This chapter describes the basics of using the QUANTA Sequence Builder application. Read this chapter to learn procedures for building and editing sequences for proteins, nucleic acids, and other polymers
In Sequence Builder, you can construct residue sequence lists for proteins, amino acids, and polymers using predefined residue libraries and structure fragments. The application can also read existing sequence files.
After a sequence is defined, it is automatically sent to CHARMm where Cartesian coordinates are constructed using CHARMm internal coordinate information. The graphical representation of the structure is then displayed in Molecular Modeling mode.
An RTF describes the molecular topology of each residue in a sequence and contains the information necessary to compute the energy of a structure as well as perform other calculations. Atom names, types, masses, and partial atomic charges are included in an RTF. Coordinates are not included.
Patch residues modify atoms after a sequence is assembled. These patches describe a deviation from the standard residue definition. For example, the RTF for cysteine generates a single cysteine residue, but does not contain information for linking two cysteine residues together via a disulfide bond. The disulfide bond is specified by applying the patch residue DISU to each of the two cysteines. A disulfide bond can also be applied to form linkages between segments.
Patches can be applied to any residue, and a residue can have more than one patch. Selecting a patch name from the palette automatically inserts the patch at the insertion point.
When a sequence of residues is specified, a secondary conformation can be applied, using conformations provided by QUANTA. Sequences can also have user-defined conformations.
The basic steps in creating a structure with the Sequence Builder are:
Sequence Builder is started from the within Molecular Modeling. To start Sequence Builder, open the pull-right Builders menu in the Applications menu and select Sequence Builder. Once Sequence Builder is started, all work in Molecular Modeling is suspended until Sequence Builder is exited.
The Sequence Builder application displays a new window over the other QUANTA windows. This window can be resized, moved, iconified, pushed, and popped in the same manner as all other standard workstation windows.
The Sequence Builder window is made up of four distinct areas. Although some window areas are the same as other QUANTA applications, the menu bar and palette are specifically customized for Sequence Builder.
The menu bar contains four menu items: Sequence Builder, Data, Edit, and Conformation. Sequence Builder menus are displayed as pulldown or sticky menus. Table 21 lists these selections and provides a brief description of each.
Tables 22 through 24 list the selections in the menus under the entries of the menu bar.
Residues available in a selected RTF are displayed in the palette. Palette selections also include repeat numbers to specify multiple residues of the same type and residue specifications based on single-letter amino acid codes.
The viewing area is used as a notepad to list residue names in sequence from left to right. The beginning of each line contains an integer designating the residue number of the first residue in that line. The name of the current sequence and segment are displayed at the top of the viewing area.
The message line provides information about current operations or directions about how to proceed.
When you open Sequence Builder, the application displays a new window over other QUANTA windows. The window can be manipulated in the same manner as other windows in QUANTA. The Sequence Builder is started from Molecular Modeling. When Sequence Builder is running, all work in Molecular Modeling is suspended.
Sequence Builder is accessed from the Applications menu opened from the menu bar in QUANTA. Select Builders from the Applications menu. Then select Sequence Builder from the Builders pull-right menu.
Sequence Builder is initialized by reading a CHARMm RTF containing the building blocks from which a sequence is constructed. RTF definition files are distributed in the directory $CHM_DATA and are accessed via the File Librarian. A File Librarian dialog box is opened whenever Sequence Builder is started.
See Appendix D of QUANTA Basic Operations for tables identifying the residues and patch residues contained in the RTF definition files.
When you start Sequence Builder, an insertion point appears in the viewing area as a highlighted dash. Residues are added to the sequence before the insertion point, thus keeping the insertion point at the end of the sequence. You can move the insertion point to a location preceding any residue. After the insertion point is moved, selected residues are added to the sequence at this new location rather than at the end of the sequence.
A sequence is comprised of one or more residues. Residues are grouped together into segments. You can add a single residue to a sequence by selecting the residue name from the palette. Several residues of the same type can be added by selecting a repeat number from the palette prior to selecting the residue name.
Patch residues modify atoms after a sequence is assembled. These patches describe a deviation from the standard residue definition. For example, patches can modify N- and C- terminal groups or form disulfide bridges within or between segments.
When a patch that links two residues is applied, the residues are counted as residues 1 and 2 in the opposite order of their selection.
Complete the following exercises to open Sequence Builder and become familiar with basic building techniques.
Display the Applications menu and select Builders. Open the pull-right menu and select Sequence Builder from the pull-right menu. The Sequence Builder window, containing a File Librarian dialog box, is displayed over the QUANTA windows.
2. Select a residue definition file.
From the File Librarian dialog box scrolling list, select AMINO.RTF and then click Open. The File Librarian is removed from the screen and residues contained in AMINO.RTF are displayed in the palette.
Select the repeat number 10 from the palette and the message line prompts:
Pick the residue you want repeated.
Select the residue ALA from the palette. Ten ALA (alanine) residues are displayed in the viewing area with a hyphen before each residue. The insertion point is positioned immediately after the tenth residue. A number appears at the beginning of each line designating the residue number of the first residue on that line.
4. Add 100 residues to the end of the sequence.
Select the repeat number 100 on the palette and then select the residue ARG.
One hundred ARG residues are added to the end of the 10 ALA residues.
5. Mark the first 10 residues in the sequence.
Move the cursor over the first ALA residue in this sequence and click the left mouse button. The residue is highlighted, indicating it is marked.
Move the cursor over the last ALA residue in this sequence and click the left mouse button again. All 10 ALA residues are now highlighted, indicating they are all selected. The insertion point, however, remains at the end of the entire sequence of ALA and ARG residues.
6. Add a copy of the first 10 residues to the end of the sequence.
Display the Edit menu and select Copy Marked Item. The 10 ALA residues are copied and inserted at the end of the sequence before the insertion point. The sequence now consists of 10 ALA - 100 ARG - 10 ALA.
Display the Edit menu and select Unmark Items. The 10 ALA residues are no longer highlighted, indicating they are no longer marked.
8. Insert 100 residues before the ARG residues.
Move the cursor to the dash before the first ARG residue and click the left mouse button. The insertion point moves to this location.
Select the repeat number 100 and then select the residue GLU. One hundred GLU residues are added between the ALA and ARG residues. The insertion point is still located before the first ARG residue. The sequence now consists of 10 ALA - 100 GLU - 100 ARG - 10 ALA.
9. Scroll through the entire sequence.
Display the Edit menu and select Scroll Down. The last section of the sequence is now displayed in the viewing area and the first section is no longer displayed.
Redisplay the Edit menu and select Scroll Up. The first section of the sequence is again displayed in the viewing area.
Mark the first 10 residues in the sequence. Then display the Edit menu and select Delete Marked Item. The 10 ALA residues are removed from the sequence.
The sequence now consists of 100 GLU - 100 ARG - 10 ALA. The insertion point is still located before the first ARG residue.
11. Delete the remainder of the sequence.
Display the Data menu and select Delete Sequence and Restart. A dialog box is displayed, requesting a name for the new sequence.
Enter peptide in the dialog box and click the OK button. Another dialog box warns that the currently displayed residue will be deleted.
Click the Yes button, indicating the current sequence is to be deleted, and the dialog box is removed from the screen. The old sequence is removed, and the new sequence name peptide is displayed at the top of the viewing area.
In QUANTA Basic Operations, the small peptide molecule, PEPTIDE.msf, was displayed. This section builds this molecule with Sequence Builder just as the original structure was built.
The Sequence Builder provides common secondary conformations that can be applied to residue sequences. The application also provides the option to assign specific dihedral angles to selected residues.
Available secondary conformations include:
When you save a sequence to a file, the file extension .seq is automatically appended to the base filename. When you exit Sequence Builder, the information in the .seq file is used to create a CHARMm command input file (filename.STR). This file is automatically created and submitted to CHARMm, where the Cartesian coordinates are built. These coordinates are then returned to the Molecular Modeling application for display and inclusion in an MSF.
Complete the following exercise to build, modify, and save a peptide to an MSF.
1. Enter residues in the new sequence.
In the listed order, select each of the following 20 residues to create the sequence. As each residue is selected, it is displayed in the viewing area.
ALA, PHE, VAL, LEU, THR, SER, ASP, TYR, SER, LYS, TYR, LEU, ASP, SER, ARG, LYS, ILE, VAL, LEU, ALA
2. Apply secondary conformation.
Display the Conformation menu and select Set Secondary Conformation . The list of residues in the palette is replaced by the instructions:
Pick residue or range of residues.
Mark the first four residues, ALA, PHE, VAL, and LEU, in the sequence and click the OK button. In a dialog box containing a scrolling list of the secondary conformation options, select the option:
Select the OK button. The dialog box is removed from the screen, the secondary conformation is applied to the marked residues, and the marks are removed from the residues.
3. Select additional secondary conformations for the rest of the peptide.
Consulting Figure 1., mark each remaining group of residues and select the indicated secondary conformation. When you have assigned all secondary conformations, click the Done button in the palette. The secondary conformation palette is replaced with a list of residues.
Figure 1. . Secondary conformation for peptide
4. Create an MSF from the sequence.
Display the Sequence Builder menu and select Return to Molecular Modeling. Since the peptide structure has not been saved, a dialog box asks if the changes are to be saved. Click the YES button and the message line reads:
Validating links between residues.
Sequence Builder checks to make sure that residues are properly assembled and patches applied appropriately.
The File Librarian is displayed. Enter mypeptide in the Filename data entry field and select the Save button. The File Librarian and the Sequence Builder window are removed from the screen, revealing the Molecular Modeling window. QUANTA automatically starts a CHARMm command input file to build the Cartesian coordinates for the sequence.
Structures created in Sequence Builder have approximate geometries (bond lengths and bond angles). These geometries need to be refined through a minimization process before continuing with other modeling operations.
Complete the following exercise to minimize the structure in mypeptide.msf.
1. Display mypeptide in the Molecular Modeling window.
When the creation of mypeptide.msf is complete, a dialog box allows the structure that was just created to be used.
Use the new molecule mypeptide.msf only
then click the OK button. The structure mypeptide is displayed in the Molecular Modeling viewing area.
Display the CHARMm menu and select the Minimization Options function. In a dialog box that contains the minimization setup options, select the option:
Number of Minimization Steps: 50
Coordinate Update Frequency: 5
Energy Gradient Tolerance: 0.000
Energy Value Tolerance: 0.000
Initial Step Size: 0.020
Step Value Tolerance: 0.000
Click the OK button and the dialog box is cleared from the screen and new minimization parameters are established.
From the Modeling palette, select CHARMm Minimization to start the CHARMm calculation. The cursor changes from an arrow to a watch and the message line displays information regarding progress of the minimization process.
Minimization is completed when the cursor is restored to an arrow. The final conformation of the structure is displayed in the viewing area. The minimized energy results are displayed in the upper-right corner of the viewing area and in the Textport.
From the Modeling palette, select Save Changes and a dialog box enables the new coordinates of mypeptide.msf to be saved.
Create New Generation of mypeptide.msf
Click the OK button. The dialog box is cleared from the screen, the textport indicates that the old structure file is renamed mypeptide.msf,001, and the minimized structure is saved in mypeptide.msf.
You also can build cyclic peptides in Sequence Builder using the same residue and patch libraries used for linear peptides. Complete the following exercise to construct the cyclic peptide, CCK7. This peptide will be used in a search exercise in Chapter 4 of QUANTA Simulation and Analysis.
Display the Applications menu. Select Builders and then select Sequence Builder from the pull-right menu that is displayed. The Sequence Builder window and a File Librarian dialog box are displayed over the QUANTA window.
From the scrolling list in the dialog box, select the file AMINO.RTF and then click the Open button. The File Librarian is removed from the screen, and the residues contained in AMINO.RTF are displayed in the Sequence Builder palette.
5. Enter the residues in the sequence.
Select the following residues (in the order shown) to create a peptide sequence:
As each residue is selected, it is displayed in the viewing area.
6. Set Secondary Conformation.
Display the Conformation menu and select Set Secondary Conformation . The list of residues in the palette is replaced by the instructions:
Pick residue or range of residues
and by the OK, Done, and Unmark buttons.
Mark the GLY and TRP residues and click the OK button in the palette. In a dialog box containing a scrolling list of available secondary conformations, select the scrolling list entry:
Click the OK button. The dialog box is removed from the screen and the conformation is applied to the two residues.
Click the Done button in the palette. The Secondary Conformation palette is replaced by a list of residues, along with the Sequence Builder window.
Display the Edit menu and select Apply Patches to Residues. The patches contained in AMINO.RTF are displayed in the palette replacing the display of residues.
8. Apply a patch to the sequence.
Select the LINK patch from the palette and the message line reads:
Please select 2 residues to apply patch to.
Mark TRY, the first residue in the sequence, and the residue is highlighted.
Mark PHE, the last residue in the sequence, and the sequence changes to indicate the link between the residues.
9. Return to Modeling and save the peptide.
From the Sequence Builder menu, select Return to Molecular Modeling . In a dialog box that offers the option to save the changes, click the YES button.
In a File Librarian dialog box, enter the name:
Click the Save button. The File Librarian and the Sequence Builder window are removed from the screen revealing the Modeling window. QUANTA automatically starts CHARMm to build Cartesian coordinates for the sequence. A dialog box asks if the just created structure is the structure to use.
Use the new molecule CCK7.msf only
Select the OK button and the structure CCK7.msf is displayed as a cyclic structure. The link patch imposes a covalent bond between the N alpha and C-terminal atoms. The cyclic conformation is created during minimization, when the lowest energy value is calculated for this bond.
10. Set up minimization for CCK7.msf.
Display the CHARMm menu and select Minimization Options. In a dialog box containing the minimization setup options, select the option:
Number of Minimization Steps: 200
Coordinate Update Frequency: 5
Energy Gradient Tolerance: 0.0100000
Energy Value Tolerance: 0.000000
Initial Step Size: 0.020000
Step Value Tolerance: 0.000000
Select the OK button and the dialog box is cleared from the screen.
11. Run a minimization for CCK7.msf.
Display the Modeling palette and select CHARMm Minimization to start the CHARMm calculation.
When minimization is completed, the final conformation of the structure is displayed in the viewing area. The minimized energy results are displayed in the upper-right corner of the viewing area and reported in the textport. The cyclic conformation is created as a result of calculating the lowest energy value of the covalent bond between the N alpha and C terminal atoms.
12. Save minimization results.
Display the Modeling palette and select Save Changes.
In a dialog box that offers the opportunity to save the new coordinates of CCK7.msf, select the option:
Select the OK button and the dialog box is cleared from the screen.
Sequence Builder is used to construct residue sequence lists for proteins and polymers using predefined residue libraries and structure fragments. Sequence Builder also can read and edit previously created sequence files. After a sequence is defined, it is automatically sent to CHARMm, where Cartesian coordinates are constructed using CHARMm internal coordinate information. Using patch residues, cyclic peptides and other modifications can be applied to linear structures.
The basic steps in creating a structure with Sequence Builder are:
1. Select a set of residue topology files.
4. Apply secondary conformations as required.
5. Exit Sequence Builder, sending the sequence definitions to CHARMm for coordinate construction and minimization.
When a structural build is complete, the molecule is ready to use in other QUANTA operations.
