ADAPT-NMR Enhancer is a tool for visualizing the tilted 2D plane data from ADAPT-NMR, for correlating peaks in the tilted planes with peaks in the reconstituted 3D spectrum, and for tracing the assignments of these peaks to atoms in the covalent structure of the protein.

BACKGROUND

Because traditional assignment packages, such as SPARKY, NMRVIEW, CARA and CCPNmr, are not designed to make assignments from tilted 2D planes data, we have developed ADAPT-NMR Enhancer for evaluating and extending the results from ADAPT-NMR.

ADAPT-NMR Enhancer can be used in the following three ways:
    1. To visualize peaks in 2D tilted planes from specific 3D NMR experiments and to correlate these with corresponding peaks in 3D space. The peaks picked by ADAPT-NMR are shown, and these can be un-picked and additional peaks can be picked.
ADAPT-NMR Enhancer can display up to six tilted 2D spectra along with the picked peaks. This enables the user to evaluate the quality of the automated peak picking and assignment by ADAPT-NMR. Peaks in 3D space are easily correlated with evidence for their existence in tilted 2D planes. Users add and remove peaks manually and in doing so modify or refine assignments.
    2. To create a modified peak list as input to an ADAPT-NMR assignment iteration.
The visualization and verification tools offered by ADAPT-NMR Enhancer enable the user to pick additional peaks, delete noise peaks, and separate overlapped peaks. The refined peak list can then be submitted to ADAPT-NMR for probabilistic assignment.
    3. To complete resonance assignments with a manual interface.
If the user wishes to improve the number or confidence level of the assignments over those achieved following peak list refinement and submission to ADAPT-NMR, the PINE-SPARKY-like tools supported by ADAPT-NMR Enhancer support the verification and extension of assignments by an atom-by-atom and peak-by-peak approach.

DOWNLOAD

 We provide the ADAPT-NMR Enhancer for Linux, Windows, and MacOSX in binary executable formats. As ADAPT-NMR itself is only provided in Linux, we also only guarantee usability of Linux version of the ADAPT-NMR Enhancer. However, the ADAPT-NMR Enhancer will be executable and good for viewing ADAPT-NMR results with some limitations and unknown errors if you do some tweaks by yourself.

The lateset version of the ADAPT-NMR Enhancer is uploaded in 12/04/13 for linux and 11/25/13 for windows.

* Download
ADAPT-NMR Enhancer for Linux [click to download]
ADAPT-NMR Enhancer for Windows [click to download]
ADAPT-NMR Enhancer for MacOSX [click to download]

* Installation (for Linux)
First, unzip the downloaded file from above.

# unzip adapt_nmr_enhancer_linux.zip
  inflating: adapt_nmr_enhancer

Then, copy the uncompressed file to the /usr/local/bin directory.
[linux 32bits] adapt_nmr_enhancer
[linux 64bits] adapt_nmr_enhancer64

# sudo cp adapt_nmr_enhancer /usr/local/bin/

When you run "adapt_nmr_enhancer", sometimes it requires necessary libraries. In that case, just install the libraries.

# ./adapt_nmr_enhancer
  ./adapt_nmr_enhancer: error while loading shared libraries: libQtGui.so.4: cannot open shared object file: No such file or directory
# sudo apt-cache search libQtGui
# sudo apt-get install libqtgui4

Required libraries for execution are different depending on system environment. Also, the way we showed finding and installing libraries is for Debian decendants such Ubuntu, Mint Linux, etc. However, you can easily figure out how to install them for the other linux distributions.

MANUAL

* Main Window Dialog

The main window is where you can see visually plotted 2D tilt planes along with orthogonal planes. Tool buttons for visual management and peak management are located on the top. Left hand side is place for list boxes- tilt planes, 2D peak list and 3D peak list. You can see 6 tilt planes max at once in the main screen. You can find relative 2D positions depending on tilt angles in the screen helping you the quality of the 3D peak construction by ADAPT-NMR.

Left-side

The list box at the top-left is showing currently visualized planes. You can check six at once to see in the screen. The label shows the experiment type and the tilt angle. For example, HNCO_59 means 59 degree tilt plane of HNCO. Sometimes the selection of the plane is vital to do some plane specific functions. If you want to change the contour level of 59 degree plane for better resolution, you should select the plane from here.


2D peaks are listed in the list box. The column indicates; 2D peak index, X position, Y position, 3D peak index. X, Y positions are in pixel units, but they are refined under a pixel by interpolation. 3D peak index indicates the constructed 3D peak linked to the 2D peak. If the 3D peak index is zero (0), it means the 2D peak is not linked to any contructed 3D peak. In that case, user should look carefully to judge whether the peak is noise or not. If user select a peak in the list, the peak will be highlighted purple in the screen. The numbers- 1,2,3,4- are actually buttons for sorting. If user clicks 1 button, the list will be sorted by the first column which is peak index. The color of the peak in the list box indicates the priority- white, the highest, green, the next, dark green, the next... Higher priority means that the peak is confidently picked, so it will be strongly used for 3D peak construction. You can change the priority by the icons introduced later.


3D peaks constructed by ADAPT-NMR are listed in the list box. The column indicates; 3D peak index, direct dimension chemical shift, primary indirect dimension chemical shift, secondary indirect dimension chemical shift, probability. The probability implies the confidence of 3D peak construction. User can select a 3D peak from this list box. When a 3D peak is selected, the 2D tilt peaks linked to (used to construct) the 3D peak in every tilt planes will be highlighted lime in the screen. The numbers- 1,2,3,4,5- are also sorting buttons just same as the 2D peak list box above. The color of the 3D peak in the list box indicates how ADAPT-NMR confidently constructed that 3D peak out of 2D tilt peaks. The color indicator is like this. White: >90%, green: >70%, dark green: > 50%, red: <50%. Also, if a 3D peak does not have any linked 2D tilt peaks, the background color of the peak is blue (because the peak is lonely and blue!).

Top panels

ADAPT-NMR Enhancer works in the ADAPT-NMR working directory. As the ADAPT-NMR sub-directories and files are formatted, open and save functions are made for getting working directory from users. However, the formatted structures between Varian (Agilent) version and Bruker version are different, so that user should let the ADAPT-NMR Enhancer knows what spectrometer was used for collecting data. You will see the radio buttons to select the spectrometer in the Open Dialog section. ADAPT-NMR Enhancer also provides a functionality to make the project the initial stage just after ADAPT-NMR run in the case of unreturnable fault by users. The undo all recover everything. The following is the Open Dialog. You can see the radio buttons for selecting spectrometer types.




Full extent will change your viewport to cover whole spectrum range. So you will see every contents from the spectrum. This is useful when you are seeing very zoomed specific area in the spectrum and you need to quickly move to totally diferent place in the spectrum. In that case, you can click this button and zoom in to the place of your interest. Redraw button is for some accidental plotting miss by ADAPT-NMR Enhancer or other reasons. If you click this buttons, the extent will be kept but drawing will be made again. Previous extent is for going back to your previous extent. When you accidentally changed your viewport, or you just want to see the previous work in the different viewport, you can just click this button to go back.


Five mouse cursor mode icons are here- pan, zoom in, zoom out, lock and add a 2D peak. The last icon is delete a selected 2D peak. Those mouse cursor mode will take an effect when you click on the screen. For example, let say you are in zoom in mode. If you want to see a certain peak on the screen more in detail, click on the peak. Then the peak will be drawn bigger and centered. The lock mode requires clicking twice on different spectra. If you select two places in different tilt planes, the calculated 3D place will be circled in all visible tilt planes. This is useful when you want to decide 3D peak construction. If there is a peak and the peak is not linked to any of 3D peaks in the list (which means 3D peak index column is zero), you click on the peak with the mouse mode. Then, you will see yellow dotted lines on the spectra. The yellow dotted lines are on the same position in direct dimension. If you click on the suspicious linked 2D peak from the other spectra after that, ADAPT-NMR Enhancer will calculate 3D peak position using those two 2D peaks. The projected positions to 2D tilt planes from the constructed 3D peak will be rectangled with purple lines in every 2D tilt planes. By these repeats, you can complete lacking 3D peak construction from 2D tilt peaks.


First two buttons are select and deselect icons. The select button is another mouse cursor mode. If you are in that mode, you can drag a rectangle on a certain place to select a peak in the screen. You can either select a 2D peak and a constructed 3D peak linked to a 2D peak in the your dragged rectagle. Right-button-drag will bring you a 2D peak selection while left-button-drag will bring you a 3D peak selection. If you want to deselect, you can click the deselect icon or drag on the empty space in the screen. Third and fourth icons are adding a peak and deleting a peak. Adding a peak is another mouse cursor mode. If you click on the screen, a 2D tilt peak will be created. This function is useful when ADAPT-NMR initially could not pick a peak due to some reasons such as overlap, shoulder peak, etc. If a noise peak is picked by ADAPT-NMR, deleting a peak icon can be useful. The function is not a mouse cursor mode, and it deletes a peak which is currently selected.


First two buttons are related to linking functions. A 2D peak which is not linked to any of 3D peaks will has a value of zero at the last column in the 2D peak list, which means there no related constructed 3D peak in the list. However, if you could figure out the peak should came from a certain 3D peak, you can make it linked to the 3D peak by clicking the first button. Otherwise, a 2D peak which is miss-linked to some 3D peak can be unlinked, too.


ADAPT-NMR Enhancer offers two viewing methods. One is overlaped view, which shows all selected tilt planes in the same screen, and the separated view shows 6 tilt planes in divided screens. The overlapped view is good to see the linked 2D tilt peaks for the 3D peak quickly- how they should be located and picked. But other functions related to the actual peak modifications and validations will be done mostly in the separated view.


If Draw Contour is not checked, contoured level of spectra will not be drawn. All you will see are circled peaks. The contour requires 4x or more zoom in scale. The 2D peak index will be drawn if Draw IDs is checked. Likewise, the peak circle color will not always be white if you check By priorities. The color will be the priority indicating color same as in the 2D peak list.


The first icon is Assignment Table. If you click the button, you will see the dialog showing current assignment status.


Second icon is adding a horizontal line by a chemical shift value of 2nd indirect dimension(13C). This is useful especially when you are doing assignment by yourself in the ADAPT-NMR Enhancer because you might want to find matching C(i) to weave H(i)-N(i) and H(i-1)-N(i-1) pieces for assignment walking strategy.
Third icon is export. Many formats are supported by ADAPT-NMR Enhancer; XEASY Prot, PINE pre-assignment, PINE probabilistic output, TALOS+ input. The export dialog will require base threshold of probability. You can manually set up the minimal probability to include the confident assignments by ADAPT-NMR. Assignment made by user will basically be considered as 1.0 probability.
The last icon is for changing probability manually for the selected 3D peak. If you are very sure about the constructed peak which has low probability, you can change it by yourself.


These two icons are for contour levels. You can lower or higher the contour level for getting rid of noise peaks or showing weak peaks.This function works for individual tilt plane. So you need to select a 2D tilt plane from the plane list box at the left.


You can move from an experiment to the other experiment by changing in this drop down list box.


At the bottom, there is a status bar shows useful information. You can see current zoom scale, cursor position, intensity on the cursor, mouse cursor mode, peak count, and current ADAPT-NMR Enhancer status.

* Pine Assignment Dialog

Pine Assignment Dialog is a similar to the Pine Graph Assigner from PINE-SPARKY supporting atom-by-atom assignment. You can see the assignment and probability on the residues. The atom color indicates the probability visually. The legend is just same as PINE overall picture. Green: >99%, cyan: 85%-99%, yellow: 50%-85%, red: less than 50%, grey: no assignment.
If you click on the experiment, the visible 3D peaks predicted by using the assignment will be shown in the candidate list box. You can double-click on the one of the candidates, then ADAPT-NMR Enhancer tries to find the peak at the position and select. If there is no peak at the place, it will ask you to create the peak on the position.

* Probable Assignment Dialog

Probable Assignment Dialog is similar to the Pine Assigner from PINE-SPARKY supporting peak-by-peak assignment. When user selects a constructed 3D peak, this dialog automatically pops up. It shows what probable assignment candidates are available for this peak based on assignment table given by ADAPT-NMR. User can double click on a candidate from the candidate list to confirm it. Also, user can manually assign a peak if there is no proper candidate offered in the dialog. By clicking Confirm button, you can give 100% probability to the assignment. The confirmation is necesary to include your assignment to the exported file afterward.

* Keyboard shortcuts
[F1] Pan
[F2] Zoom in
[F3] Zoom out
[F4] Lock
[F5] Add 3D peak by current lock
[F6] Select by rect
[F7] Add a new peak
[F8] Link a selected peak
[F9] Unlink a selected peak
[Del] Delete an existing peak

[o] Open...
[s] Save...
[u] Undo All
[q] Exit program

[f] Zoom to full extent
[r] Redraw screen
[<] Zoom to previous extent

[^] Increase a priority level of a selected peak
[v] Decrease a priority level of a selected peak
[i] Increase color level.
[d] Decrease color level.
[e] Export assignmnets

// Not yet implemented
[+] Zoom in *2
[-] Zoom out /2
 

EXAMPLES

 In this page, a simple process to use ADAPT-NMR Enhancer on practical ADAPT-NMR outputs will be shown.

First of all, you launch ADAPT-NMR Enhancer, and it will bring up ADAPT-NMR Enhancer program on the screen.
# adapt_nmr_enhancer [ENTER]

Click Open button,

and you will see Open Dialog.


Click Browse button and set your working directory. Don't forget to set the spectrometer vendor you used to collect data by ADAPT-NMR.



When opening is done, set up viewing options for better investigation on quality of peak picking.
For example, this 121 degree tilt plane had a peak picked at a bit shifted place from the likely place. This happens frequently on the shoulder peaks or overlaps. You should focus on peaks with lower priorities. The peaks will be colored darker green in both on the screen and the peak list box.

To move it to the more reasonable place, use icons below.

 

Select a peak (1st icon), delete a peak (4th icon), and add a peak (3rd icon).


You can see a new peak on the different place from the previous.

Sometimes, you will find a totally noise peak that should be removed without moving.

At the center, dark green peak looks a noise. You can judge by using some other convenient tools in the ADAPT-NMR Enhancer to compare between tilt planes, and if you are sure that it is a noise,

delete a peak to help ADAPT-NMR reiteration.

You should do it throughout experiments. Change the experiment type.


Sometimes, you will find a place where needs a peak that didn't picked.


Add a peak. And if you are very sure about the peak, increase the priority to make it white circle. The line of the circle is now broken, because it is not linked to any 3D constructed peak. However, disregard it at this moment, because re-iteration of ADAPT-NMR will make new constructed peaks and links.


After this work, click Save button.


Then, run ADAPT-NMR.

# $ADAPT_NMR/exe/run_hifi_II.sh $ADAPT_NMR your_working_directory [ENTER]

And, wait for about an hour for ADAPT-NMR to recalculate everything. Then, re-open ADAPT-NMR Enhancer.
# adapt_nmr_enhancer [ENTER]
Click Open, and set working directory just like before.




After re-iteration, you will complete the assignment. First, you will look at Pine Assignment Dialog to find assignments with lower probabilities, or no assignments. If probability of the assignment is low, check the candidates for the atom. Double-click on the candidate will make a selection of the constructed 3D peak which is the nearest to the double-clicked candidate.


If there is no 3D peak matching with the candidate, ADAPT-NMR Enhancer will ask you whether a new peak on the place of candidates on the spectra.


If you choose yes, you can see a 3D peak is added at the bottom of 3D peak list. So, you can see the new peak on the spectra whether the peak makes sense or not.


If you are very sure that the new peak, or selected peak is fine and it came from the atoms, you can make an assignment in the Probable Assignment Dialog.


Double click on the candidate and click Confirm button will add the selected candidate into the Confirmation list.

If the peak is not from the candidate list, and you know the assignment, you can click Manual Assign button and make an assignment by yourself.

After finishing the assignment, click the export button.


And export the assignment in various formats. You can use the assignment to collect dihedral dstraints by TALOS+, or you can use these data in PINE-SPARKY by using PINE probabilistic outputs. If you have 13C-NOESY, 15N-NOESY data, and you collected sidechains with ADAPT-NMR, you can use XEASY prot to calculate protein 3D structure by using CYANA or PONDEROSA.

CITATIONS

Lee W, Bahrami A, Markley JL.
ADAPT-NMR Enhancer: complete package for reduced dimensionality in protein NMR spectroscopy.
Bioinformatics. 2013 Feb 15;29(4):515-7. doi: 10.1093/bioinformatics/bts692. Epub 2012 Dec 7.