scan files. The program will open a file browser to help locate the scan files.
• The save option has been superseded by the automated generation of output files. The program will prompt for the output file name and will write out a single bip file containing the fluorescence counts, processed counts and f" and f' values for each energy.

Excitation Scan

While MAD scan measures the fluorescence counts from a single element by changing the energy, the excitation scan measures the fluorescence counts from any element present in the sample with an absorption edge below the excitation energy. Excitation scans are very useful for the identification of heavy elements in a crystal. They take less time than the MAD scans and thus are a faster way to determine the presence of a heavy atom derivative/ligand in the sample.

[Fig 5. Excitation Scan Mode]

• Select the element for scan from the periodic table and press the Scan button. The energy will automatically change to an appropriate value.
• After collecting an excitation scan, Blu-Ice will automatically search for peaks and attempt to match them to the emission energy for all stable elements. If it finds a possible match, the element symbol will be added to the list of elements displayed below the scan in the first position. The most common elements found in proteins, either introduced (Se, Hg, Pt) or occurring naturally in metalloproteins are also displayed, even if a matching peak has not been found.

  

• Hovering with the mouse over the element name in the list will display between one and three vertical lines on the scan plot, corresponding to all the emission lines for that element that can be excited at the scan energy. If you see peaks that coincide with all the predicted lines, it is possible that the sample contains that element. Note that this is always not 100% certain, as there may be another element or elements emitting at the same or similar energy responsible for the peaks you see. See Pitfalls and caveats for more information.
• You can permanently mark the peak by left-clicking on the element name. This will add a brown line in the plot which does not disappear as you move the mouse. In Fig. 6 the Zn peak is labeled permanently. Right-clicking on the line marker displays the emission energy and allows changing the line color and width.
• To display the emission lines for elements not listed under the figure, use the drop-down menu to the right of the element names, and select the desired element name.



[Fig 6. Excitation Scan of Sample Containing Zinc and Selenium.]

• Only alpha emission lines are marked in the plot, as they are usually the most intense ones; however, when an element is present in a high concentration, there may be a smaller beta peak to the right of the alpha peak. For example, in the scan displayed above, there is a peak corresponding to the Zn K-beta emission which is not labeled. In cases like this, you can confirm the identity of the peak by checking the Emission Line Tables. The right-most peak in the scan does not arise from emission, but from elastic and inelastic scattering (they occur at different energies, but they cannot be fully resolved with the fluorescence detector). This peak is not labeled either. For a full interpretation of a typical scan, see Fig. 7
• Excitation scan plots can be saved, zoomed in, etc. in the same way as fluorescence scans.

[Fig 7. Fully interpreted excitation scan. The elastic and inelastic peaks are not separated because of the detector resolution. The K-beta peak for Se is relatively small in this sample and it disappears into the tail of the next peak at higher energy.]

Pitfalls and caveats

Sometimes emission peaks for different elements will overlap. In this case, you need to use common sense to identify the element. Could one of the elements be present in your crystal as a derivative, ligand or part of the buffer?. If not, the chances are that the element is something commonly found in proteins (the right-most elements listed under the plot) and not a rare-earth or a heavy transition metal.

If you find many different strong emission peaks that you don't expect (Cu, Ni, Fe), double-check that you are not hitting the metal pin. Very weak peaks may also originate from a source other than the sample (beamline components or sample holders; for example, Zn emission has been observed from empty loops, Pb from glass capillaries; Fe can be present in the fluorescence Be window. It is a good idea to repeat the excitation scan in absence of the sample and verify that the peaks disappear too.

Appendix: Absorption edge and main emission line energies

Strongest emission lines for the most common elements present or introduced in biological macromolecules. (Emission and excitation energies in eV).