Xsight

Density fitting

The ability to manipulate a model relative to an electron density map constitutes one of the most important activities that a macromolecular crystallographer performs. The following tutorials teach you some of the basic techniques that can be used with the Xfit program.

In Lesson  7:Making maps and basic fitting techniques, you will learn to calculate electron density maps and compare them with a refined structure.

The topics covered in this lesson are:

• Creating a phase file.
• Executing Xfit, the model fitting module.
• Calculating structure factors.
• Calculating OMIT maps.
• Executing basic map fitting commands.

In Lesson  8:De novo map fitting, you will learn to trace the alpha carbon (CA) chain through an electron density map.

The topics covered in this lesson are:

• Skeletonizing a map.
• Marking CA positions in a map.
• Manipulating the electron density in the display.
• Turning certain elements of the display on or off.

Lesson 7:   Making maps and basic fitting techniques

The scenario: You have just collected a new native data set of C. vinosum cytochrome c' (cvccp) and you want to calculate electron density maps and compare them with your currently refined structure. In particular you would like to check the validity of some questionable residue positions using OMIT maps and map fitting techniques.

1.   Preparing the tutorial directory

To run the map-making tutorial you must set up a directory which contains the files ccp.cool3.pdb and ccpniau1.fin, i.e., a coordinate file and a reflection file.

Enter the following commands, in the order shown, at the command prompt: > mkdir tutorial_fit > cd tutorial_fit > cp $XSIGHTTUTORIAL/ccp.cool3.pdb . > cp $XSIGHTTUTORIAL/ccpniau1.fin .

2.   Getting started

At the system prompt, issue the command: > insightII and press <Enter>.

Wait a few moments while Insight II loads.

3.   Creating a project

Go to the Xsight module. Now select the Utilities/Project command. When the command parameter block appears, type in the name of the project (for example, tutorial_fit) and select Edit. Select cvccp from the Crystal_List and enter the Project_Directory based on the directory that was created in Step 1. (You must use the absolute path for this directory, ~usr is not accepted in this box; for example, /net/machine/usr/people/pns/tutorial_fit.) Select Execute.

The Xsight module commands are now displayed on the lower menu bar.

4.   Creating a phase file

This tutorial is written from a point of view that assumes that you already have your processed reflection file in a .fin format, the standard XtalView reflection format. To calculate electron density maps within Xfit, it is necessary to create a phase file (.phs) before starting the program. The.fin file only contains h, k, l, F_o, and (F_o) information, so the .phs file that is created will not contain any values of F_c or . In reality, this step only involves formatting existing data (stored in a .fin file) into a .phs type file.

The file formats are shown here as an aid to understanding the information flow. Since this is a native data set, F1 and F2 are the structure factor amplitudes associated with Friedel mates, and can be averaged when generating the value of F_o in the .phs file.

.fin format: h, k, l, F1_o, (F1_o), F2_o, (F2_o)
.phs format: h, k, l, F_o, F_c,

Select the Data_Control/Import_Data command. Select the hkl_F1_SIG1_F2_SIG2 option as the Input_File_Format. Enter ccpniau1.fin as the Input_File_Name parameter. Select the Phase(.phs) option as the Output_File_Format. Make sure that ccpniau1.phs is entered as the Output_File_Name parameter. Select Execute

5.   Starting Xfit

Select the Model_Building/Density_Fitting command. Make sure that Pass_Molecule is set off and Pass_PDB_File is set on. Now click PDB_File to display the list of available .pdb files in the PDB_File_List, and select ccp.cool3.pdb from the list.

Now select the One_Phase_File option for the Pass_Phase_Files parameter. Click the Phase_File parameter window and select ccpniau1.phs from the value-aid. Select Execute.

Within a few moments the Xfit program is spawned from Insight II.

Four windows appear when Xfit begins execution. The first window to appear is the Xfit Tools menu bar. The next window to appear is called the canvas. It is the graphics output window for Xfit and the contents of the .pdb file are displayed here. Since you loaded a .phs file at startup, the Fast Fourier menu appears in anticipation of Fourier calculations to come. Finally, the main Xfit window appears.

6.   Calculating F_c and

In the main Xfit menu, click the SFCalc button to open the structure factor calculation menu.

You will use this menu to calculate F_c and values based on the model that was read into Xfit. In addition, it will be necessary to scale the F_o and F_c values together. It is usually a good idea to use all reflections in map calculations in order to reduce series termination effects. For that reason, leave d min and d max set to their default values.

Click the Calculate All and Scale button in the StructureFactors menu to start the structure factor calculation.

When the structure factor and scaling calculations are finished, the menu disappears and you are returned to the main menu. The text window at the bottom of the menu contains a summary of the calculation. The R factor may seem high at this point (28%) but that is due to the fact that the low resolution reflections are included in the calculation.

A Fourier map is automatically calculated at the end of the structure factor calculation, and displayed in the canvas graphics window. However, the default coefficients are different (F_o) from what you want in this lesson. For this reason, the Fourier calculation must be repeated.

7.   Calculating a Fourier map

Now you calculate and display a SigmaA weighted map using the Fast Fourier menu that was displayed initially.

Using MB3, click and hold the Coefficients button in the Xfit-Fast Fourier menu. A popup appears that contains a selection of Fourier coefficients that can be used. Select 2mFo-DFc(SigmaA) and then click Apply.

This initiates the Fourier calculation, displays the map in the canvas window, and makes the Fast Fourier menu disappear.

8.   Selecting and centering a residue

Suppose that you now want to re-fit the density of a residue that you are unsure about. There are two things that you need to do: position the residue to the center of the canvas window and display the electron density at that point.

There are two ways to position a specific residue to the center of the screen: selecting an atom with the mouse from the canvas window and selecting an atom from a list in the Model menu.

To select an atom from the canvas, use MB1 to select an atom in the molecule. The atom label appears on the canvas and the label also appears in the Stack list of the main Xfit window. Click and hold MB3 while the cursor is inside the canvas and a pop-up menu appears. Select the icon labeled center@ in the popup to move the selected atom to the center of canvas.

It is not always easy to find the residue you are interested in by looking at the molecule, so an alternative method is available for selecting the residue to center on.

When picking an atom in the canvas window, you must hold the mouse stationary. If the mouse moves during the clicking operation, Xfit interprets the event as a rotation request, and does not draw a label.

As an alternative to the previous step, click the Model button on the main Xfit window to make the associated menu appear. This menu contains two scroll lists: a list of all residues in the current structure and a list of all atoms in the current residue. You can position a specific residue by first finding it in the Residue scroll list and then selecting it with MB1. All of the atoms associated with that residue then appear in the Atoms in Selected Residue scroll list. Select one of the atoms from this list using MB1, followed by pressing MB3 while the cursor is inside the canvas and selecting the icon labeled center@ from the pop-up menu that appears.

For the purpose of this tutorial, use the Model menu to put residue PHE 91 in the center of the screen, that is, select PHE 91 from the residue list, select atom CB from the atom list, generate the canvas pop-up menu by clicking and holding MB3 in the canvas window, and click the center@ icon. Residue PHE 91 should now be in the middle of the screen with the CB atom located in the middle of the screen.

9.   Controlling the map

When you moved the residue of interest to the center of the screen, the electron density was automatically re-centered on that residue. The Fourier map for the full unit cell was calculated when you ran the Fast Fourier routine but only a small portion of it is displayed at any one time and it may be necessary to move the density independent of the molecule.

Click the Contours button at the top of the Xfit window to open the corresponding menu. The size, position, and display characteristics of the electron density maps are controlled by this menu. By default, the electron density is displayed as a cube that encloses a volume of the specified Cube/Sphere Radius in angstroms. Thus, to increase the size of the density region, increase the value of the radius. When the Auto Contour on Scroll button is toggled on (the default) density is automatically recontoured around the center of the screen. To display the density centered on the selected residue when this option is toggled off, click the Apply button.

If you only want to generate the density at the center of the screen and the auto contour function is disabled, it is more convenient to use the canvas window pop-up menu (click and hold MB3) and select the Contour icon. This contours the map at the center of the screen using the current parameters in the Contour menu.

10.   Calculating an OMIT map

Now you want to remove the Phe 91 residue from the model, calculate an OMIT map, and check the position of the model vs. the new map densities.

First you must select the residue for removal. The CB atom of residue Phe 91 should still be on top of the stack, however, if it is not on the stack, select the CB atom again in the Model menu.

Click the Residue button in the Xfit window and the PHE 91 residue turns green. (In Xfit, green indicates "current" atoms.)

Click on the SFCalc... button in the main Xfit menu. Go to the SFCalc menu and click the button labeled Omit Current Atoms. This generates new Fc and values but retains the scale factor that was calculated with the full model.

A SigmaA weighted 2F_o- F_c map is automatically generated at the end of the structure factor calculation, if the FFT menu still has the 2mFo - DFc (SigmaA) option set. The new map replaces the old map in the canvas window; it should be slightly different. There will be less electron density around the Phe 91 sidechain.

11.   Adjusting torsion angles

Now you can try moving the phenyl ring to a different position and recalculate the map with the new model. This requires first learning how to change torsion angles. When only one residue has been selected as "current", moving around the ₁, ₂, ₃, ₄, and ₅ torsion angles is very easy.

To activate rotation about the 1 torsion angle, press the <1> key on your keyboard while the cursor is in the main graphics canvas. While clicking and holding MB2, move the cursor in the canvas window from left to right to vary the torsion angle. In a similar fashion, 2, 3, 4, and 5 angles can be changed after first pressing <2>, <3>, <4>, and <5>.

Next press the <1> key and rotate the phenyl ring far away from the current density. Using the SFCalc menu, calculate a new set of structure factors that removes the old atom positions for the phenyl ring and includes the new rotated atom positions. You do this by clicking the Update Current Atoms button.

After clicking this button, the new position of the phenyl ring disappears and then reappears. This follows the course of calculational events that are taking place, that is, first the contributions to the structure factors from the old atom positions are calculated and subtracted, then the contributions from the new atom positions are calculated and included. Note that the current residue (the one contributing to the model in this calculation) is still green, and the original residue position is represented by the original color scheme.

A new map is automatically calculated and displayed. The resulting map should still show density around the original phenyl ring position and no new density at the rotated position. This is a verification that the map fits well at this residue and that there is not much phase bias.

12.   Accepting or rejecting the "current" model

At this point you can save the current residue position, go back to the original residue position and continue adjusting the fit, or accept the original residue position as correct and go to another residue.

To accept the current model and delete the old residue position, click the Apply Fit button (in the main Xfit menu). To reset the current model to the original residue position but remain in current residue mode, you click the Reset button. To exit the current residue mode and retain the original residue position, click Cancel.

For the purpose of this tutorial, click the Reset button in the main Xfit menu and remain in current residue mode so that you can try some of the other basic map fitting commands.

13.   Basic map fitting commands

Xfit is designed to make map fitting as easy as possible. The popup (accessed with MB3) that is associated with the canvas window gives you access to the necessary map-fitting tools without having to look away from the graphics region. It is often more efficient to use this popup than to fumble around with a dialbox or to glance over at a side-bar menu. If you want to make the items from the popup permanently visible, then you can use MB3 to click the Canvas Tools button in the floating Xfit Tools menu bar and select the `pin' at the top of the popup. This provided a vertical menu containing the canvas tools options.

MB2 changes the mode during the fitting process. The active mode type is listed at the bottom right corner of the main Xfit window. The different functions that are available include:

• Centering the display.
• Translating the current atoms.
• Rotating the current atoms.
• Rotating torsion angles.
  

  Center Mode: Select the center icon from the popup. While pressing MB2, move the cursor within the canvas window. (Note that center and center@ are different buttons with different functions.)

The full display translates with this movement.

Move mode: Select the translate icon from the popup, using MB3. With MB2 pressed, move the cursor within the canvas window. The current atoms are translated relative to a stationary model. If red bonds are drawn between current atoms and the rest of the model, the bonds are more than 10% longer or shorter than the starting bond lengths.

 

Rotate mode: Select the rotate icon from the popup. With MB2 pressed, move the cursor within the canvas window. The current atoms are rotated relative to a stationary model. If red bonds are drawn between current atoms and the rest of the model, the bonds are more than 10% longer or shorter than the starting bond lengths.

Torsion mode: Torsion mode is different from the Center, Move, and Rotate modes because you need to specify which torsion angle you want to vary. If a single residue is current, then you trigger Torsion mode for a specific angle by pressing the <1>, <2>, <3>, <4>, or <5> keys. These keys will trigger Torsion mode only if they are applicable to the current residue.

If more than one residue has been selected as current, torsion angles must be specified by selecting the two central atoms within the torsion angle sequence and then selecting the torsion icon from the popup. The atoms attached to the second selected atom will become a group that can be rotated about the selected bond. As before, the torsion angle is varied by clicking and holding MB2 while moving the cursor in the canvas window.

To try this, first clear the current atoms by clicking the cancel button in the main Xfit menu. This returns the green residue to the original color scheme. Click the Clear Stack button in the main Xfit menu to remove all atoms from the stack. Use the Model menu to pick ALA 88 and ARG 89 (remember--to select a residue it is also necessary to pick one atom of the residue!). There should now be one atom from each residue contained in the Stack list. Click the Residues button in the main Xfit menu (as opposed to the Residue button), and both residues turn green.

Place the CG atom of ARG 89 at the center of the canvas window (click the CG atom in the Model menu, then click and hold MB3 and select the center@ icon) and position the density map at the center (click and hold MB3 and select the contour icon). Calculate an OMIT map (SFCalc menu with the Omit Current Atoms option) for the two current residues.

14.   Adjustment of the model

Now optimize the 5 torsion angle manually by selecting atoms ARG 89 NE and ARG 89 CZ with the mouse or by using the Model menu. Click and hold MB3 and select the Torsion mode. The 5 torsion angle can now be manually optimized by clicking and holding MB2 within the canvas window and moving the cursor from left to right.

The Refine menu contains a convenience function to aid you in adjusting the torsion angle to generate the best fit of the density to the model.

Click the Refine button on the main Xfit menu and then select the Update R-density while fitting option.

With this option selected, the R density is displayed and updated in the lower left corner of the canvas window as the torsion angle is adjusted. The R density is a function of maximum overlap between atoms and electron density with a penalty for atoms that do not have overlapping densities. The higher the R-density value, the better the fit of the model to the map.

You can also have the program automatically find the position of maximum R density by clicking the Torsion Search button in the Refine menu.

15.   Conclusion

In this tutorial you have learned how to calculate a phony .phs file, start the Xfit program, perform basic model and map control operations, calculate an OMIT map, and perform basic map-fitting operations.

16.   Ending the lesson

To quit Xfit, double click the quit button in the main Xfit menu.

To quit Insight II, type quit on the command line, and press <Enter>. Press <Enter> again at the next prompt.

Lesson 8:   De novo map fitting

This tutorial uses the following commands, accessed from the specified Xsight modules:

The scenario: You have phased your native data set of C. vinosum cytochrome c' (cvccp) using two heavy-atom derivatives. You now want to begin tracing the alpha carbon (CA) backbone.

Tracing the chain is one of the trickiest and most difficult aspects of solving a protein structure. It is difficult for a beginner to visualize the possible paths of the main chain and to recognize these features in the map. Looking for large pieces of secondary structure can greatly speed up map fitting. Helices are the easiest features to recognize because they often show up as long tubes of density disconnected from the rest of the structure. The skeletonized representation of the map is sometimes helpful in providing an aid to the pattern recognition of these features. This tutorial does not address the process by which you first select a region to fit.

The purpose of this lesson is to teach you how to position residues in the electron density once you have identified a region to fit. Therefore you are going to deliberately cheat in this lesson by loading an existing model file to position to a specific residue. This allows everyone running the tutorial to start from the same position in the electron density map.

1.   Preparing the tutorial directory

To run the de novo map fitting lesson, you must set up a directory which contains the files ccp.cool3.pdb and ccp.calc.phs, i.e., a coordinate file and a phase file.

Enter the following commands at the system prompt: > mkdir tutorial_fit > cd tutorial_fit > cp $XSIGHTTUTORIAL/ccp.cool3.pdb . > cp $XSIGHTTUTORIAL/ccp.calc.phs .

2.   Getting started

At the system prompt issue the command: > insightII and press <Enter>.

Wait a few moments while Insight II loads.

3.   Creating a project

Go to the Xsight module and select the Utilities/Project command. When the parameter block appears, enter the name of the project (e.g., tutorial_fit) and select Edit. Select cvccp from the Crystal_List and enter the Project_Directory based on the directory that was created in Step 1 (you must use the absolute path for the directory. Select Execute.

The Xsight module commands are now displayed on the lower menu bar.

4.   Skeletonizing a map

It is frequently helpful to create a skeleton representation of the electron density map to help guide model fitting. The skeletonized representation of the map provides a tool for tracing possible paths through the electron density and allows large regions of the map to be displayed.

Select the Model_Building/Skeletonization command. Select ccp.calc.phs as the Phase_File parameter. Enter ccp.calc.bones as the Bones_File parameter Select Execute.

The Xskel menu will appear. For this tutorial lesson you will calculate a Fobs map and apply the default settings.

Select the Make Ridgelines button in the XSkel menu.

The program will take just a few seconds to complete. Now leave this menu.

Select the Quit button in the XSkel menu and then click Yes.

5.   Begin model fitting

Select the Model_Building/Density_Fitting command. Make sure that Pass_Molecule is off and Pass_PDB_File is on. Now click PDB_File to display the list of available .pdb files, and select ccp.cool3.pdb from the list.

Now select the One_Phase_File option for the Pass_Phase_Files parameter. Click the Phase_File parameter window and select ccp.calc.phs from the value-aid. Toggle the Pass_Bones_File option on and select ccp.calc.bones for the Bones_File parameter. Select Execute.

The Xfit program should now be spawned from Insight II.

Four windows appear when Xfit begins execution. The first window to appear is the Xfit Tools menu bar. The next window to appear is called the canvas. It is the graphics output window for Xfit, and the contents of the .pdb file and the .bones file are displayed there. Since you loaded a .phs file at startup, the Fast Fourier menu appears in anticipation of Fourier calculations to come. Finally, the main Xfit window appears.

Next you select a specific residue and a specific atom within that residue so you can find a good position to begin fitting density.

6.   Selecting a starting point

Click the Model button to activate the Active Model menu. Use the slider associated with the Residues list to help locate PHE 18, then click the PHE 18 label within the Residues list to select the residue. After the atoms associated with Phe 18 are listed in the Atoms in Selected Residue list, click the CA label in that list.

Atom CA of residue Phe 18 is now loaded on the stack.

When an atom is loaded on the stack it becomes active for further operations. The labels for atoms currently on the stack are listed in the Stack window, which is located in the main Xfit menu.

Position atom CA of PHE 18 in the center of the display by clicking and holding MB3 with the cursor in the canvas graphics window. Select the Center@ icon and the center of the display is positioned at atom CA of PHE 18. To delete the Active Model menu, double-click the upper left button.

Removing the molecule from the canvas window makes the tutorial more interesting and realistic. (For a real structure determination problem there would not be a model.).

Click the Show button to activate the Show and Hide Objects menu. Remove the model by clicking on the highlighted filename in the Models scroll list (this deselects the model as an object to be displayed). Once the canvas graphics window is empty except for the skeleton, double-click the button in the upper left corner of the menu to remove the Show menu.

You must now select a new model number to store the residues that you are going to fit to the density.

Use the arrows to enter a value of 2 for the Fit (Active) Model # parameter located in the main Xfit menu.

7.   Calculating a Fourier map

It is now time to calculate an electron density map and display it in the canvas graphics window. The FFT menu was generated when Xfit was started, and can now be used to calculate an electron density map. You want to calculate a Fourier map based on F_o coefficients and MIR phases.

The default setting for the Coefficients parameter is already set to do the correct calculation, so you only need to click the Apply button.

The Apply button is shaded while the calculation is being performed to signify "calculation in progress". Information about the calculation is listed in the output window of the main Xfit menu.

8.   Basic display manipulation

The electron density is displayed in the canvas graphics window when the Fourier calculation is completed. It can be rotated and translated using the same standard mouse operations that are used for manipulating the molecule.

Rotation: Click and hold MB1 while the cursor is in the canvas graphics window, then move the mouse to rotate the display. Horizontal motions of the mouse when the cursor is in the bottom 80% of the canvas window result in rotation of the display about the vertical axis. Horizontal motions of the mouse when the cursor is in the upper 20% of the canvas window result in rotation of the display about an axis perpendicular to the screen. Vertical motions of the mouse when the cursor is in the canvas window result in rotation about the horizontal axis.

Translation: Click and hold MB2, then move the mouse while the cursor is in the canvas graphics window to translate the display.

Zoom: Click and hold MB1 and MB2 simultaneously to activate the zoom control. While holding both buttons, move the cursor from the bottom to the top of the screen to zoom in on the display. Move the cursor in the opposite direction to zoom out.

9.   Manipulating the skeleton

Sometimes the skeletonized representation of electron density map shows too much or too little connectivity.

Select the Ridgelines... button in the main Xfit menu. By moving the Ridgeline Level slider you can adjust the threshold for displaying the skeletonized map. Try adjusting the Ridgeline Level to about 20, where the alpha helical trace of the density will be very apparent. Now iconize the Ridge Lines menu by clicking the small square in the upper right of the menu.

10.   Placing CA markers

The center of the display is still coincident with the position of the CA atom of residue Phe 18. Notice that the actual maximum of electron density does not correspond with that atomic position. To select the first CA, you first translate and rotate the map until the electron density maximum is centered on the white cross.

The tools used in fitting residues to electron density maps are located in the Active Model menu. Click the Model button in the main Xfit menu to open this menu. Once the menu is displayed, click and hold the Type menu button with MB3 to display a list of all possible residues. Select the MRK residue (near the bottom of the list) for the purpose of marking CA positions, set Autonumber to On, and set Name to a value of 1.

Click and hold the Insert Res button with MB3. The resulting popup is "pinned" to the screen when you move the cursor to the "pin" icon in the top left corner of the popup and release MB3. You need this menu many times during the fitting procedure, so it is useful to have it available at all times.

You should now select the first CA position.

Move the density of the most probable CA position to the center of the display (marked by the white cross) using a combination of rotation (MB1) and translation (MB2).

Moving the most probable CA position to the center of the display is analogous to aligning a crystal on a goniometer head. First adjust the "height" with the vertical translation and then use a combination of 90º rotations about the vertical axis and horizontal translations to center the density.

Click the New model button in the Insert menu and a yellow cross is drawn at the center of the display. This also generates a residue named MRK1 and lists it in the Residues scroll list of the Model menu.

11.   Placing additional CA atoms

You should now select a second CA position. Since CA atoms are separated by approximately 3.8Å along the polypeptide backbone, you should look for a second possible position that is approximately 3.8Å from MRK1. Distances can be estimated by aligning the next potential CA position horizontally with MRK1. Pressing <Shift>-<+> produces a distance aid that has marks of 1.5, 2.3, and 3.8Å. If you want to remove this scale bar, press <.>.

Select the second CA position by rotating and translating the most probable CA position to coincide with the white cross in the center of the screen. Click the Insert After Selection button to mark the position.

If the separation between the two markers is less than 4.5Å, a line is drawn between the two markers. A new marker, MRK2, is added to the list and is automatically selected for further extension of the backbone.

Now select another eight CA positions to gain practice with the method.

To summarize the steps of adding another CA position:

1.   Locate a probable CA position by analyzing the map in a radius of approximately 3.8Å from the previous marked position.

2.   Rotate and translate the possible CA position until it coincides with the white cross in the center of the canvas window.

3.   Click the Insert After Selection button.

The contour maps are drawn with a default radius of  5Å. In some cases you might need to view a larger portion of the map when making decisions about selecting the next CA position. The display characteristics of the maps, for example, radius, sigma levels, and colors, are modified in the Contour menu.

12.   Save the CA markers

After marking ten CA positions you should save the markers to a .pdb file.

Click the Files button in the main Xfit window to activate the associated menu. Enter the name of a file (for example, calpha.pdb) in the Output PDB text box and click the Save Model button to save the CA positions.

13.   Conclusion

In this lesson you marked the positions of a a few CA atoms in the electron density map. As an alternative to the procedure given in Step 11 of this tutorial you may want to try using the semi-automated `baton' placement method, accessible from the AutoFit menu in the floating menu bar.

After the CA-chain trace is completed for as much of the map as possible, the next step would be to convert this chain trace to a more complete model. Useful tools for carrying out this conversion are found in the AutoFit and Active Model menus.

14.   Ending the lesson

To quit Xfit, double-click the quit button in the main Xfit menu.

To quit Insight II, type quit on the command line and press <Enter>. Press <Enter> again at the next prompt.