Processing NMR Data With OS X/Linux Freeware

Volume 2: A User’s Guide to NMRView

with NvAssign

by Josh Kurutz, Ph.D.

Technical Director of the Biochemistry & Molecular Biology NMR Facility

University of Chicago Last revised: August 22, 2006 Disclaimer: This guide is written in good faith by a user for other users, but the author cannot take responsibility for problems associated with using it. Be advised that the contents may not be error-free or comprehensive, though the author has attempted to make them as useful as practical.

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Table of Contents. 1) Overview ..........................................................................................................................................4

A. Purpose .........................................................................................................................................4 B. General Procedure.........................................................................................................................4 C. Helpful Concepts...........................................................................................................................4 D. Platforms & Program versions.......................................................................................................4 E. Clicking ........................................................................................................................................5 F. Guide Nomenclature .....................................................................................................................5

2) Viewing and Plotting a 2D spectrum .................................................................................................5 A. Presumptions.................................................................................................................................5 B. Start NMRView ............................................................................................................................5 C. Orient yourself ..............................................................................................................................6 D. Set Preferences (optional)..............................................................................................................7 E. Create a new window ....................................................................................................................9 F. Bring your spectrum into the working memory..............................................................................9 G. Orient yourself with respect to the window menu and its options.................................................11 H. Draw your spectrum in the window you created ..........................................................................13 I. Familiarize yourself with cursor functions and zooming in & out ................................................15 J. “Plotting” your spectrum.............................................................................................................16

3) Picking and Assigning Peaks in a 2D spectrum ...............................................................................18 A. Call up the peak picking window.................................................................................................18 B. Adjust the appearance of your spectrum ......................................................................................19 C. Select your peak picking parameters............................................................................................19 D. Pick the peaks .............................................................................................................................20 E. Orient yourself with the Peak Panel.............................................................................................21 F. Save your peak list as an .xpk file................................................................................................23 G. Couple multiple peaks that should be one peak............................................................................24 H. Manually add and/or delete peaks to/from the peaklist.................................................................24 I. Compress and Degap...................................................................................................................25 J. Manually assign residue identities to your peaks (optional) .........................................................25

4) Overlaying two 2D spectra..............................................................................................................26 5) Measuring chemical shift differences between two 2D spectra ........................................................26 6) Obtaining distance restraints from a NOESY spectrum ...................................................................26 7) Examining 3D spectra .....................................................................................................................26 8) NVAssign: Coordinating 3D Experiments to Achieve Total Assignment.........................................26

A. Overview ....................................................................................................................................26 B. Important details .........................................................................................................................27 C. Preparation..................................................................................................................................28

Process the necessary spectra and get them in NMRView format. ...................................................28 Pick peaks in each of these spectra ..................................................................................................29 Change your default paths...............................................................................................................31 Review and edit your picked peaks in each spectrum.......................................................................31

D. Open NVAssign ..........................................................................................................................31 E. Read in your data ........................................................................................................................32 F. Filter your peaks..........................................................................................................................37 G. Cluster your peaks.......................................................................................................................38 H. Edit your clusters ........................................................................................................................40

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I. Link/Match your clusters.............................................................................................................44 J. Assemble your fragments of linked clusters.................................................................................46 K. Inspect your assignments.............................................................................................................46

Table of Figures. Figure 1. Xterm window after launching NMRView.................................................................................6 Figure 2. NMRView Main Toolbar ...........................................................................................................6 Figure 3. NMRView TkCon window ........................................................................................................7 Figure 4. Main toolbar pulldowns .............................................................................................................7 Figure 5. The NMRView Preferences Panel..............................................................................................8 Figure 6. The NMRView Paths and Filenames Preferences panel..............................................................8 Figure 7. The Windows...Add panel..........................................................................................................9 Figure 8. Your new blank window with is title..........................................................................................9 Figure 9. The "Open Dataset" window ....................................................................................................10 Figure 10. The "open dataset" window with .nv choices..........................................................................10 Figure 11. The TkCon window after opening a dataaset ..........................................................................11 Figure 12. Open Dataset error window....................................................................................................11 Figure 13. The window menu..................................................................................................................12 Figure 14. The Attributes window...........................................................................................................12 Figure 15. The Attributes window pulldowns..........................................................................................12 Figure 16. Window menu pulldowns.......................................................................................................13 Figure 17. The window dataset manager window....................................................................................13 Figure 18. The window daaset manager window, post-add......................................................................14 Figure 19. Window with spectrum, initial display, Lvl=0.1. ....................................................................14 Figure 20. Left/black and Center/red crosshairs.......................................................................................15 Figure 21. Expanded portion of the spectrum..........................................................................................15 Figure 22. Zoomed spectrum at Lvl>1.0..................................................................................................16 Figure 23. The plot window....................................................................................................................17 Figure 24. The revised plot window........................................................................................................17 Figure 25. Updated file name with full path ............................................................................................18 Figure 26. Terminal window with postscript spectra ..............................................................................18 Figure 27. The peak-picking window. .....................................................................................................19 Figure 28. Spectrum after initial peak-picking.........................................................................................20 Figure 29. TkCon window after peak-picking .........................................................................................20 Figure 30. The peak panel.......................................................................................................................21 Figure 31. Peak pulldown menus ............................................................................................................21 Figure 32. The peak panel with data........................................................................................................22 Figure 33. The Peak panel "Write File" panel..........................................................................................23 Figure 34. The .xpk file...........................................................................................................................23 Figure 35. The Peak Couple panel...........................................................................................................24 Figure 36. The TkCon window after peak coupling.................................................................................24 Figure 37. Example files for NVAssign in a common directory...............................................................28 Figure 38. Displayed spectra required for NVAssign (3D spectra shown as 2D projections)....................29 Figure 39. Spectra for NVAssign with peak assignments (3D spectra shown as 2D projections) .............30 Figure 40. Location of the NvAssign menu selection ..............................................................................31 Figure 41. The NvAssign window...........................................................................................................32

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Figure 42. NvAssign menu pulldowns.....................................................................................................32 Figure 43. Error message for clicking "examine peaks" prematurely .......................................................33 Figure 44. The NvAssign Preferences panel............................................................................................33 Figure 45. The NvAssign Preferences panel, properly completed............................................................34 Figure 46. NvAssign Prefs "Set PeakLists for exporting data" panel, filled in properly. ..........................34 Figure 47. NvAssign error due to improper axis labels in Nvassign prefs ................................................35 Figure 48. The NvAssign "Examine Peaks" display ................................................................................35 Figure 49. Correspondence of HNCO and NvAssign "Examine Peaks" display.......................................36 Figure 50. The "Filter Peaks" display after selecting appropriate peaklists ..............................................37 Figure 51. TkCon window displaying results from peak filtering ............................................................38 Figure 52. The TkCon window displaying results from post-filtering compression & degapping.............38 Figure 53. The "Cluster Peaks" display ...................................................................................................39 Figure 54. The TkCon window after intraLinking peaks .........................................................................39 Figure 55. The TkCon window after interLinking peaks into clusters......................................................39 Figure 56. The "Edit Clusters (HN-C)" display, with the "nvassignCntrl" panel ......................................40 Figure 57. The AddPeaktoCluster panel ..................................................................................................41 Figure 58. The "Save Clusts" dialog window. .........................................................................................42 Figure 59. The "Save Clusts" dialog window, properly completed ..........................................................42 Figure 60. The NvAssign Cluster Editor .................................................................................................43 Figure 61. A .xpk file prior to clustering .................................................................................................44 Figure 62. A .xpk file after clustering......................................................................................................44 Figure 63. The NvAssign "Match Clusters" display (sans spectra) ..........................................................45 Figure 64. The "Edit Matches" panel, with spectrum display...................................................................46

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NMRView Processing Guide

1) Overview

A. Purpose NMRView is a sophisticated program for analyzing NMR data that has been processed by other programs. It has some capabilities to convert and process raw data from the spectrometer, but we will ignore that for the purposes of this guide. We will assume you’ve processed your data using NMRPipe and converted it to NMRView format already. Our aim is to provide a reliable path that should enable a scientist unfamiliar with these programs to transform raw data into something scientifically useful. Alternate paths to the final result definitely exist, but we are only going to cover this one that we know works well.

B. General Procedure NMRView is must be initiated from a Linux/Unix/X11 command line, but, unlike NMRPipe, you will not need to go back to the command line to execute commands. It is different from most other programs in that there is no central window with a common working area. Instead, a profusion of control bars, panels, and windows are opened and closed as necessary. In theory, this means you can put away controls you don’t need. In practice, you end up with a desktop full of windows and control panels that all belong under the NMRView umbrella. A later edition of this User’s Guide should include a diagram showing the process of viewing a plotting a 2D spectrum, but the system is so nonlinear, this may not be helpful.

C. Helpful Concepts Working with NMRView is easier if you understand a couple of key concepts. First, there is what I’ll call the “working memory.” To get data into NMRView, you must first “read” spectra, protein sequences, peak lists, etc., yet these initial readings will have no immediately apparent consequence. For something meaningful to happen, you must open a “window,” which at first will be blank. Clicking within the window will give you a range of actions you can perform, some of which enable you to bring in data from the working memory. Thus, you can bring one spectrum into two windows by creating two windows and reading the same spectrum into each from the working memory. You can also bring multiple spectra in to one window, and keep other windows open with the individual spectra, if you choose. The system is very flexible, and is limited largely only by the number of windows you can tolerate on your desktop. If you open multiple windows, you will probably soon discover that they talk to one another. Moving the cursors in one window can move the cursors in the other windows, enabling the spectral positions of the cursors to be the same in all windows. This is a very nice feature when, say, comparing TOCSY and NOESY data to see which peaks are shared in the two spectra. Please be patient with NMRView. Its flexibility is very powerful, but it does cause delays. I suggest putting on some music to enhance your experience.

D. Platforms & Program versions NMRView has been revised and updated approximately a zillion times. It seems like every lab is using a different version, and established NMR labs actually use older versions that they know work well or at least with predictable bugs. It’s been ported between Linux, SGI, Sun, and Mac OSX platforms, and it’s

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unclear if the program behaves the same way on each of them. The latest version was written in Java so it can run on all platforms, but it seems to have unknown problems and known ease-of-use issues, so we’re sticking with this older version. The version we’ve been distributing to our facility’s users (and no farther, please! ) is NMRView 5.0.4, and works in the X11 environment. Actually, it may be v.5.0.16, like its compressed filename suggests it is, but when you run it you’ll see a message saying you’re running 5.0.4. I’ve also used NMRViewAqua, with is native to OS X 10.3, and requires no X11 environment of command-line startup. I’ve found that it’s slower than the X11 version, but it’s missing an obscure bug found in 5.0.4 so I have to use it occasionally. They both seem to work the same, except for the speed and bug (in the protein sequence reading function, FYI). Everything reported here was done with 5.0.4 in the X11 environment on Mac OS 10.3, but everything should work the same in the Linux environment.

E. Clicking Most actions in NMRView are effected by left-clicking. Right-clicking and center-clicking are necessary for many essential functions. If you’re using a Mac with one button, as you would on a laptop, left-click normally, option-click for center-clicking, and command-click for right-clicking. Or pop for a $15 two-button + center scrollwheel/button USB mouse. You’d think that there would be a convention like “right-click to open a menu and left-click to select a menu item,” but the rule appears to vary depending on whether you’re using a toolbar or a window or maybe even which toolbar you’re using.

F. Guide Nomenclature I’ve tried to stick to consistent usage for the words “toolbar”, “menu”, “window” and “panel.” In my scheme, a “panel” is an object that appears on the screen into which you can enter text, make choices, effect adjustments, etc. A “window” is an object primarily for display, such as that used for showing spectra. A “toolbar” is a row of buttons, usually horizontal, which enable access to menus, which are usually vertical. A toolbar may be its own object on the screen, like the NMRView Main Toolbar (Figure 2), or a horizontal row of buttons atop a panel. A menu is a simple vertical set of choices that can be selected by clicking; menus are most commonly accessed by clicking toolbar buttons.

2) Viewing and Plotting a 2D spectrum

A. Presumptions We assume you’ve somehow transformed your raw data into NMRView format. If you only have a transformed NMRPipe spectrum, go to your command line, cd to your fid directory, and type: “pipe2xyz –in test.ft –out ./test.nv –nv” *!* If you’re looking at multiple spectra, you will find it handy to place all of your .nv files into one new directory. It will also probably be useful to rename your files with meaningful names, such as “protG_Nhsqc.nv”

B. Start NMRView In your command line, type “nv5”. (For NMRView Aqua, simply double-click the NMRView Aqua icon; no terminal command line interface is required.) Four things should happen:

1. Some introductory information covering your NMRView version should appear in your X11 window, which now gives you a ‘%” prompt with no information behind it. (Figure 1)

2. The main toolbar should appear in the upper left corner of your screen. (Figure 2) 3. The TkCon window should appear in the lower left of your screen. (Figure 3)

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4. A window should appear with red text in it that says “NMRView, by Bruce Johnson,” and some other stuff. This should stay on your screen for about ten seconds before it spontaneously disappears.

I encourage you to minimize – not close – the X11 window from which you launched NMRView. There’s really nothing NMRView needs you to do with it, and you can’t use it as a normal terminal while NMRView is on. If you close it, you’ll kill NMRView. Minimizing it will give you one less window to manage on your desktop.

C. Orient yourself Here are what those windows should look like:

Figure 1. Xterm window after launching NMRView

Figure 2. NMRView Main Toolbar

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Figure 3. NMRView TkCon window

The TkCon widnow’s main utility is its display of error messages when they occur and acknowledgement that certain processes have completed when no otherwise apparent effect is observed. For instance, when you read in a protein sequence, its molecular weight will appear in the TkCon window, but nothing else will happen. If you read the sequence again trying to get some effect, you’ll just see a new MW in the TkCon window that’s double the correct MW. You can also enter commands at the prompt in the TkCon window. This enables NMRView to be scriptable, which can be very handy. This feature, however, is not covered in this guide. Here is what the pulldown menus of the main toolbar look like:

Figure 4. Main toolbar pulldowns

You access these pulldowns by left-clicking on the toolbar buttons. You also select your menu choice by left-clicking.

D. Set Preferences (optional) NMRView is most efficiently operated when it has good default locations in which to look for files of different types. Otherwise, you will need to hunt down your files every time you look for them, wasting lots of time going from directory to directory. This is especially exasperating with NMRView Aqua, which, if I recall correctly, begins every search in the Applications directory. You can tell NMRView where to look for files of different types by setting its preferences. Alternatively, you can figure out which directory the program defaults to looking in and creating aliases or shortcuts from there to your data. To set these locations, go to the Main Toolbar (Figure 2) and click “File…Prefs.” You’ll see this window:

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Figure 5. The NMRView Preferences Panel

Click the ‘Paths and Filenames Button to get to the paths preferences:

Figure 6. The NMRView Paths and Filenames Preferences panel

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The default paths for NMRView will appear as “/home/user/something” or “/home/user/nmrview/somethingelse.” This will be useless to mac users, for there is no /home directory on the mac. The ones I’ve modified here reflect actual paths on my computer. I’ve partitioned my hard drive so I have one partition devoted to NMR data (JK_scratch), but this is not my startup partition, in which NMRView must be installed. To access the other partition, my path specifies it with “/Volumes/JK_scratch.” On a Linux/UNIX system, it might be “/home/jkurutz,” and on a mac with a standard arrangement, it would be “/Users/jkurutz.” You can specify different directories for different filetypes in this panel, but you need to create the directories in your home OS environment, not NMRView. For this to work, you must check the “Use Paths…On” button. Otherwise, NMRView will default to the directory from which you launched NMRView. If this button is checked, but you don’t have a good name for the path, like the one for VNMR files above (Figure 6), you’ll be shunted to the default directory and you’ll have to click your way to where you want to go. If you’re using NMRView Aqua and you turned off the ‘Use Paths” feature, your default directory is your home user directory, e.g., “Users/jkurutz.”

E. Create a new window Left-click on the ”Windows” button of the main toolbar, then click on the “Add” menu item. The following panel should appear:

Figure 7. The Windows...Add panel

This allows you to partition a window into sub-windows, governed by the “rows” and “columns” features. For most purposes, you can ignore this. Type in a descriptive short name for your window after “Window Name”. Here, I typed “hsqc.” After you hit your return key, a new blank window will appear, and the title you gave it will be printed in its top border:

Figure 8. Your new blank window with is title

F. Bring your spectrum into the working memory Now that you’ve established your workspace, you need to get some data with which to work. In the main toolbar (Figure 2), left-click “Dataset” and left-click to select “Open Dataset.” The following window should appear (with files matching those in your directory, of course):

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Figure 9. The "Open Dataset" window

To find your .nv file, you’ll need to move around among the directories a bit. The “Current Directory” line shows your current path name, and you can edit that manually if you wish. You can also double-left-click on “..” to move up one directory and double-left-click on any of the directories in the left-hand column to move into them. If you have a number of related spectra to work with, you may find it useful to move them all into one directory. For this to work, the files must all be given meaningful names instead of “test.nv.” This is what the corresponding window should look like:

Figure 10. The "open dataset" window with .nv choices

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Either double-left-click on your selection in the right-hand column or highlight it and left-click the “Open” button. Not much will appear to happen – just a few new lines will appear in the TkCon window (Figure 11). I’m not sure where you can find out what all these numbers mean, but the main points of interes are the peak labels (on the far right) and the frequencies at the centers of each dimension (third column from the right). When opening multiple spectra for comparison, be sure that these numbers and labels are consistent and accurate before proceeding. If they don’t, edit you NMRPipe fid.com script and reprocess the spectra.

Figure 11. The TkCon window after opening a dataaset

If you double-click again, you’ll get an error window that looks like this:

Figure 12. Open Dataset error window

To manage your error, just click the “OK” button and forget about it; you’ve read your spectrum successfully. If you’ve going to be examining several spectra in this session, you can read all of the ones you’re working with into the working memory now. Just keep on adding datasets to the pool from which you can select for drawing into windows. Click the “Close” button to exit.

G. Orient yourself with respect to the window menu and its options Right-click within the blank space of the window you created. You’ll get a menu that appear like this whose selections contain menus of their own:

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Figure 13. The window menu.

Selecting “attributes” from this menu calls up a new window, which governs the content and appearance of the spectrum in the window. It has its own pulldowns, which are shown below ().

Figure 14. The Attributes window

Figure 15. The Attributes window pulldowns

Most of the window menu’s selections will come up with sub-menus when clicked (left-, right-, or center-clicked). Here are the sub-menus:

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Figure 16. Window menu pulldowns

H. Draw your spectrum in the window you created In your blank window (Figure 8), right-click to get your window menu (Figure 13), and click “attributes” to get your Attributes window (Figure 14). Left-click on the “File” button, then click “Dataset.” You’ll get a new dataset manager window that looks like this:

Figure 17. The window dataset manager window

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Double-click on your spectrum in the left column, or single-click it to highlight it then click the “Add” button. Your window should now look like this:

Figure 18. The window daaset manager window, post-add

The “replace” and “remove” buttons act on data in the right-hand column. Congratulations! Your spectrum is now an object that can be drawn in the window. Return to the Attributes window (Figure 14). You can click the “Draw” button now to get a spectrum in your working window (Figure 19).

Figure 19. Window with spectrum, initial display, Lvl=0.1.

If you wish, you can have NMRView determine a suitable level at which to draw your spectrum by clicking “Misc” then “AutoLvl” (Figure 15). You’ll see the number in the “Lvl” display of the Attributes window change. I’ve found that this makes the spectrum a bit noisy, so I suggest clicking the up-triangle button a couple of times after invoking Autolvl. (The convention here is that higher numbers bring you farther from the noise, and lower numbers closer to noise.) Now click “Draw.”

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I. Familiarize yourself with cursor functions and zooming in & out NMRView gives you two sets of cursors. Left-click-hold on the spectrum in your working window, and you should get a set of black crosshairs. Center-click-holding should give you a set of red crosshairs.

Figure 20. Left/black and Center/red crosshairs Note that the chemical shift positions of the black line-cursors are shown (in ppm) in the upper left corner of the working window, and that the positions of the red cursors are shown just to the right of them. The frequency difference (in Hz) between parallel cursors is shown in the upper right portion of the working window. Each cursor can be moved independently. Both cursors in a crosshair can be moved if you click near their intersection. If you click near one of the lines, but far from their junction, you’ll just move the one you’re near. Go ahead and practice this for a minute so you get used to it. Zooming in: Move the crosshairs so that they surround a portion of the spectrum you wish to examine more closely (Figure 20, right). Right-click in the working window to call up the window menu, left-click ‘View,” then click on “expand.” Your spectrum will be redrawn such that the region you selected occupies the whole visible spectrum in the working window, but the cursors will remain in their same positions relative to the window:

Figure 21. Expanded portion of the spectrum

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You can reverse this expansion by right-clicking to get your window menu, left-clicki “View,” then clicking “Previous.” One very handy feature of NMRView is that you can zoom in on something, zoom in again, then then back out one step without returning the full spectrum, as you must do with VNMR and NMRPipe. If you don’t like the level at which your spectrum is plotted, you can go back to your Attributes window at any time, change the level, and redraw the spectrum:

Figure 22. Zoomed spectrum at Lvl>1.0

J. “Plotting” your spectrum Quotes are placed around “plotting” because you can’t actually send anything to the printer from NMRView. Why? Ask Bruce Johnson, the program’s author. Anyway, chances are good that when you want final output, you’ll want it in electronic format, so it won’t matter for most circumstances. NMRView does do a good job of making postscript files from spectra, and that’s what is meant by “plotting.” In your working window, right-click to get your window menu (), left-click on “Misc,” then click on “plot.” The following window should appear:

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Figure 23. The plot window

The main line you wish to pay attention to is the one in the lower left, where it reads “PlotFile:.” The line that currently reads “ps.dat” can and should be modified to something meaningful so you know what this lot is all about. I strongly suggest you use the “.ps” suffix to identify this file as being one in postscript format. Even though the space looks tiny, you can type an entire path name here. If you don’t type a path name, the file will be written in the default directory for plotting, as set in the NMRView Main toolbar File…Prefs…Paths panel – NOT the directory you launched NMRView from. Either alter your path preferences or be prepared to hunt around for your postscript files. Leave the “Postscript” and “Fix Postscript Output” boxes checked. You’ll want to reduce the “height” to 6.5 (in) for a square plot. For some reason, even though the given height is larger than the width, reading this file into Adobe Illustrator puts the “width” in the vertical direction and the “height” in the horizontal direction, and manages to cut parts of the spectrum off (just the peaks, not the border, mind you) if you don’t reduce the “height” value. You can play with the numbers used, but I’ve gotten acceptable results with a 6.5x6.5 square, so I’m sticking to that. Keep or change the “title” value, you can change it later in Illustrator. When set up, your plot window should look like this:

Figure 24. The revised plot window

Click the “Plot” button. Your postscript file will be written, and its full path will be identified in the lower portion of the plot window:

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Figure 25. Updated file name with full path

You can go to the terminal window to see your new file:

Figure 26. Terminal window with postscript spectra

To view the postscript file, you’ll need a separate program capable of reading it. My favorite for this purpose is Adobe Illustrator, but you can probably insert the spectrum directly into Word or PowerPoint. The main advantage to Illustrator is that it will treat separate parts of your plotted file as separate postscript objects. For instance, you can select your axis labels and change their fonts and point sizes without changing anything else in the spectrum. Importantly, you can select the lines in the spectrum and change their line width. Further discussion is beyond the scope of this User Guide.

3) Picking and Assigning Peaks in a 2D spectrum If all you wanted was a fingerprint spectrum, then you’d be done when you plotted it. To get a list of crosspeak frequencies, you’ll need to pick your peaks and generate a peaklist. In NMRview, this is commonly known as an “xpk” file because these end with the .xpk extension. You do not need to assign all your peaks when you pick them. If you’re not working with a protein, you might be better off working manually with an unannotated peak list. If you’re working with a protein and its spectrum has already been assigned, but not for this particular spectrum, the following “assignment” protocol is for you.

A. Call up the peak picking window Right-click on you working window (), left-click on ‘Peak,” then click on “Pick.” The peak-pciking window will then come up.

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Figure 27. The peak-picking window.

Note that the text in the title bar of the window corresponds to the title of the working window. This helps you know which working window you’re dealing with when you’ve got several of them open.

B. Adjust the appearance of your spectrum Adjust the scale of your spectrum so that you no longer observe noise peaks, but the real peaks are not too small. Obviously, this involves some judgment, so you may have to try this a few times until you get suitable results. The goal here is simply to pick all the real peaks but not the noise peaks. Peaks/noise at the borderline may need to be picked/deleted manually. Also, zoom in on your region of interest so you don’t include residual solvent traces or undesired backbone areas; inclusion of either of these can give you hundreds of extra useless peaks.

C. Select your peak picking parameters First, type a name for your peak list in the “List name:” box. If you’re appending or replacing an existing list in the working memory, click “select” and menu of open lists will appear; click on one to select it. The default parameters () are usually good, so clicking the “Pick” button will usually work fine after giving your list a name. Select your pick region. ‘Window” selects everything displayed in your working window, and “Box” selects everything bounded by your cursors. I’ve never worked with “Filter,” so I have no authority to help you with it. Select your pick mode. “New” creates a new list, “Replace” replaces the named existing list in the working memory, and “Append” adds peaks to the named existing list in the working memory. Note that none of these actions actually writes anything to your hard drive. That comes later. The peak picking described here only affects what’s going on in the NMRView working memory. Apparently, NMRView can pick peaks in up to four dimensions, and each dimension’s picking parameters are organized in columns here. I don’t know what “wrapping” is, but I’ve never used it and have had no problems. “Min” and “Max” describe the bounds for expected crosspeak sizes, in Hz. You can adjust the default parameters if you like, but they work well for most proteins. Your main problem will probably arise from double-picking of peaks that are split by scalar coupling. Handling this will be covered shortly.

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D. Pick the peaks Click “pick” to pick your peaks. Your spectrum should be redrawn with boxes containing crosshairs around your peaks; numbers corresponding to peak number should appear next to the boxes:

Figure 28. Spectrum after initial peak-picking.

You should also get some messages in your TkCon window:

Figure 29. TkCon window after peak-picking

The important thing to view here is the total number of peaks picked. The meanings of “good” and “bad” peaks is unclear, and it hasn’t proven itself to be useful in practice. It may have to do with shape and symmetry of the peak. Also don’t worry about the dimensions of the peaks or other messages in this window. The total number of peaks picked should approximate the number you’d expect from your protein for this type of spectrum. While the number of peaks in TOCSY and NOESY spectra will be hard to predict, the number in an 15N HSQC spectrum should be nearly equal to your number of residues, plus twice the number of glutamines and asparagines, minus one for the N-terminus NH3+, which you don’t expect to observe because of rapid chemical exchange. Feel free to close your peak picking window.

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E. Orient yourself with the Peak Panel Now that you have a list, you’ll want to write it to the hard drive so you don’t lose it. In your NMRView main toolbar (), left-click “Assign,” then click “Peaks” (). You should now see the “Peak Panel”:

Figure 30. The peak panel

Orient yourself with the Peak panel. Here are the pulldowns for the peak panel:

Figure 31. Peak pulldown menus

Left-click on “List” then click to activate the desired peak list. You should see many of the boxes in your peak panel fill with numbers and text:

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Figure 32. The peak panel with data

Orient yourself with respect to the displays and controls. In the box at the top, you’ll find the number of the peak whose descriptors are on display; this number corresponds to the number you see on your spectrum in the working window. The “down” and “up” triangles enable you to scroll up and down the list of peaks. You can always type a number into the box to get to a particular peak. The cute symbol toggles the peak between being “alive” and “dead.” If is’ black, the peak is alive. If you click on the black , you target the peak for removal in a later “cleaning up” step, and the should turn red. The lock button “locks” the peak’s data in so you cannot change it without “unlocking” it first. The “Lab” line contains peak labels. The “0” and “1” here refer to the peak label number and total number of peak labels for this peak. This feature is helpful when you have overlapped or ambiguous peaks. The < and > buttons scroll through the labels associated with this peak. Each column of boxes corresponds to the parameters associated with the peak in one dimension. I think the order in which these appear horizontally (HN, N, for example) corresponds to the order displayed vertically in the Attributes window (Figure 14). The top-line boxes containing “?” symbols are where you type in your peak assignment labels, which bear the form “24.HN”, corresponding to residue number 24 and the amide H atom (more on that later). The RName boxes will automatically display the three-letter name of the residue assigned to this peak in this dimension with this label; NMRView knows what the residue name is based on the residue number when you’ve told it what your protein’s sequence is (more on that later). The “Center” line shows you the frequency of the center o your peak in each dimension, in ppm. The “Width” boxes display the estimated line widths, in Hz, of the peak in each dimension, and the “Bounds” boxes display the widths, in Hz, of the box used to define the peak in each dimension. If your peak is split by scalar coupling, it looks like NMRView tries to estimate the apparent coupling constant, which is displayed in the “J” boxes. The “LinkA”, “LinkB”, and “Cluster” boxes correspond to parameters associated with advanced processing, and will be covered in a later section ofthis guide. As far as I can tell, you can ignore the “user” and “error” boxes. At the bottom, you can associate a comment with a peak. In the “Int” box, you’ll find a value for crosspeak intensity. In the Vol. Box, you’ll find the volume of the crosspeak, but it will show up as “0.00000” until you measure the crosspeak volumes under the peak panel “Int”…”Get Volumes” function.

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F. Save your peak list as an .xpk file After you’ve loaded your peak list from the working memory into the Peak Panel (Figure 32), left-click on the peak panel’s “File” button (), and click on “Write List.” Yet another window will appear:

Figure 33. The Peak panel "Write File" panel

This panel will default to writing into the directory specified in the NMRView paths preferences panel (main toolbar…File…Prefs…Paths and Filenames). You can change the directory in which you wish to save your current file, but you cannot change your default directory here. Where it reads “File Name:” type in a file name ADDING THE “.xpk” EXTENSION and click the “Save” button. The grayed-out “files of type” selector appears to suggest you should be saving your file with a “.seq” extension, but this is wrong. When it comes time to read your peak list, you’ll need the .xpk extension. After you click “Save,” your crosspeak data will be written to a space-delimited text file. If you open it up in vi editor, it will look like this:

Figure 34. The .xpk file

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G. Couple multiple peaks that should be one peak It frequently happens that your peak picking routine will choose two or more “peaks” where there is clearly just one in the spectrum. This is clearly the case when you have lines sharp enough to resolve your NH-Ha scalar coupling, which manifests itself by exhibiting two peaks in the 1H dimension for your H-N crosspeak. The simplest way to take care of this is to use the “Couple” routine in NMRView. To couple your peaks, go to your Peak Panel (Main Toolbar…Assign…Peaks, Figure 30), and pull down Edit…Couple (Figure 31). You’ll get the Coupling panel, which looks like this:

Figure 35. The Peak Couple panel

The Min and Max numbers here refer to the minimum and maximum frequency differences for peaks that are to be combined into one peak. The different columns refer to your different dimension, as defined in the Peak Panel. The minimum difference should be set to 0 for both 1H and 15N. A good set of maximum values to start with would be 3 (1H) and 10 (15N). Click “couple.” You should get a message in your TkCon window telling your how many peaks were eliminated and how many remain:

Figure 36. The TkCon window after peak coupling

If this coupled too few peaks, i.e., you have too many left, then increase your maximum value; I’d start by setting the 1H difference to 5 (Hz).

H. Manually add and/or delete peaks to/from the peaklist Some of the peaks picked may actually represent noise. Some objects that you know are peaks may not have been picked automatically. The picked peaks you observe may have bounds that don’t correspond to the actual spectral peaks’ dimensions. To correct these problems, you can use the tools “Peak Adjust”, “Peak Add,” and “Peak Delete” in the Window Menu’s “Cursor…” menu (Figure 13, Figure 16).

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Note that these tools can be DANGEROUS! Manually adjusting peaks must be governed by strict ethical principles! With these tools, it would be trivial to insert a picked peak where there really is none or deny the existence of one that really is there. Be careful! The functions of the add and delete tools are self-explanatory. With the “Peak Adjust” tool, left-clicking on a peak moves that peak around, and center-clicking enables you to adjust its size. Be careful! Left-clicking on an area where there is no peak may bring the nearest peak to your current cursor position, causing you much grief. This is especially bad when you’ve selected the “Peak Delete” tool, as there’s no way to “undelete” the peak short of reading in your .xpk file all over again. The “Peak Add” tool adds a peak to the nearest spot where it thinks there’s a peak. You can’t add a peak to empty space, nor can you do it to a shoulder. If no suitable peak is nearby, nothing will happen when you left-click to add the peak. Youd best course of action is to add a peak to a nearby obvious peak, switch to “Peak Adjust,” then drag your new “picked peak” box to your desired location and adjust its size appropriately.

I. Compress and Degap After you have marked peaks for deletion, which occurs when you use the “Peak Delete” button or click the button, you need to expunge them from your peaklist. To accomplish this, use the “Edit…Compress” tool in your Peak Panel (Figure 31). This simply eliminates peaks from the peaklists. Your .xpk file will not reflect the changes until you write it over with the new peaklist contents. If you just use the ‘Compress” tool, your peak numbers will be the same before and after compression. If you wish to renumber your peaks so that there are no numerical gaps in your list, click “Edit…Degap” in your Peak Panel (Figure 31). The ‘Compress” and “Degap” functions are combined in the “Compress&Degap” tool (Figure 31). So if you have a set of 100 peaks, 0-99, and you mark #54 for deletion, your peaklist will still have 100 entries. Invoking “Compress” will eliminate #54 from your list, leaving 99 peaks, but the sequence will be, “0…51, 52, 53, 55, 56, 57…99.” Invoking “Degap” will renumber the peaks so they’ll be, “0…51, 52, 53, 55, 55, 56….98.” It is a good idea to save your peaklist to the hard drive after compression and degapping. You may wish to save it under a different name in case you eliminated peaks that should have been retained.

J. Manually assign residue identities to your peaks (optional) Ultimately, you need to correlate your picked peaks with the atoms that gave rise to them. If you are working with a large set of spectra, you should go directly to the Nvassign module for peak assignment. If you have a set of homonuclear spectra, such as a TOCSY and NOESY from an unlabeled peptide, you’ll need to proceed with this manual assignment routine. This guide’s purview does not include methods of assigning homonuclear spectra. For that, please see Cavanagh (Cavanagh, J., W. J. Fairbrother, et al. (1996). Protein NMR Spectroscopy: Principles and Practice. San Diego, California, Academic Press, Inc.) and/or Wührich (Wüthrich, K. (1986). NMR of Proteins and Nucleic Acids. New York, John Wiley & Sons, Inc.) When you are prepared to declare a frequency’s association with an atom, type the entry into the “Lab” box of the peak panel (Figure 32), replacing the question mark. You can type whatever you like in here and it will be saved with your peaklist, but it is most useful to follow the standard NMRView syntax. For the amide proton of residue #24, type “24.HN” in ther “Lab” box. If you’ve opened up a protein sequence under the “(main menu) Molecules…Read Topology…Sequence” routine, NMRView will know which of the 20 amino acids is in position #24. If you update the peak panel display by clicking the up triangle to

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select the next peak up in the sequence, then click the down triangle to go back, the three-letter code for the residue in question should appear in the “Rname” box. One nice feature of using the standard NMRView syntax is that NMRView will compare your atom’s frequency to others of its type for the same residue in its chemical shift database, then tell you whether you assignment is probable or not. To check your assignments this way, open up the assignPanel by clicking from the Main Toolbar (Figure 2) Assign…Peaks. In the assignPanel, click File…Get Shifts From Peaklists, then select your peaklist. If you’ve already opened this peaklist in the assignPanel, but you’ve made changes to it in the Peak panel, then you need to update the entries by clicking the “Update” button. TO check the probability of your assignments, click “File…Check Shifts.” The results will appear in your TkCon panel. If you have assigned a peak to a residue whose normal chemical range is incompatible with your peak, a message to this effect will appear in the TkCon panel. If there are no conflicts, you won't get such an error message.

4) Overlaying two 2D spectra

5) Measuring chemical shift differences between two 2D spectra

6) Obtaining distance restraints from a NOESY spectrum

7) Examining 3D spectra

8) NVAssign: Coordinating 3D Experiments to Achieve Total Assignment

A. Overview NVAssign is a module that operates within NMRView that aids and partially automates spectrum assignment from standard 3D 1H/13C/15N data. It will not work for homonuclear assignment, nor will it be helpful for working with 1H/15N (no 13C) proteins. The module, which originates from NIH, is installed after nmrview is already installed. It replaces an older tool, CBCA Analysis, with a more streamlined processing pathway and more complete features. The NMRView manual does not have a discussion of NVAssign, but it does have one for the CBCA Analysis. The authors of NVAssign have not provided a manual for its use, but have a set of notes describing its differences from the CBCA tool and a few general points of interest. The goal of NVAssign is to derive a list of frequencies that can be associated with the backbone atoms HN, N, CA, CB, and C. You will read in peaklists, but these will not be annotated with your residue names and numbers, as you would have if you had assigned peaks manually. Your goal is simply to go to the Main Menu…Assign…Peaks to call up the assign panel, see that all the backbone and CB atoms have frequencies assigned to them, then save the frequency list.

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To work with NVAssign you must have results from the following 3D experiments: HNCO, CBCA(CO)NH, and HNCACB. These must be peak-picked and their peaklists must be in the working memory before entering NVAssign. It will also be necessary to read your protein’s sequence into working memory before beginning NVAssign if you wish to complete your assignments. You can save the work you’ve done within the NVAssign window, so can close the program and come back to your state later. The general strategy here is to compare peaks in your HNCACB and CBCA(CO)NH experiments to identify peaks corresponding to residues i-1 (which are in both spectra) and residues i, which are only in the HNCACB spectra. The set of peaks (six peaks, unless you have a glycine in the i and/or i-1 positions) comprising the CA and CB peaks from residue i-1 and the CA and CB peaks from residues i-1 and i constitute a “cluster”. After analyzing your peaklists, NVAssign will show you sets of peaks that are probable clusters of peaks, and you stare at them, evaluate the likelihood that they’re valid, then declare them a genuine cluster. Because the “i” resonances in one cluster are the “i-1” resonances in another cluster (unless the residue is at the polypeptide terminus or adjacent to a proline), the clusters can be “linked” to form “fragments” corresponding to stretches of consecutive residues. Once you’ve got your automatically assigned fragments, you can iteratively re-evaluate your clusters and links, then reassemble fragments to improve the extend of your assignment. Once you’re happy with the assignments and all of your assignments are made, write your peak assignments from your assign panel and congratulate yourself for completing the NVAssign routine.

B. Important details NVAssign insists that several simple criteria be satisfied. If you do not meet them early in your processing labor, you may observe no immediate consequence, but you will be digging yourself into a deep hole, requiring you to start from scratch when the chickens finally come home to roost. So if you don’t want to start all over when you’re nearly finished, you MUST follow these rules:

1) All axes must have consistent names in all experiments! Your 15N dimension, for instance, should be labeled “N15” in all your spectra. If your HNCO uses “N15” and your HNCACB uses “15N” you won’t be able to use NVAssign. For consistency, we recommend you use “HN” for your 1H axis, “N15” for your 15N axis, and “C13” for your 13C axis. Don’t get fancy and label the 13C axis of your HNCO “CO” and the 13C axis of your CBCA(CO)NH “CBCA.” If you do this you will be doomed to reprocess your spectra. If you commit the error, you’ll need to reconvert your Varian data with an edited version of “fid.com” and reprocess with “process2d.com.” Use your favorite text editor to edit the axis label terms in fid.com, then invoke both scripts. There’s no need to change the process2d.com script. If you’ve already picked your peaks, you’ll need to either repick them or edit your .xpk files to make all their axis labels consistent.

2) All axes must be referenced the same way in all experiments! Nvassign will fail miserably if the center of your 1H dimension in your HNCACB is 4.70 ppm when it’s 4.76 ppm in your CBCA(CO)NH. If any of your spectra are referenced incorrectly and/or inconsistently, fix your fid.com file, reprocess the spectra with fid.com and process2d.com, and, if need be, repick your peaks.

3) Keep the same file names throughout your NVAssign process! It may be tempting to save changes to your .xpk files under new names so you don’t overwrite files that worked well in the past. NVAssign and NMRView will let you do this without giving you any warning and will show no apparent problem for awhile. BUT, when you close NVAssign and NMRView, then get back to your project at some later time, NOTHING in NVAssign will work properly, even if you’re simply loading in your old state. If you want to back up a

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working .xpk file or cluster file, go to a terminal window and copy the working file to a file with a different name, like “cp protG_hncacb.xpk protG_hncacb_bckp.xpk”.

4) Load your .xpk files and sequence file into your working memory every time you start NMRView. You can save your NMRView layout and identity of the spectra in the working memory with “save state,” and reload them later with “Load State,” but the .xpk files and sequence file are not saved as part of the state. I don’t know exactly what happens if you assign everything to clusters without the sequence data being loaded up, but it can’t be good. Ultimately, the fragments will be assembled and assigned to residues in your sequence, so NVAssign will need to know your sequence. make sure to look in your TkCon window when you load your sequence. Your proper MW should appear in the window. If you observe a multiple of your expected MW (e.g., 12000 instead of expected 6000), you’ve read your sequence multiple times and you have to exit NMRView and restart it to clear the sequence from working memory.

5) Be sure to have ~1.5 - 2GB free on your computer’s startup drive (or startup partition). NMRView running NVAssign seems to write a lot of data to scratch space your hard drive as part of a “virtual memory” scheme. I had cleared out my hard drive so I had about 1.5 GB of free space left on my bootup partition, but over the course of a day or two with NVAssign, I started getting warnings that my drive was almost full. Quitting the program and rebooting cleared the problem.

C. Preparation

Process the necessary spectra and get them in NMRView format. You’ll need the 2D 15N HSQC, 3D HNCO, 3D HNCACB, and 3D CBCA(CO)NH. Make sure all the axes are labeled consistently and the chemical shifts area referenced consistently. It will be handy to make sure all these spectra are in the same directory:

Figure 37. Example files for NVAssign in a common directory

It will probably be handy to keep all these spectra open. This is a state you can save and load, so once it’s set up, you shouldn’t have much trouble restoring it:

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Figure 38. Displayed spectra required for NVAssign (3D spectra shown as 2D projections)

Give your spectra a once-over. Make sure all are displayed with negative peaks showing up red. Your HNCACB spectrum should contain both negative and positive peaks, with negative peaks (beta carbons) in the low and high 13C ppm ranges (Figure 38). The high-ppm negative peaks correspond to beta carbons of serine and threonine.

Pick peaks in each of these spectra In each of those four spectra, pick peaks with a reasonable threshold and save them as .xpk files with short filenames in one directory devoted to peaklists.

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Figure 39. Spectra for NVAssign with peak assignments (3D spectra shown as 2D projections)

Before sorting through individual peaks, you should do a reality check and estimate the number of peaks you’d expect for your spectra and compare that number to the number of peaks actually picked for each spectrum. The best you can hope for is that you get only 5-10% more peaks than you expect. For a protein with X residues, P prolines, G glycines, N asparagines, Q glutamines, and W tryptophans, your HSQC should have X-P-1+W+2N+2Q peaks and maybe more for the histidine side chains. (You don’t expect to see the N-terminus, hence the “-1”.) For an HNCO, you should expect X-P-1+2N+2Q peaks. For the CBCA(CO)NH, you should expect 2*(X-1)-G peaks, and for the HNCACB, you should expect 2*(X-P-1)-G+2(X-1)-G peaks (note that you can observe proline CA and CB resonances as i-1 signals but not i signals). Make sure to display your HNCACB experiment with the negative peaks in red – these will correspond to your CB resonances. If you have picked too few, you either have substantial overlap problems and/or your threshold was too high (when you picked, your display was too far away from including noise). If overlap is not your problem, repick your peaks while displaying your spectrum at a level closer to the noise. If you continue to have too few peaks even when some of your picked peaks are random nose, and you know you don’t have overlap problems, you should probably reacquire your spectrum with a greater nt values to improve your signal-to-noise ratio and/or reassess the quality of your sample. If you picked too many, your display may have been too close to the noise level, or you may have included a stripe of T1/T2 noise, which happens because of the severe truncation of sharp signals. You should try repicking your peaks while displaying less noise, being sure to click the “replace” button of your peak-picking panel. If you have many spurious peaks associated with T1/T2 noise ridges or poorly suppressed solvent, you should try placing your cursors around peaks you wish to select, avoiding known

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noise peaks, and picking peaks with the “box” box selected instead of the “window” box checked. If you have about twice as many peaks as you expect, your lines may be sharp enough for you to see splitting of the HN peak due to 1HHN/1Hα scalar coupling. In this happy event, you should consolidate the coupled peaks with the “” function in the “” menu of the Peak Panel. Alternatively, you could reprocess your spectrum with a 1H window function that broadens your lines and reduces noise.

Change your default paths While this is not strictly necessary, it will make things easier if you tell NMRView to save all your peaklists and other files in the same places.

Review and edit your picked peaks in each spectrum Go through each spectrum and eliminate spurious peaks. This task is a bit tedious, so I suggest putting on some music. In principle, you could look at regions of every plane in the 3D spectra, hunting for peaks, but this is inefficient and incompletely effective. The best method here is to look at peaks one at a time. Right-click in your spectrum to call up the menu, left-click “Peak…Peak Attributes.” in the row labeled “”, check the “” box and click “Draw.” This should revise your spectrum display so that you see only one peak at a time. Go to your Peak Panel and scroll up and down with the up and down arrow buttons to scroll through the different picked peaks. I suggest starting at peak 0 and working your way up, reviewing every peak. When you encounter a peak you wish to unpick, click the skull and crossbones button (), which should then turn from black to red. This will flag it for deletion and should change the color of the peak boundary, crosshairs, and label in the spectrum; you may need to scroll up once and down once to see the updated display. This peak is still part of the peak list, but you should eventually eliminate it and renumber the peaks using the “compress and degap tool in the peak panel. Common problems you see at this stage are the inappropriate picking of noise and side-lobe peaks. Sometimes, you’ll get a completely blank screen that you can scroll to as if it were a legitimate peak. this is a noise peak that was picked with no peak width, so the frequency on one side equals that on the other side. Click the to get rid of it. Sometimes you’ll see negative peaks in the HSQC, HNCO, and CBCA(CO)NH spectra; these experiments should give you positive peaks only, so click the to get rid of a negative peak in these spectra. Sometimes you’ll get weak peaks that may or may not be legitimate, but may actually be side-lobe peaks. You can usually identify such peaks as spurious if you see a large peak next to it with another peak on its other side. Click the to eliminate this peak, but be very careful that you’re not actually getting rid of a legitimate, but overlapping, peak.

D. Open NVAssign

Figure 40. Location of the NvAssign menu selection

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Figure 41. The NvAssign window

Figure 42. NvAssign menu pulldowns

E. Read in your data Hold your horses! I know you want to click “examine peaks,” but more needs to be set up before you can do that. If you click that, you’ll get this message:

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Figure 43. Error message for clicking "examine peaks" prematurely

You first need to tell NvAssign which files you’re examining by entering their names into the NvAssign Prefs window. In the NvAssign main window, click “File…Preferences,” and the following window should appear:

Figure 44. The NvAssign Preferences panel

Your choices are basically self-explanatory, but you must take care to enter everything in correctly. You’ll see that this working with this panel is easier if you’ve organized your files into handy directories and told NMRView where to find them in the NMRView paths preferences. Your reference peaklist should be your HNCO (.xpk file), your interResidue filename should be your HNCACB spectrum (.nv file) and its peaklist should be your HNCACB peaklist (already loaded into the working memory by picking peaks or reading an .xpk file). Likewise, the intraResidue filename and peaklist must correspond to your CBCA(CO)N. You need to change your MatchScript from MatchCBCA4 to MatchCBCA2. You also need to change your spectrum axis labels to the ones you used when processing your spectra; for discussion here, we’re using “HN,” “N15,” and “C13.” This is what your completed NvAssign preferences panel should resemble when you’ve filled in all this information:

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Figure 45. The NvAssign Preferences panel, properly completed

You also need to set additional preferences by clicking the “Set PeakLists for exporting data” button. This ensures that you cluster data will be written into your peak lists, which you must save occasionally during the clustering process, by the way. Here is the panel that comes up after clicking:

Figure 46. NvAssign Prefs "Set PeakLists for exporting data" panel, filled in properly.

OK, you can tell that this is an early version of this manual right now because the peak lists in () are different from those in (), where they should be the same. At some point, I’ll update the figures to have consistent filenames, but we’ll just have to make do for now. This discrepancy is actually how I learned how important it is to keep the same filenames throughout your whole NvAssign procedure, and I wasted much time figuring this out. Now you don’t need to! Note that you should leave the noesy entry as “none.”

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If you think you’ve gone through all this, but you haven’t entered things right, you’ll get an error message. If you failed to enter in the proper axis labels, you’ll see this sort of an error when you click “Examine Peaks”:

Figure 47. NvAssign error due to improper axis labels in Nvassign prefs

If at least your prefs were set correctly, you should see (without the blue annotations) something like:

Figure 48. The NvAssign "Examine Peaks" display

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Orient yourself with respect to this display. the left two strips correspond to the CBCA(CO)NH experiment, and the right two strips correspond to the HNCACB experiment. each of these panels will respond to right- and left-clicking as it it were a regular spectrum-containing window; left- and center clicking will control the cursors, and right-clicking will call up the window menu (Figure 13).The vertical axis for all four strips is C13, and has the same scale in each in order to effect horizontal alignment of peaks with common 13C frequencies across the strips. The horizontal axes of the outer strips correspond to N15, and those of the inner strips correspond to HN. The C13 axis always represents the full spectral width in that dimension, but the horizontal axes are narrowed to focus only on the peaks of interest. This set of peaks is identified in the “Peak” box at the top of the panel, and its number corresponds to the picked peak number in the HNCO experiment. To help you visualize what’s going on, examine the following figure, which shows an HNCO spectrum 2D projection with crosshairs placed on peak #1, and the NvAssign “examine Peaks” display with HNCO peak #1 as the subject:

Figure 49. Correspondence of HNCO and NvAssign "Examine Peaks" display

Now you need to review the overall quality of NvAssign’s presentation. You should be seeing picked peaks that match up well: probably two positive peaks in each of the CBCA(CO)NH windows, corresponding to the CA and CB resonances of residue i-1; and probably four resonances, two positive and two negative, in each of the the HNCACB windows, corresponding to the CA and CB resonances of residues i-1 and i. In each strip, the peaks should be centered on the horizontal axis. Each peak label should correspond to the peak’s number in the peak list of that strip’s experiment; in the figure displayed here (Figure 48), the HNCACB strips show peaks #0, 1, 2, 3, and 4 of the HNCACB peak list, and the CBCA(CO)NH strips show peaks #0 and 1 of the CBCA(CO)NH peak list. Note that these peaks are all now associated with HNCO peak #1 due to their common HN/N15 frequencies.

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If your peaks are centered in some strips, but not in others, you may not have referenced your 15N and/or 1H dimensions consistently in all spectra. If this is the case, start over by correcting your fid.com files, reprocess the necessary spectra, repick their peaks, and re-check them for reasonableness. Go through all of your peak sets by scrolling up and down with the up and down arrow buttons. If things look basically OK, proceed to the next step. If most peaks don’t provide rational displays, you should probably clean up your peak lists, then start NvAssign again.

F. Filter your peaks The actions at this stage filter out peaks in your HNCACB and CBCA(CO)NH peaklists that do not correspond to peaks in your HNCO peaklist. Click the “Filter Peaks” button at the left of the NvAssign panel. (Alternatively, you could click “Mode…Filter Peaks.”) You should see a display that looks like the “Examine Peaks” display, but you’ll have a section below the buttons at the left entitled “Tolerances,” and a section at the top for selecting peak lists. The default tolerances for filtering are pretty good, so you shouldn’t need to change them. For the “Ref PeakList” selection, click the “Select” button and choose your hnco peak list. For the “Peaks Filtering” box, click the “Select” button and choose either your CBCA(CO)NH or HNCACB peaklist. After selection, your display should look something like this:

Figure 50. The "Filter Peaks" display after selecting appropriate peaklists

Click the “Filter Peaks” button. Your TkCon window should tell you about peaks NvAssign has marked for deletion from the peaklist:

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Figure 51. TkCon window displaying results from peak filtering

At this stage, these peaks are only marked for deletion, but they aren’t actually gone from your peaklist yet. You should go to your spectrum’s window (not the one in NvAssign) and display the peaks in question, assessing the validity of NvAssign’s suggested deletions. If a chosen peak should be kept, unmark it for deletion by toggling the button to black. Once you have decided that all the � marked peaks should be deleted, go to your Peak Panel (with the proper peak list selected) and run the “Compress&Degap” routine. Your results should show up in the TkCon panel something like this:

Figure 52. The TkCon window displaying results from post-filtering compression & degapping

Perform your reality check, as described above during the peak-picking discussion. If you have fewer peaks than you expect, you may be in trouble. If you have a couple too many, you’ve probably included all you need plus a couple of noise peaks you can refuse to cluster later. repeat your procedure with the peaklist you haven’t filtered yet, i.e., the HNCACB list if you just filtered the CBCA(CO)NH, or vice versa. Note that this does nothing to your .xpk file. It only modifies the peaklist in your working memory. At this stage, you should open up a terminal window, copy you .xpk files to .xpk files with new names, then save your new peaklists under the same .xpk filenames you opened them with.

G. Cluster your peaks The next stage in NvAssign is to group your peaks into “clusters,” that is, you get NvAssign to associate individual peaks into groups corresponding to residues i and i-1. The process places flags in your peaklist to identify clusters, but most of the data are must be saved as what I’ll call a “cluster file<’ which we’ll discuss once you’ve got a set of clusters to save. To start. click on the “Cluster Peaks” button on the left side of the NvAssign panel. Your display should look something like this:

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Figure 53. The "Cluster Peaks" display

At the lower left, you can set tolerances that NvAssign will use to group crosspeaks into clusters. We presume that the “IntraList” and “InterList” labels correspond to grouping peaks within your two peaklists and between the two, respectively. Click the “Link intraList” button, then click the “Link interList” button. You should see the following appear in the TkCon window:

Figure 54. The TkCon window after intraLinking peaks

Figure 55. The TkCon window after interLinking peaks into clusters

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That’s about all there is to the peak clustering job. Figuring out how well this worked and cleaning up the results are part of the next stage.

H. Edit your clusters The NvAssign software’s great achievement is that it automates the grouping of related peaks and does the bookkeeping for you. However, its automation routines are imperfect, and you can expect that you’ll need to fix 10-30% of the clusters it has automatically generated. Thankfully, NvAssign realizes this and not only provides you with its best guesses for peak associations, but, when in doubt, it also provides second- (and third- etc.- if necessary) best guesses to help you out. Before initiating this stage of analysis, get mentally prepared. You’ll be looking at lots of peaks, visually lining them up and doing rather right-brain work. get some coffee or another favorite beverage and put on some music. All the facility computers have headphone jacks, so put them to use. Click the “Edit Clusters (HN-C)” button. I don’t know what’s supposed to happen if you click the “Edit Clusters (HN-N)” button, as I got an error message every time I tried using it. I was able to assign protein G without doing that routine, so it’s at least not strictly necessary. A new panel entitled “nvassignCntrl,” pop up. Your display should look something like this:

Figure 56. The "Edit Clusters (HN-C)" display, with the "nvassignCntrl" panel

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Orient yourself with respect to the new features of the NvAssign panel. Your peak strip displays represent the same spectra and axes as observed in the “Examine Peaks” display. The cluster you’re examining, labeled by the corresponding HNCO peak, is identified in the top line’s “Cluster” box. You can select which cluster you are examining by typing in the box and hitting “return,” or you can scroll up and down with the up and down triangle buttons. The “Update Spectra” button should be checked – why would you want outdated spectra? The display for “HSQC peak” should really be labeled “Reference Peak.” The syntax for this display label is peaklist.peak number, and your HNCO peaklist name should be appearing here, not your HSQC peaklist. Frankly, I don’t know why this HSQC thing is here, but I haven’t had recourse to make any reference to the HSQC peaklist in NvAssign. I haven’t discovered a use for the “Toggle Peak Label” or “Unify” buttons. The “11” button will redisplay spectra at a level of 11.0, which always rendered my peaks unviewable. As far as I can tell, you’ll be just fine if you ignore these three buttons. The peak labels may appear as cluster number by default, but they can be changed to their peaklist number by right-clicking in the window to get the window menu, selecting “Peaks…Peak Attributes,” checking the “peak number” box for the “label by” choice instead of the “cluster” box, and clicking “Draw” in the Peak Attributes panel. Orient yourself with respect to the nvassignCntrl panel. The left-hand side shows a cluster number in the top box and a column of peaks in that cluster. Each peak follows the peaklist.peak number syntax described above. Note the correspondence of the peak numbers in this column and the numbers displayed as peak labels in the spectra of the NvAssign panel (Figure 56). The right-hand side of the nvassignCntrl panel corresponds to an alternative cluster. NvAssign defaults to placing a blank cluster here so you can construct a new one from rejected fragments of the old. The buttons at the bottom are used for editing. Editing your clusters is straightforward – even fun! – once you get the hang of the editing protocols. If you think a peak belongs in the cluster, double-left-click its crosshairs in the NvAssign strip where it appears (I’m not sure what this does, but there’s some discussion of the procedure in the help guides for the CACB module, on which NvAssign is based.) You move peaks from one side of the nvassignCntrl panel to the other with the “<” and “>” buttons. Clicking on a peak title to highlight it and clicking ‘Remove” removes the peak without moving it to the other column. If you think the peak you’d like to include is nearby, you can click the “Neighbor” button to retrieve an assortment of probable alternatives to the peaks listed. You can add a peak not displayed in either column by clicking the “add” button, which will initiate a short dialogue that requires you to select the peaklist in which your intended peak resides, and type in the number of that peak. Here is the panel for the “Add” dialogue:

Figure 57. The AddPeaktoCluster panel

When you are happy with your grouping, or at least feel like you wish to save this group for later, click the “Save Clusts” button. If you click the NvAssign panel’s up or down arrows to view a different cluster before you’ve saved this one, all of your changes will be lost without warning. Clicking the “save Clusts” button brings up the following window (yes, yet still another window):

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Figure 58. The "Save Clusts" dialog window.

Before saving your cluster, you need to specify the atomic identities of each of your peaks. This is where it will help to have your peak labels show your peaklist numbers, not your cluster numbers. Also note that you should really have shortish names for your peaklists, or the numbers for your peaks will be cut off and you risk confusion over where you labels go (as seen for the bottom two entries in this panel). To save typing and confusion, the first thing to do here is click the “guess” button, which should fill the boxes with the most probable identities for each peak:

Figure 59. The "Save Clusts" dialog window, properly completed

Do a quick reality check to see if these assignments are reasonable. All CA peaks must be postitive, and all CB peaks must be negative. The iminus1 peaks should be in all four strips of the NvAssign panel, and the “i” peaks should only be in the right two strips (HNCACB). You may have fewer than six peaks if you have overlap, or are dealing with a glycine. To distinguish between the two, keep in mind that glycine CA’s show up at ~45 ppm and that the presence of two negative peaks always means you do NOT have a glycine as either residue i or i-1. If you have one peak in each of the left-hand strips (CBCA(CO)NH), your i-1 residue is probably a glycine (unless your CA and CB peaks overlap, which is unlikely). If you have two peaks at the left, you cannot have a glycine in position i-1. If you have four peaks in the right-hand strips (HNCACB), of it you have two negative peaks on the right-hand side, you cannot have a glycine in position i. If you have two positive peaks and one negative peaks in your HNCACB strips, you either have a glycine in position i or your i and i-1 CB resonances overlap.

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The guesses seem like they should depend on the “Fragment Preferences” being set correctly (CA positive, CB negative), but the Guess button seems to get the answer right most of the time whether you’ve set this or not. Guess does seem to have a problem with glycines, though, assigning them to reside i-1 instead of i, or vice versa. When satisfied with your assignments, click the “Save” button of the “Save Clusts” panel. You’ll be prompted for a filename, which does not require an extension. Do not give it the same name as any of your other files. Put it in a handy place so later, after you’ve exited and restarted the program, you can read the file back into NvAssign. If you put peaks in the right hand column to make a new cluster, you need to click the “Save Clusts” button, just as you would to save the left-hand column entry. Be patient! You’ll first be met with a “Save Clusts” window for the left-hand column, with which you need to proceed normally. When done with that, a new “Save Clusts” panel will pop up, silently requesting that you fill in its boxes with guesses or knowledge-driven atom assignments. You can recall your cluster work by going to the NvAssign Panel, clicking “File…Restore,” and selecting your cluster file (Figure 42). NvAssign includes a cluster file editor, but I haven’t found a use for it yet. You can access it by clicking “File…Cluster Editor” in the NvAssign Panel (Figure 42). The Cluster Editor panel looks something like this:

Figure 60. The NvAssign Cluster Editor

OK! There you go! You have the rules, now it’s time to cluster, cluster, cluster!

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At various stages of your clustering process, be sure to save you peak lists back to their original .xpk files. Your atom assignments will be written to them this way. Here’s a comparison of the first few lines of a .xpk file before and after clustering:

Figure 61. A .xpk file prior to clustering

Figure 62. A .xpk file after clustering

I. Link/Match your clusters This part is even yet still more fun than assigning clusters. You can start to see how some assignments will come out just by seeing who’s linked to whom. Start by clicking the “Match Clusters” button on the left of the NvAssign Panel. Your display should look something like this:

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Figure 63. The NvAssign "Match Clusters" display (sans spectra)

OK, this display is supposed to have spectra in it, but I just called in this image quickly for the purposes of writing the guide. It’ll be replaced in a later version of the guide. The actions performed in this panel are straightforward and non-interactive. Simply click “Match” to match up your clusters. You can click the handy “Save Cluster Data” button here, but as I far as I can tell, it does the same thing as “File…Save.” The other buttons clearly indicate you can export this data for other programs, but that’s beyond the scope of this guide.

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Figure 64. The "Edit Matches" panel, with spectrum display

J. Assemble your fragments of linked clusters

K. Inspect your assignments