The first step in using Autoscreen to study protein-ligand interactions is to set up the project. The Autoscreen module organizes a research project in the FELIX database management system into a group of entities. Using the Project menu items, you can build a new project, open an existing project, add, delete, or display experiments, and define molecule files for the protein and ligands.
The project entities are built (or loaded if they exist) using the Autoscreen/Project (<Alt>-np) menu item. The entities contain information about the NMR experiments and associated ligand structures, parameters for processing, scoring and displaying spectra, scoring results, and other data. The project entities are organized in a database directory with the same name as that of the project.
When you select this menu item, it prompts you to specify a project name if an Autoscreen project does not exist. This name is used to create a directory (in the database) in which several project entities are also created.
If a project already exists (that is, if it was built in a previous session) this menu item opens it.
When creating a new project, FELIX tries to use the parameters for spectrum display and scoring that were used in the most recent project in the same session. If these are not available, it assigns default values for them. (But note that parameters for spectrum processing are not inherited from project to project.) The spectrum-display parameters related to the display limits, references, and threshold are reset based on the control spectrum so that they are, in effect, not strictly inherited either.
When creating a new project, the VERIFY DIRECTORIES control panel allows you to define the paths used to access or save the following files:
You can click Browse next to any of the paths to select a directory interactively, or you can type in the directory directly. When you select OK to close the control panel, an empty Autoscreen Experiments Table opens.
Once the project is built, you can import the NMR experimental data and molecular data. You can use the Autoscreen/Experiment pullright menu or the menu items on the Autoscreen Experiments Table to add, delete, and display experiments, or to define project paths.
Table 26 shows the types of raw or processed data that can be used by Autoscreen. If a processed datamatrix is used, it is converted (or simply copied, if it is a FELIX matrix) to a FELIX matrix with a filename the same as the experiment ID. The symbol is used in the Autoscreen Experiments Table or a file list for indicating the datatype.
| Data file type | Symbol | ||
|---|---|---|---|
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1Although various types of data can be imported into an Autoscreen project, you are advised to stick to one of them for all experiments in a particular project instead of mixing them.
|
Although various types of data can be imported into an Autoscreen project, you are advised to stick to one of them for all experiments in a particular project instead of mixing them.
This menu item allows you to add experiments to the project with a file list. A file list is an ASCII file that specifies the experiments and associated information to be added to the project. Each line in the file specifies the experiment ID, datafile, file type, structural filename (optional), and comments (optional), as explained below:
1. An experiment ID is one word used as the identification of the experiment. It is also used as the filename of the processed (or converted) FELIX matrix.
2. A datafile is the filename of the time-domain data (or processed data) prefixed by an absolute path or a path relative to the project path forpfx.
3. The type of the raw or processed data is specified as in Table 26.
4. A structural filename can be a .pdb, .car, or .mol file. Any other filename is taken as a list of structural files. A filename can have an absolute path or have a path relative to the project path xyzpfx. If you do not have a molecule but want to input comments, enter null as the filename.
5. The optional comment can be one or more words; FELIX concatenates them with underscores (_).
The following sample file list specifies information for five experiments:
Control 1/ser ser demo control
Test1 2/ser ser test.car test-1
Test2 3/ser ser mols.txt
Test3 4/ser ser
Test4 5/ser ser null The last experiment
This menu item allows you to add all Bruker experiments in a certain directory to the project.
This menu item allows you to add experiments to the project interactively. You can also specify other information for each experiment, such as the filename of the associated structure.
This menu item allows you to verify and change the important paths used by the project. See the Autoscreen/Project section above for more detail.
This menu item displays the Autoscreen Experiments Table if it is not open yet.
This menu item is equivalent to Autoscreen/Experiment/Add From File List. See the Autoscreen/Experiment section above for more detail.
This menu item is equivalent to the Autoscreen/Experiment/Add All Files menu item. See the Autoscreen/Experiment section above for more detail.
This menu item is equivalent to Autoscreen/Experiments/Add One. See the Autoscreen/Experiment section above for more detail.
This menu item deletes the highlighted experiment(s) in the Autoscreen Experiments Table from the project.
This menu item is equivalent to Autoscreen/Experiments/Verify Directories. See the Autoscreen/Experiment section above for more detail.
After adding experiments to the project, you can process the control spectrum, pick or import peaks, import assignments, set up scoring parameters, and score the test spectra. The relevant menu items are found on the Autoscreen menu and in the Autoscreen Experiments Table. Two menu items in the Peak Displacements Table also allow you to manually edit scoring results.
This menu item is used to interactively correct systematic errors of peak locations. To do this, zoom in on a spectrum region so that a peak and a peak label are clearly visible. Then select the menu item. Click the peak label, drag the cursor to the correct location, and release the mouse button. The displacement defined by the mouse movement is added to all control peaks. You can repeat this step until you are satisfied and then press <Esc> to exit the calibration.
This menu item is used to import resonance assignments. The files can be in FELIX spin system, Insight resonance file, or BMRB assignment table format.
The Autoscreen/Setup Scoring menu item is used to set up the scoring parameters and create a score matrix, if necessary, where the results will be saved. A score matrix usually has a filename score_DB.mat, where DB is the name of the current database. After you perform this action, a new score matrix is generated if it does not exist, if you've added or deleted control peaks, or if you're switching to a new control-peak table.
When you select this menu item, the 2D SCORING PARAMETERS control panel appears, which allows you to define the basic parameters for scoring. If you click the Advanced button, you can define more advanced parameters in the ADVANCED PARAMETERS FOR 2D SCORING control panel. The parameters are explained below. For more details. please refer to Chapter 1, Theory, in the FELIX User Guide.
Basic parameters, which are controlled via the 2D SCORING PARAMETERS control panel, include:
Advanced parameters, which are controlled via the ADVANCED PARAMETERS FOR 2D SCORING control panel, include:
All other parameters in this section are the same as for standard 2D peak picking. The same values as for the control spectrum are recommended for them.
The toggles Use Peak Widths and Use Peak Heights, if checked, allow the peak widths and peak heights, respectively, to be used for calculation of shape similarity between a control peak and a test peak.
If peak shape is used, Minimum Shape Similarity is a threshold for two peaks to match. This is an extra category for two peaks to match in addition to the Peak Displacement Limits in the basic parameters group.
Search Methods provides two alternative algorithms for searching for a best match between the test and control peaks. If Tree Search, the default method, is selected, a heuristic depth-first search method is applied. Since this can be time-consuming, you can limit the CPU time spent on each test spectrum by defining a value for CPU Time Limit (default value is 10 s). If Simulated Annealing is selected, the stochastic method is applied. The latter is usually fast (so CPU Time Limit is not used), yet does not guarantee a best match. The latter method is recommended when the spectra are so complicated that tree searching does not give satisfactory results in a reasonable amount of CPU time.
For each unmatched control peak, if Fit to Test is selected, it is fitted to the test spectrum using the peak-optimization function if the percentage of unmatched control peaks has not exceeded the Maximum (%). (For details please see Peaks/Optimize in Chapter 4., Processing, visualization, and analysis interface (1D/2D/ND) in the FELIX User Guide.) If the fitting is successful, the optimized peak is taken as their matched test peak. Otherwise it remains unmatched, and a Penalty (default 0.60) contributes to the score of the experiment
For unmatched test peaks, Selection allows you to select the method to define them. If None is selected, such peaks are ignored. If Close to Control Peaks is selected, only those that are close to at least one control peak, namely with displacements no larger than the Maximum D1 (or D2) Peak Displacement Limits, are included. Otherwise, if All is selected, all peaks are included. When determining if it is a legitimate test peak, the peak widths and height of a test peak are compared with the statistics for all the matching test peaks. The parameter Num.of RMSD (default 2.0) allows you to define a tolerable deviation from the average peak widths and height. The Penalty is the contribution of each unmatched test peak to the score of the experiment. The default (0.2) is smaller than that for an unmatched control peak (0.6), because automatic peak picking of test peaks is usually less reliable than picking of control peaks, which is normally done manually where refinement is possible.
This menu item allows you to process the highlighted experiment as a control spectrum. The processing parameters are saved and are later used for automatic processing of all other test experiments.
This menu item allows you to select a processed experiment as the control spectrum. This is useful when you are using processed data to set up an Autoscreen project (hence you do not need to process a control spectrum) or when you want to switch to another control spectrum after processing a control spectrum and some test spectrum. In the latter case, you are warned about losing the current scoring results, if any.
This menu item processes the highlighted experiment(s) as test spectra. The same processing settings as for the control spectrum are used. When a spectrum is successfully processed, its status is updated to 1 in the Autoscreen Experiments Table.
This menu item scores the highlighted experiment(s). If a highlighted experiment is not processed, it processes the experiment before scoring. You must set up scoring parameters before using this menu item. When a spectrum is successfully scored, its status is updated to 2 in the Autoscreen Experiments Table, and the test spectrum and control spectrum are displayed as an overlay of contours. Arrows are displayed over the spectra showing the displacements of peaks. The Peak Displacements Table is updated with scoring information such as the contributions of individual peaks.
Using this menu item processes all nonprocessed test experiments and scores all nonscored experiments. If an experiment is already processed or scored, it is not processed or scored again. This menu item is useful when you trial-processed and -scored a few experiments and now want all the remaining experiments to be scored in batch mode.
Upon completing this action, a histogram of scores vs. experiment is displayed.
This menu item scores all processed experiments, whether or not they were previously scored. This menu item is useful when you have changed ROI peaks and want the changes to be reflected in the scores.
Upon completing this action, a histogram of scores vs. experiment is displayed.
This menu item processes and scores all experiments in batch mode, no matter what their status.
Upon completing this action, a histogram of scores vs. experiment is displayed.
After the Autoscreen/Setup Scoring action is successfully completed, a Peak Displacements Table is displayed. This table shows the control peaks and their contributions to the score of a current scored experiment. The ID of the current experiment, if any, is displayed next to the title of the table. This table is automatically updated after you select Action/Score Selected Spectra. You can also update it to reflect the scoring of a certain experiment by double-clicking the experiment in the Autoscreen Experiments Table or by displaying its peak contribution histogram. If the table is closed, you can select Autoscreen/Show Displacement Table to open it.
The Peak Displacements Table contains the columns shown in Table 27:
This menu item allows you to click a certain control peak to remove its displacement. This peak is then scored as an unmatched control peak. This process can be repeated until you press <Esc> to exit.
This action is equivalent to that of the Remove Displacement icon on the tool bar of the same table.
This menu item allows you to change an existing displacement or add a new one. Click a control peak and drag to a destination test peak. When you release the mouse button, a violet arrow shows the new displacement. You can repeat this process as often as you want to, then press <Esc> key to exit.
A user-defined displacement is scored in the same way as an automatically determined one, except that there are no minimum and maximum limits for the displacement. The destination is not checked either, so it can be anywhere in the spectrum. In addition, peak shapes are not considered when calculating their contributions to the score. All the changes you've made are reported in the text window and updated in the Peak Displacements Table.
This action is equivalent to that of the Remove Displacement icon on the tool bar of the same table.
By default, the displacements of all control peaks are used for scoring. In practice, you may be interested in only a subset of peaks that are particularly informative for the study. This subset, designated region of interest (ROI), can be defined and displayed using several methods offered by the Autoscreen/Define ROI pullright menu of the Edit menu of the Peak Displacements Table.
As displayed in the Peak Displacements Table, an ROI peak has a weight greater than zero. When a peak is included as ROI, its weight is set to 1.0. When a peak is removed from ROI, its weight is set to zero. In addition to using the following menu items, you can always directly modify the weight of a peak in the Peak Displacement Table.
This menu item displays a cross-hair cursor, allowing you to add ROI peaks one-by-one by clicking each peak. To exit this mode, press <Esc> or click a blank area.
This menu item includes all displayed peaks as ROI.
This menu item allows you to drag out a rectangle around a set of peaks to include all peaks in the rectangle as ROI.
This menu item allows you to include ROI on the basis of their assignments. In the FIND 2D ROI PEAKS BY RESIDUE NUMBERS control panel, enter the residue numbers, as individual numbers, ranges, or a combination of both, separated by commas (for example, 1,3,6-12).
This menu item also allows you to include ROI on the basis of their assignments. In the Find control panel, all assigned peaks are displayed in a list box. You can select one of them and select OK to include it as ROI.
This menu item displays a cross-hair cursor which allows you to remove ROI peaks individually, by clicking a peak at a time. To exit this mode, press <Esc> or click a blank area.
This menu item allows you to drag out a rectangle so that all peaks in the rectangle are excluded from ROI.
This menu item allows you to exclude peaks from ROI on the basis of their assignments. In the FIND 2D ROI PEAKS BY RESIDUE NUMBERS control panel, enter the residue numbers as individual numbers, ranges, or a combination of both, separated by commas (for example, 1,3,6-12).
This menu item designates all peaks as nonROI peaks.
This menu item highlights the ROI peaks in yellow by default. You can change the color by using the Autoscreen/Define Display menu item.
The ROI peaks are automatically highlighted when you draw a spectrum with an Autoscreen menu item. However, sometimes redrawing the spectrum (for example after you resize the spectral window) turns off the ROI peak highlighting. You can select this menu item again to highlight them.
This menu item enhances the display of ROI peaks by reserving a separate window for each one. This focuses the display directly on the interesting areas.
Peak Displacements Table menu items:
Edit/Add To ROI - This menu item adds the currently highlighted peaks (rows) in the Table to be included to the ROI.
Edit/Remove From ROI - This menu item removes the currently highlighted peaks (rows) in the Table from the ROI.
Autoscreen allows you to display and print spectra in single or overlay mode. You can set the display limits and other display options and save them along with the project. The relevant menu items are found on the Autoscreen menu and in the Autoscreen Experiments and Peak Displacements tables.
This menu item saves the following display settings in the database. These are usually changed using the general FELIX menu items, as detailed below:
These settings are used for display, hardcopy, and scoring of all experiments in the project.
This menu item allows you to select some frequently used display options in a control panel. You can save the changes in the database by selecting OK.
The options include the following for displaying the contours of control and test spectra:
For test spectra, you can select Automatic or Define as described above, or select Control so that the threshold is the same as that used for the control spectrum. If you select Automatic and the spectrum has been scored, the value in the thresh column in the Autoscreen Experiments Table is used.
The options also include the following for displaying control peaks:
This menu item allows you to choose options for printing with the Print icon (see the Print icon section below for more details). The options in the control panel are similar to those in File/Print, except for the following:
Clicking this icon displays the contours of the highlighted experiment in the Autoscreen Experiments table. If you select multiple experiments, they are displayed in turn. If the control spectrum is included, the picked peaks, if any, are displayed. Otherwise no peaks are displayed. If you select a nonprocessed test spectrum (that is, one with a status less than 1), it is automatically processed before it is displayed.
Many display attributes can be changed. See the Autoscreen/Setup Display and Autoscreen/Setup Print sections for more information.
The Draw Next icon displays the contours of the experiment next to the currently highlighted one in the Autoscreen Experiments Table. If you select multiple experiments, the one next to the first selected one is displayed.
This icon displays the contours of the experiment previous to the one currently highlighted in the Autoscreen Experiments Table. If you select multiple experiments, the one previous to the first selected one is displayed.
This icon overlays contours of the highlighted test experiment on top of those for the control experiment. If you select multiple experiments, the first test experiment is used. If you select only the control experiment, FELIX ignores the menu item. If control peaks and displacement arrows exist, these are also displayed.
If you have scored the selected test, this menu item also updates the Peak Displacements Table.
Many display attributes can be changed. See the Autoscreen/Setup Display and Autoscreen/Setup Print sections for more information.
Clicking this icon overlays the contours of the test experiment next to the currently highlighted experiment over those of the control experiment. If you select multiple experiments, the experiment next to the first selected experiment is used.
Clicking this icon overlays the contours of the test experiment previous to the currently-highlighted experiment over those of the control experiment. If you select multiple experiments, the experiment previous to the first selected experiment is used.
Clicking this icon overlays the contours of the one or more highlighted test experiments over those of the control experiment. If the control experiment is highlighted, it is ignored.
For each highlighted test spectrum, this icon displays the histogram of contributions vs. peaks if it is scored. This menu item also updates the Peak Displacement Table.
In the PEAK CONTRIBUTION HISTOGRAM OPTIONS control panel, the X Coordinates can be set to Peak IDs or Residue Numbers. If Peak IDs is selected, the item numbers of the peaks in the Peaks Table are used as the x coordinates. Otherwise the numbering of residues assigned to the peaks is used and unassigned peaks are ignored.
The five choices for Y Coordinates are:
This icon displays the molecule in Insight II. The molecule file can be a PDB, CAR, or MDL (.mol) file or a list of such files.
Depending on your setup, this menu item does one of the following:
See the Autoscreen/Setup Print section for more information about printing setup.
This menu item sorts the peaks in descending order of their contribution to the score. This action is equivalent to that of the Sort Contributions icon on the toolbar in the same table.
This menu item sorts the peaks in ascending order of their item number. This action is equivalent to that of the Undo Sort Contributions icon on the toolbar in the same table.
This menu item zooms the overlay spectrum display into highlighted peak(s) in the Peak Displacements Table, providing a closer view of the displacement of peak(s) that you are interested in.
This action is equivalent to that of the Zoom on Peaks icon on the toolbar of the same table. If you are interested in one particular peak, you can simply double-click that row to zoom in on it.
This menu item zooms the overlay spectrum display into the peak next to the currently highlighted one in the Peak Displacements Table. If you highlight multiple peaks, only the first one is considered.
This action is equivalent to that of the Zoom Next icon on the toolbar of the same table.
This menu item zooms the overlay spectral display into the peak previous to the currently highlighted one in the Peak Displacements Table. If you highlight multiple peaks, only the first one is considered.
This action is equivalent to that of the Zoom Previous icon on the toolbar of the same table.
This menu item highlights, in the Peaks table, the corresponding peaks of the highlighted peaks in the Peak Displacements table, providing a straightforward way to locate control peaks in the Peaks table.
This action is equivalent to that of the Locate in Peak Table icon on the toolbar of the same table.
The scoring results can be presented in various ways for visualization and export, using menu items on the Autoscreen menu and on the Peak Displacement Table, as described below.
This menu item shows the Peak Displacements Table, which displays the scoring information of the current test spectrum. This table is automatically displayed when you set up scoring parameters and is updated when you display the overlay or score histogram of a test spectrum.
This menu item displays a histogram of scores vs. the experiments. It is useful for visually identifying highly displaced experiments, which usually correspond to high-affinity ligands.
This menu item groups experiments that share common displaced peaks, providing a way to identify individual binding subsites in a protein. The VIEW CLUSTERS control panel allows you to define a threshold, so that peaks with displacements smaller than that value are ignored. Some clustering details are displayed in the text window.
This menu item opens the EXPORT ACTIVITY control panel, allowing you to export the scoring results in several formats:
This menu item exports the contributions of the assigned peaks to a text file that can be used by Insight II or Cerius2 to color the residues in the protein, providing a way to visualize the subsites that have close contacts with the ligand.
This menu item plots a histogram of contributions vs. experiment for the highlighted peak in the Peak Displacements Table, providing a way to see the affinity of different ligands to a particular residue.
This action is equivalent to that of the Titration icon on the toolbar of the same table.
This icon highlights the corresponding control peak in the Peaks Table for the one currently highlighted in the Peak Displacements Table. This facilitates viewing the details of a selected control peak.