Xsight - Tutorial

Overview of tutorial lessons

The purpose of these lessons is to introduce you to the commands and functionalities of the Xsight module of the Insight II molecular modeling program. You will perform several common procedures normally performed in macromolecular crystallographic analysis. This should acquaint you with the most commonly used features and calculation strategies of Xsight. Lessons illustrating MIR phasing (Isomorphous replacement), Phase determination from MAD data, Density modification, Density fitting, Molecular replacement, Refinement, Completing the atomic model and Visualization are included in this set of tutorials.

Throughout the tutorial sessions that involve XtalView modules, the following conventions are observed when discussing mouse actions:

MB1 refers to the left mouse button.
MB2 refers to the middle mouse button.
MB3 refers to the right mouse button.

Hardcopy tutorial lessons

All of the Xsight tutorial lessons are provided in this user guide (there are no pilot tutorials).

This user guide contains the following lessons:

Isomorphous replacement

: Lesson 1:Merging native and derivative data
Lesson 2:Finding heavy atom sites for the first derivative
Lesson 3:Adding a second derivative

Phase determination from MAD data

: Lesson 4:MAD phase determination

Density modification

: Lesson 5:Solvent flattening and histogram matching
Lesson 6:Non-crystallographic symmetry averaging

Density fitting

: Lesson 7:Making maps and basic fitting techniques
Lesson 8:De novo map fitting

Molecular replacement

: Lesson 9:Calculating a self-rotation function
Lesson 10:Generating structure factors for the search model
Lesson 11:Ordinary cross-rotation function
Lesson 12:Translation search
Lesson 13:Applying the solutions

Refinement

: Lesson 14:Rigid-body refinement
Lesson 15:Multi-rigid-body refinement
Lesson 16:Simulated annealing refinement
Lesson 17:Least-squares minimization

Completing the atomic model

: Lesson 18:Generating ligand dictionaries
Lesson 19:Locating ordered water molecules

Visualization

Lesson 20:Richardson diagrams
Lesson 21:Display of atomic models with electron density maps
Lesson 22:Display and creation of symmetry related molecules

Setting up to run tutorials

Before starting to use Xsight, you must carry out the following setup steps. For a more detailed description of the directory structure referenced by the $CRYSTALDATA environment variable, see Chapter 3, Implementation.

1. Create a directory that will be referenced by the $CRYSTALDATA environment variable by typing the UNIX command:


>	mkdir $HOME/crystal_info

2. Copy the crystals and projects, and the cvccp files, from the $XSIGHTTUTORIAL directory to the $CRYSTALDATA directory:


>	cp $XSIGHTTUTORIAL/crystals $HOME/crystal_info

>	cp $XSIGHTTUTORIAL/projects $HOME/crystal_info

>	cp $XSIGHTTUTORIAL/cvccp $HOME/crystal_info

3. Make sure that your crystals and projects files are writeable.


>	chmod +w $HOME/crystal_info/crystals

>	chmod +w $HOME/crystal_info/projects

4. Edit your .cshrc file to include two environment variables:


setenv CRYSTALDATA $HOME/crystal_info
setenv CRYSTAL cvccp

5. Log out and log back in.

Each time you start a new project or create a new crystal, the information will be updated in the $CRYSTALDATA directory. The cvccp crystal file is used for many of the tutorials. You will want to change your $CRYSTAL environment variable, depending on which crystal you most frequently use.

Do not do your work in the $CRYSTALDATA directory. This directory is only for storing information about crystals and projects. Define your working directory through the Utilities/Project command in the Xsight interface.

Isomorphous replacement

Isomorphous replacement is one of the key techniques used in solving protein crystal structures. The following lessons lead you through the process of merging native and derivative data, calculating Patterson maps, finding heavy atom sites, and combining phases from multiple derivatives.

In Lesson 1:Merging native and derivative data, you learn to convert processed data into the .fin file format. You then merge and scale a native data set with an Au derivative. Finally, the lesson takes you through the different statistical plots that you can generate to evaluate the quality of the derivative.

In Lesson 2:Finding heavy atom sites for the first derivative, you calculate and contour a difference Patterson map for the Au derivative. You then locate the two major Au sites using an automated search method. You verify these positions against the difference Patterson map. Finally, you refine the Au sites and calculate SIR phases.

In Lesson 3:Adding a second derivative, you merge and scale an Ir derivative with the native data set. You then calculate a cross-Fourier using the SIR phases from the Au derivative and the isomorphous differences between the Ir data and the native data. From this map you locate an Ir site in the Ir derivative crystal. Finally, you refine the Ir site and calculate the MIR phases using the Au and Ir positions.

Lesson 1: Merging native and derivative data

This lesson uses the following commands, accessed from the Xsight module:

Xsight module

Utilities

Data_Control

MIR

Project

Import_Data

Merge_Data

Statistics

Xsight module
Utilities	Data_Control	MIR
Project	Import_Data	Merge_Data
		Statistics

The Scenario: You have just collected a putative derivative data set of C. vinosum Cytochrome c' (cvccp) and you want to merge it with an existing native data set. You then want to calculate statistics that will indicate whether you have been successful in producing a derivative.

1. Preparing the tutorial directory

In order to run this tutorial (and the other associated MIR tutorials), you must set up a directory which contains the ccp_raxis_au1.dat, ccp_raxis_ir1.dat, and ccp_raxis_n1.dat files, that is, two derivative data sets and a native data set.

Enter the following commands, in the order shown, at the command prompt:
> mkdir tutorial_mir > cd tutorial_mir > cp $XSIGHTTUTORIAL/*raxis* .

Enter the following commands, in the order shown, at the command prompt: > mkdir tutorial_mir > cd tutorial_mir > *cp $XSIGHTTUTORIAL/raxis* .**

The following steps are only necessary if you do not already have a cvccp crystal file. If this is the first Xsight tutorial that you have run, then you probably need to enter these commands. If you have already completed these steps once before, there is no need to repeat them.

Use your favorite text editor to add the following line to the crystals file:
> cvccp
Alternatively, you can add the line with the cat command by typing the following lines at the UNIX command prompt:
> cd $CRYSTALDATA > cp $XSIGHTTUTORIAL/cvccp . > cat >> crystals
Type cvccp in the textport, even though there is no UNIX prompt, then press <Ctrl-D> to exit the cat command mode.

The following steps are only necessary if you do not already have a cvccp crystal file. If this is the first Xsight tutorial that you have run, then you probably need to enter these commands. If you have already completed these steps once before, there is no need to repeat them. Use your favorite text editor to add the following line to the crystals file: > cvccp Alternatively, you can add the line with the cat command by typing the following lines at the UNIX command prompt: > cd $CRYSTALDATA > cp $XSIGHTTUTORIAL/cvccp . > cat >> crystals Type cvccp in the textport, even though there is no UNIX prompt, then press <Ctrl-D> to exit the cat command mode.

The >> symbol allows you to append lines to an existing file. Once you type the cat command, all typed entries in the textport are appended to the crystals file. For this lesson, you simply want to add the cvccp crystal to the file and exit from the cat append mode. Be sure not to type anything else in the textport while you are in the cat command mode.

2. Getting started

Invoke the Insight II software.

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

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 pulldown by clicking the MSI logo and selecting Xsight from the list that appears (the Xsight module commands are now displayed on the lower menu bar).

Select the Utilities/Project command and type in the name of the project (for example, mir).

Toggle on Edit.

Go to the Xsight pulldown by clicking the MSI logo and selecting Xsight from the list that appears (the Xsight module commands are now displayed on the lower menu bar). Select the Utilities/Project command and type in the name of the project (for example, mir). Toggle on Edit.

Click the Crystal parameter field, and select cvccp from the Crystal_List, then enter the name of the Project_Directory you created in Step 1. (You must use the absolute path for this directory, since ~usr is not accepted in this field--for example: /net/machine/usr/people/pns/tutorial_mir.)

Select Execute.

Click the Crystal parameter field, and select cvccp from the Crystal_List, then enter the name of the Project_Directory you created in Step 1. (You must use the absolute path for this directory, since ~usr is not accepted in this field--for example: /net/machine/usr/people/pns/tutorial_mir.) Select Execute.

The dialog box disappears automatically.

4. Importing and converting a processed data file

This lesson assumes that you have your processed reflection files in a format other than the .fin reflection format. In particular, it assumes you are starting with three reflection files in the standard R-AXIS II format. In this lesson, you use the native data set and one of the derivative data sets. The second derivative data set is used in Lesson 3:Adding a second derivative.

First, you will convert the native data set to the .fin file format.

Select the Data_Control/Import_Data command

Select the hkl_F1_SIG1_DELANOM option for the Input_File_Format.

Enter ccp_raxis_n1.dat in the Input_File_Name parameter field.

Select Fin(.fin) in the Output_File_Format parameter field.

Enter ccp_n1.fin in the Output_File_Name parameter field.

Select Execute.

Select the Data_Control/Import_Data command Select the hkl_F1_SIG1_DELANOM option for the Input_File_Format. Enter ccp_raxis_n1.dat in the Input_File_Name parameter field. Select Fin(.fin) in the Output_File_Format parameter field. Enter ccp_n1.fin in the Output_File_Name parameter field. Select Execute.

5. Converting the Au derivative data set

Now you will convert the Au derivative data set to a .fin format.

Select the Data_Control/Import_Data command.

Select the hkl_F1_SIG1_DELANOM option for the Input_File_Format.

Enter ccp_raxis_au1.dat in the Input_File_Name parameter field.

Select Fin(.fin) in the Output_File_Format parameter field.

Enter ccp_au1.fin in the Output_File_Name parameter field.

Select Execute.

Select the Data_Control/Import_Data command. Select the hkl_F1_SIG1_DELANOM option for the Input_File_Format. Enter ccp_raxis_au1.dat in the Input_File_Name parameter field. Select Fin(.fin) in the Output_File_Format parameter field. Enter ccp_au1.fin in the Output_File_Name parameter field. Select Execute.

6. Merging the native and Au derivative data files

You now want to scale and merge the native and Au derivative data files so you can evaluate the quality of the derivative.

Select the MIR/Merge_Data command.

Enter ccp_n1.fin as the Fin_File_1 parameter.

Enter ccp_au1.fin as the Fin_File_2 parameter.

Enter ccp_n1.ccp_au1.fin as the Merge_Output_File parameter.

Select Execute.

Select the MIR/Merge_Data command. Enter ccp_n1.fin as the Fin_File_1 parameter. Enter ccp_au1.fin as the Fin_File_2 parameter. Enter ccp_n1.ccp_au1.fin as the Merge_Output_File parameter. Select Execute.

This displays the Xmerge window

In the Xmerge menu, set Scaling Type to Anisotropic.

Click Scale to begin the merging procedure.

In the Xmerge menu, set Scaling Type to Anisotropic. Click Scale to begin the merging procedure.

After a few moments, two graphs are generated at the end of the merging procedure: merging R-factor versus resolution and the average |F_p-F_ph| versus resolution. These two graphs help you evaluate your derivative data.

As a general rule of thumb, a merging R-factor above 10% indicates that you have a derivative crystal with significant phasing power. An R-factor above 7% may have significant phasing power. An R-factor below 7% indicates a crystal that cannot be used for phasing.

The |F_p-F_ph| differences should decrease as a function of resolution. If they do not, it is a symptom of non-isomorphism. In addition, the centric differences should be larger than the acentric differences.

Select Quit, then click Yes to confirm.

Select Quit, then click Yes to confirm.

7. Generating more graphs

This is an optional step in which you may replot the comparisons between the native and derivative data.

The graphs that are generated from the Xmerge program are not optimal because reflections are not evenly distributed into resolution bins. A better analysis can be made using the Xstat program.

Select the MIR/Statistics command.

Set Input_File to ccp_n1.ccp_au1.fin.

Select Execute.

Select the MIR/Statistics command. Set Input_File to ccp_n1.ccp_au1.fin. Select Execute.

The Xfinstat window now appears.

In the Xfinstat menu toggle on R-factor vs. resolution and |F1-F2| vs. resolution.

Click Graph it.

In the Xfinstat menu toggle on R-factor vs. resolution and \|F1-F2\| vs. resolution. Click Graph it.

After a few moments, two graphs are calculated and generated using an even reflection distribution between bins. These plots can be easily sent to a PostScript printer, if you desire.

At the top of either graph, there is a parameter field labeled Printer/File. Enter the command that you normally use to print a PostScript file, except do not include the filename.

Click Print.

The graphs can be deleted by double-clicking the top left button of each graph window.

At the top of either graph, there is a parameter field labeled Printer/File. Enter the command that you normally use to print a PostScript file, except do not include the filename. Click Print. The graphs can be deleted by double-clicking the top left button of each graph window.

Now exit the Xstat program.

Select Quit, then click Yes to confirm.

Select Quit, then click Yes to confirm.

8. Ending the tutorial

You can now continue to the next tutorial to locate and refine the heavy atoms of the first derivative, or you can quit.

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

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

Lesson 2: Finding heavy atom sites for the first derivative

This lesson uses the following commands, accessed from the Xsight module:

Xsight module

Utility

MIR

Project

Calc_Fourier

Contour_Map_3D

Find_Heavy_Atoms

Calc_Phases

Xsight module
Utility	MIR
Project	Calc_Fourier
	Contour_Map_3D
	Find_Heavy_Atoms
	Calc_Phases

You need to have run lesson 1 before doing this tutorial lesson.

The Scenario: In Lesson 1:Merging native and derivative data, you merged an Au derivative data set of C. vinosum Cytochrome c' (cvccp) with a native data set. The intensity statistics indicated a derivative with good phasing power. In this lesson, you will locate and refine the positions of the two major heavy atom sites for this first derivative.

1. Starting the tutorial

If you have just finished Lesson 1:Merging native and derivative data, and you are still running Insight II, you can skip to step 3.

If you have run Lesson 1:Merging native and derivative data, but need to restart Insight II carry out step 2.

2. Getting started

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

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

Insight II takes a few moments to load.

Go to the Xsight pulldown by clicking the MSI logo and selecting Xsight from the list that appears.

Select the Utilities/Project command and enter mir as the Project_Name parameter.

Select Execute.

Go to the Xsight pulldown by clicking the MSI logo and selecting Xsight from the list that appears. Select the Utilities/Project command and enter mir as the Project_Name parameter. Select Execute.

3. Calculating a difference Patterson map

In the last lesson you created a data file that contains scaled and merged reflections from a native data set and an Au derivative data set. You now want to calculate a Patterson map using coefficients based on the differences between the two data sets.

Select the MIR/Calc_Fourier command.

Enter ccp_n1.ccp_au1.fin for the Phase_File parameter.

Make sure that ccp_n1.ccp_au1.map is entered as the Map_File parameter.

Select Execute.

Select the MIR/Calc_Fourier command. Enter ccp_n1.ccp_au1.fin for the Phase_File parameter. Make sure that ccp_n1.ccp_au1.map is entered as the Map_File parameter. Select Execute.

Since you are calculating a Patterson map, you need to generate the Fourier coefficients from the observed differences between the native and derivative data sets. Your input file to the Fourier program therefore comes from a .fin file (structure factor amplitude file) rather than a .phs file (phase angle file).

4. Start the Patterson calculation

When the Xfft menu appears you will need to change a few of the default parameters. You should type <Enter> after altering the numerical values in order to ensure that your changes are properly entered.

Set the following parameter values in the Xfft menu:

Map Type: Fo*Fo (Patterson)

Resolution filter: 999.999

to: 5.0

Outlier Filter: differences> 100

Set the following parameter values in the Xfft menu: Map Type: Fo*Fo (Patterson) Resolution filter: 999.999 to: 5.0 Outlier Filter: differences> 100

Using MB3, click and hold the Phase File Type button. Drag the cursor to the Extra for Pattersons only selection and then drag to the right. (An additional pulldown menu appears.)

Select the fin(Fp s(Fp) Fph s(Fph)) option.

Using MB3, click and hold the Phase File Type button. Drag the cursor to the Extra for Pattersons only selection and then drag to the right. (An additional pulldown menu appears.) Select the fin(Fp s(Fp) Fph s(Fph)) option.

The label Patterson map from h,k,l,fp,s(fp),fm,s(fm) should now be loaded next to the Phase File Type button.

The other parameters should remain at their default values.

Click Calculate to create the difference Patterson map.

Click Calculate to create the difference Patterson map.

The calculation takes a few moments. The message FFT finished tells you when it is completed.

Click Quit when you are finished, then click Yes to confirm.

Click Quit when you are finished, then click Yes to confirm.

5. Display the difference Patterson map

Now you want to contour the Patterson map and look at the Harker sections for significant peaks. These peaks correspond to self-vectors between crystallographically related copies of bound heavy atoms and confirm the bounding of the heavy atoms in the derivative crystal.

Select the MIR/Contour_Map_3D command

Check that Contour_Operation is set to Setup.

Set the Map_File_Type to Patterson_Map.

Set Map_File to ccp_n1.ccp_au1.map.

Check that the Contour_Sigma_Level is set to 3.0, the Two_Level_Contour option is toggled on, the High_Sigma_Level is set to 6.0 and the Input_Atom_Sites option is toggled off.

Select Execute.

Select the MIR/Contour_Map_3D command Check that Contour_Operation is set to Setup. Set the Map_File_Type to Patterson_Map. Set Map_File to ccp_n1.ccp_au1.map. Check that the Contour_Sigma_Level is set to 3.0, the Two_Level_Contour option is toggled on, the High_Sigma_Level is set to 6.0 and the Input_Atom_Sites option is toggled off. Select Execute.