Because of the success of our NOAH/DIAMOD program suite(1-6) in automated protein ND NOESY spectra assignment and 3D structures determination for crambin (S22/I25, 46 residue), scorpion neurotoxin (CsE-v5, 60 residues), myeloid progenitor inhibitory factor-1 (MPIF-1, 77 residues) and aplysia attractin (58 residues), we have recently started implementing a Graphic User Interface (GUI) to facilitate the use of our programs by NMR experimentalists. By using our GUI, users need not to understand the basics of stiff FORTRAN I/O formats and shell scripting to prepare or edit the lengthen NOAH/DIAMOD input and shell scripting files that are currently used to run NOAH/DIAMOD programs. Under a few mouse clicks by our user-friendly GUI design, the preparation of the input files and parameter selections are done automatically. The GUI could also import 2D, N15 or C13 HSQC NOESY peak lists from NMR data processing programs such as FELIX, VNMR and NMRView for direct assignment of the peaks, and export assigned NOESY peak lists back to those programs for on screen visualization for further analysis. User could also use our GUI to do assignment analysis without reading the FORTRAN output files to dig out necessary assignment information.The DIAMOD program alone beneath the GUI can be used easily for structure calculations such as in homology modeling if all its input constraint files are determined by other means instead of NMR methods. 

Abstract

InputNecessary:

Sequence; Chemical shifts list; one or more NOESY cross peaks list: 2D 1H-1H, 3D 15N-HSQC, 3D 13C-HSQC

Optional:3J-Coupling constants; Stereo specific assignments;

H-Bonds; S-S BondsSecondary structure topology

NOAH•Check previous assignments•Generate new assignments

consistent with the current structures

DIAMOD•Generate new structures consistent with current set

of assignments

Output•Bundle of Structures consistent with

the constraints

•List of cross peak assignments

Top : Superposition of 20 NOAH/DIAMOD structures to their mean after the FANTOM energy minimization

Bottom : Superposition of the energy minimized X-PLOR (thick line) and NOAH/DIAMOD mean structures.

Comparison of automated and conventional structure determination

using 2D-data

Top : Stereo view of the NOAH/DIAMOD NCH bundle of structures and the mean structure from the manual assignment procedure (Rajarathnam et al., 2001) (red line).

Bottom : Stereo view of mean structure from the NCH calculation (thick line) and the mean structure from the manual assignment procedure (thin line).

Comparison of automated and conventional structure determination

using 2D- and 3D-data sets

Top : Stereo view of energy minimized bundle of structures (mean structure drawn in thick line)Bottom : Stereo view of the mean structure (S22/I25) (thin line) superimposed on

crambin(P22/L25) isoform (thick line)

Ab initio structure determination of crambin (S22/I25)

Top : Bundle of 20 energy minimized structures Bottom : Ribbon representation of the mean structure

Ab initio structure determination of Aplysia

The Design of GUI

• We have used Perl/Tk programs as our GUI design language as it is a very popular GUI development tool on both UNIX and Window based computers.

• The GUI Should completely eliminate the large number of tedious FORTRAN formatted input and shell scripting preparations that are needed for running NOAH/DIAMOD programs.

• Our GUI should import necessary experimental NMR data from any other popular data processing software packages and the imported data can be used directly by NOAH/DIAMOD.

• All NOAH/DIAMOD parameters can be selected by mouse clicking or multiple choices to minimize the user input errors. It also provides simple editing features for making changes of GUI generated input files and save the edited files for a new NOAH/DIAMOD calculation.

• It must be easy for new users to use, but powerful for experienced users and it should not crash the FROTRAN codes if user makes mistakes while using the GUI tool.

• The GUI must provide detailed assignments analysis and export the assigned peaks to other programs such as (FELIX .etc) for data visualizations.

GUI Main Window

The main window has 6 pull down menus and 2 buttons

1) The File menu for easy file selecting, opening, editing and saving.

2) The Project menu for I/O directory handling, project naming and input file creating etc.

3) The Noah INP menu for creating NOAH input parameters.

4) The Diamod INP menu for creating DIAMOD input parameters.

5) The Run Noah menu for different types of NOESY spectra assignments.

6) The Run Diamod button for constrained structure calculation.

7) The FLXFMT button for creating FELIX formatted NOESY peak lists.

GUI Screen Capture 1

This screen shot shows two pull down menus and a sub-menu

1) The Project menu and it’s sub-menu: Import C13 peaks.

2) The Run Noah menu. It has 7 buttons for different type of 2D or 3D HSQC spectra assignment.

GUI Screen Capture 2

A screen shot from Noah INP pull-down menu, which gives user options of selecting proper NOAH input parameters. The user can use either default parameters or an experienced user can go into details to fine tune any given parameters.

GUI Screen Capture 3

The two menu widgets are from Project pull-down menu. If CYSS pairs are known to form disulfide bonds, or if stereo-specific assignments of some residues are known then constraints can be automatically generated via a mouse clicking.

GUI Screen Capture 4

Two menu widgets from Diamod INP pull-down menu used for creating input parameters in DIAMOD structure minimizations. All parameters have been in default values. User can simply click Select button to select default values or change some of them.

GUI Screen Capture 5

These two widgets show Find and Find/Replace editing features if a file is loaded on to the main text window from any one of the main pull-down menus.

A partial plot of labeled FELIX NOEY peaks assigned by NOAH/DIAMOD programs that were exported back to FELIX via our GUI for on screen visualization so that the assignments can be examined visually by users.

Conclusion•The NOAH/DIAMOD suite is able to determine 3D protein structure using any combination of 2D homonuclear, 3D 15N-edited and 13C-edited heteronuclear spectra.

•The addition of GUI will completely eliminate the manual editing of NOAH/DIAMOD input files, parameter files as well as shell scripting files and therefore greatly reduce the errors and time for input data preparation.

•All NOAH/DIAMOD commands are done by mouse clicking therefore it is very suitable for users who have no computer experience to do automated structure determination.

•The use of GUI makes it much easier for importing experimental NOESY peaks directly from other data processing software packages such as (FELIX .etc) for peak assignments and structure calculations.

•The implementation of GUI made NOAH/DIAMOD program suit a even more powerful tool for high throughput structure determination in genomics projects.

•The DIAMOD part alone can be easily adapted as a protein homology modeling tool.

Acknowledgement: This work was supported by grants from NFS (DBI9714937) and DOE (DE-FG03-00ER63041) to WB.

References

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2 Xu, Y., Schein, Ch. and Braun, W., Combined automated assignment of NOESY spectra and three-dimensional structure calculation of proteins, Biological Magnetic Resonance Vol. 17, pp 37-79 (1999), L.J. Berliner & N. Rama Krishna, Kluwer Academic/Plenum Publishing Corporation, New York, NY.

3 Mumenthaler, Ch., Güntert, P., Braun, W. and Wüthrich, K., Automated combined assignment of NOESY spectra and three-dimensional protein structure determination, J. Biomol. NMR. 10, 351-362(1997).

4 Xu, Y., Wu, J., Gorenstein, D. and Braun, W., Automated 2D NOESY assignment and structure calculation of crambin(S22/I25) with the self-correcting distance geometry based NOAH/DIAMOD programs, J. Mag. Res. 136, 76-85 (1999).

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7Ravindranath, G., Xu, Y., Schein, C.H., Rajarathnam, K., Painter, S.D., Nagle, G.T. and Braun, W.  NMR solution structure of attractin, a water-borne protein pheromone from the mollusk Aplysia californica.  Biochemistry, 42(33):9970-9979, 2003.