Solid-state NMR Course - Part Two

6. Load a More Complete Project

In the second part of the course we move on from a starting project with few resonance assignment to examples with near complete assignments. Firstly, with a project populated with data we will illustrate how to access various charts and tables that illustrate the assignment status of the project. With his in mind we will make some strip plots at various positions in the sequence, which is an ideal way of presenting the evidence for assignments. Then we look at how knowledge of chemical shifts can allow us to make synthetic peak lists, which allows for the efficient interpretation of some spectra. Lastly, we will look at using through space-peak lists and chemical shifts to make restraints that may be used in a structure calculation.

If CcpNmr Analysis is not already open, start it up by double clicking its icon or on the command line by typing:

-> analysis

Then from the main menu select M:Project:Open Project and choose CcpnCourseMas2a as the CCPN project to load.

Resonance and Chemical Shift Tables

Once the project loads, we will have a look at some of the ways that you can see the assignment status of the project. Firstly look in the M:Resonance:Resonances table, where you will see a list of all the resonances in the project, and their associated chemical shift values. Look at the 'All Peaks' column and note that some resonances are assigned to several peaks, and others are assigned to none; the latter is because not all of the spectra have been included in this project. For one of the resonances associated with several peaks, e.g. number 223 - 269Gly Ca, click on the resonance row and then press [Info], under the table to the right. Resize the resulting Resonance: resonance Info table so you can see all its contents. This popup window illustrates how the chemical shift for the resonances is calculated, as an average of all the peak positions (in a given dimension) listed in the Peak Assignments table. Note that by default all spectrum dimensions contribute equally to calculate the chemical shift average, but this may be adjusted at M:Experiment:Spectra {Tolerances} - adjusting the Shift Weighting column.

Another way to look at this kind of information is at M:Resonance:Spin Systems where resonances are displayed in groups that relate to a residue (or residue fragment). In this popup go to the {Shifts} tab and you will see the chemical shift values that go with each of these groups. There is a much prettier way of looking at the same chemical shifts, i.e. for printing and making figures, at M:Chart:Chemical Shifts Table. Open this popup and toggle the [CB] button at the to on to see how particular atom types can have their shifts listed in neat columns. You can export this display as a PostScript file for printing or manipulation in a graphics program; click [Export PostScript] and set an appropriate file name, e.g. ChemShifts.ps.

Assignment Status and Quality Reports

The chemical shift and resonance tables naturally give you an overview of what is assigned in a CCPN project, but there are a couple more informative displays that you can look at.

The chart at M:Chart:Assignment Graph gives a graphical overview of the assignments in the context of atom sites in the molecule. Note how the black circles indicate atoms sites that have been assigned to resonances. Go to the {Options} tab and click on some of the Spectra buttons and then return to the {Assignment & Connections Chart}. For each of the selected spectra you will see lines connecting different atom sites; these connections result from the peak assignments that are made, linking the resonances. Return to {Options} and enlarge the table if you cannot see the Isotope Labelling panel, then set Pattern in the lower panel to 13Glycerol. Now in the {Assignment & Connections Chart} tab you will see colour coding indicating which atoms sites are expected in the selected labelling scheme, although for these spectra the sample is actually uniformly labelled.

The option M:Assignment:Quality Reports will show you a stable of statistics relating to the number of assignments generally and the number of through-space connections. Also note that the {Resonances} and {Peaks} tabs in this popup are specially designed to illustrate where errors might be in the project. These tables look at the same kind of issues, but one presents the information from a resonance point of view, and the other from a peak point of view. Most notably the assignments are checked for the following criteria:

• • An assignment to a given atom is duplicated.

  • The standard deviation for a chemical shift is large.

  • If a peak position is far from the chemical shift average (irrespective of a shift's deviation).

  • A resonance has an impossible number of covalently bound partners: e.g. an amide N should only have a 'onebond' relationship to one CA resonance.

  • The chemical shift for an atom assignment is unlikely given the known chemical shift distributions (BMRB).

  • If peaks have unusual intensities or sign.

When something is potentially wrong, then cells of the tables are coloured red. A highlighted cell doesn't mean that something is definitely wrong, just that it warrants an explanation or further investigation. To find the peaks responsible for any aberration, simply click on the row in the table and click [Show Peaks]. In the resulting table you can assign or find any of the peaks in the various spectrum windows.

7. Making Strip Plots

In this section we will look at how to display particular assignments as strip plots within the spectrum windows. This is especially handy to generate an overview of connectivity within a given spin system or between sequential residues in the protein (or nucleotide) sequence.

There are several ways of making strip plots in Analysis, including doing things manually which can be somewhat tedious. Here we illustrate building strips based on peak positions and based on resonance positions. Using peaks is perhaps easier, but is more limited because you can only make strips for the peaks you have. With resonances getting the correct intersections of ppm locations is more fiddly, but you have more control and can go to any combined resonance based locations.

Note that spectrum window strips in Analysis can either be vertical (typical for solution NMR) or horizontal (typical for solid state). For this example we have pre-set the windows to make horizontal strips, but if this is not already set the strip direction can be toggled between the two orientations; in the [Strips] section of a window using the button to the right of the green arrows. - For horizontal strips these arrows will be up/down and likewise for vertical strips they will be left/right.

Making Peak-based Strips

To make strip plots based on peak positions you first need to go to the peak list table. Select M:Peak:Peak Lists and select the {Peak Table} tab. Set the Peak List in the top pulldown menu to CANCO:1:1. Next we will put the peaks in the table into sequence order, so for the Assign F2 column (15N) click on the column heading so that a small triangle pointing down appears (clicking again on the heading will sort in the opposite direction). Now scroll down to the row for 262AsnC, 263SerN, 263SerCa which corresponds to peak number 36. Then hold the <Shift> key and click on the row four below corresponding to 267ValN. This operation should highlight five rows for C-N-CA peaks in five sequential residues.

To make the strip plots first set the Window pulldown menu toward the top right to "window3". Then click [Strip Selected], which is also above the table. If window3 is not visible select it from the M:Window menu. In window3 go to [Spectra] and make sure that the CANCO and NCACX spectra are both visible. You may need to zoom in/out to an appropriate level, but these strips should show the N-CA strip locations for the selected peaks in the carbonyl region of the spectra (X axis). Hopefully it is clear from the name of the experiments that the CANCO spectrum shows peaks for the carbonyl of the residue which is before the backbone nitrogen (i.e. crossing the peptide bond) and the NCACX spectrum shows peaks for the carbonyl that is in the same residue as the nitrogen. Lining these locations up as strips allows us to illustrate the "backbone walk" between the peaks as their assignments go from one residue to the next, i.e. from a given blue CANCO peak the same N-CA strip location shows a red NCACX peak which matches the carbonyl ppm of the CANCO peak for the next residue in the strip immediately below.

Making Printable Spectra and Strip Plot PostScript

Keeping these strips in window3 we will now look at making printable PDF or PostScript files from the spectrum view in Analysis. Accordingly go to M:Window:Print Window and set the Window pulldown at the top to window3. Set the File entry line to "StripsExample.pdf" (or similar). Set both the Tick Font and Using Font to Helvetica 7, the Format to be PDF and leave all other settings at their default values. Click on the {spectra} tab and make sure that only the NCACX and CANCO contours are drawn (select "Use below settings when printing" and double click to toggle in the table). Also look in the {Peak Lists} and {Region} tabs to see that you can have fine control over which peaks and regions are printed. In the {Peak Lists} tab select "Use below settings when printing" and change the Peak Font column for the displayed spectra to Helvetica 7. With all settings adjusted click the green [Save Print File] at the top to make the PDF document. Use a PDF viewer program to check that the file was made and that it looks sensible.

Making Resonance-based Strips

To make strips based on resonance locations, first open the M:Resonance:Resonances table. In this example we will make carbonyl based strips. To help do this we will filter the table to only display the carbonyl rows. With the mouse over the table right click and select R:Filter. In the resulting Filter Options popup set the Filter column to Assign Name, check the Regular expression option and in the entry line type "C$" (the dollar sign means end of line and will exclude matching "Ca" etc.) then click [Filter Include]. Hopefully the resonance table will now only show carbonyl rows.

Click on the Residue column of the Resonance Table to put the rows into sequence order. With the <Shift> key held down click to select residues 262 to 267 (same as before). In the upper Navigation & Marks section set the Window pulldown menu to window5 and finally click [Strip Selected]. In window5 toggle the green NCOCX spectrum on (others can be off) and move the X axis, if you are not already looking at the aliphatic carbon region ('CX') beween about  70 and 20 ppm. Hopefully you will see the aliphatic carbon peaks in N-C strips for the selected sequential residues.

8. Making and Using Synthetic Peak Lists

For this last section we will be using a CCPN project that once again uses data from the alpha-spectrin SH3 domain.  If CcpNmr Analysis is not already open, start it up on the command line by typing:

-> analysis

from the main menu select M:Project:Open Project.  Navigate to find and select the CcpnCourseMas2b project, then click [Open].

You might get a warning that various files have moved location. You might also get a dialog with a list of spectra paths (because those also have moved location). If the paths are all in grey then just click the [All Done!] button at the bottom. If any path is in red then Analysis cannot find the corresponding spectrum data file, so either you need to tell Analysis where it is (by double clicking the path cell and navigating to the correct location) or accept that that particular spectrum will not have its contours displayed. When the project data is loaded select M:Window:CC PDSD and 1,3-13C glycerol labelled (blue) and 2-13C glycerol labelled (red) spectra will hopefully appear. These spectra have been recorded with a long mixing time and many of the peaks have been picked (but not assigned). We can use the crosspeak information to generate 13C-13C distance restraints, which we will cover in the next section. This CCPN project also carries assigned chemical shift information, although the spectra used in this assignment have been removed. We will use these assignments to look at how we can make synthetic peak lists, i.e. predict where peaks will occur, considering chemical shift values, isotope labelling  and experiment type.

Making Labelling Aware 13C-13C peak Lists

Firstly we will make some peak predictions for connections between adjacent residues. This will account for a significant proportion of the signals in the PDSD spectrum. This prediction will naturally need to consider any isotope labelling pattern in the sample for which the spectrum is recorded. This labelling information is already setup for this project, in the manner described in the first part of the tutorial.

To demonstrate the procedure we will make a synthetic peak list for the PDSD500_2C:sh3 spectrum, i.e. the one with 2-13C glycerol labelling. In the PDSD window toggle off all the spectra in the [Spectra] section, except for PDSD500_2C, so that we can more clearly see what is happening. To make the synthetic peak lists go to M:Peak:PeakLists and select the {Synthetic Lists} tab. Here you will see that there are four different ways of making/predicting synthetic peak lists. These options will make peak position (not intensity) predictions using the respective methods:

• • Positions at assigned resonance intersections considering the experiment type and limiting through-space transfers to a given number of sequential residues and/or bonds.

  • Positions at assigned resonance intersections where atoms connected by a through-space transfer are within a given distance in a 3D structure (or model), and also considering the experiment type for other magnetisation/coherence transfers.

  • Reciprocal positions where peaks in an experiment that has two axes of the same isotope type. This is equivalent to flipping peaks to the opposite side of a homonuclear diagonal line.

  • Estimating through-space peak locations given the pairs of close atoms that are listed in a distance restraint list and the chemical shift values of the atoms' resonances.

For this exercise we will demonstrate the first two methods.

In the top "From Shift Intersections" panel set the Spectrum pulldown to be PDSD500_2C:sh3, set Min Isotope Fraction to be 0.1 and the Through-space bond limit to 6. Leave all other settings at the default values. Then simply press the large green [Predict from Shifts] to the left and wait for the process to complete, which may take a few minutes to make about 1000 peaks.

Looking at the PDSD window you should see some new, fully assigned peaks appear on the spectrum. Look at these new peaks in isolation by turning the original, unassigned peak list off; do this by pressing [Peaks] in the window and toggle the first red button. See how the predicted locations can explain many, but not all, of the signals in the 2-13C glycerol labelled PDSD spectrum. Some signals are missing in the spectrum, but this is to be expected given imperfect spectra, imperfect labelling predictions, diagonal artifacts and the structural conformation. Despite these limitations look at the group of ValCb-Ca peaks around 35, 60 ppm and see how the predictions can help to explain a large mass of signals, even when they are overlapped.

Assigning Ambiguous Peaks Using Synthetic Peak Information

So that we can distinguish peak lists more clearly in the M:Peak:Peak Lists popup go to the {Peak Lists} tab and set the colour for the new, synthetic peak list to be blue (or perhaps skyblue). Then back in the PDSD window toggle on the first peak list in the top [Peaks] section. You can copy any assignments you wish from the predicted shift list to the 'real' picked peaklist using the M:Assignment:Copy Assignments system described in the Analysis beginners' course. Alternatively you can assign the black peak list in the usual way; press the <a> key over the peak. Do this for the black peak at 33.7, 59.2 ppm, which is near two predicted peaks. The locations of the predictions suggest that the black peak maximum may actually be from two superimposed signals, thus assign the peak ambiguously; select 22GluCb & 53ValCb for the first dimension and 23ValCa & 52PheCa for the second dimension.

Ambiguous peak of this kind provide useful information for structure calculations; a program like ARIA or CYANA will allocate different weights to the assignment possibilities when using ambiguous distance restraints. To help clarify the ambiguity of the situation we can specify how to pair up the ambiguous assignment possibilities, i.e. 22GluCb goes with 23ValCa and 53ValCb goes with 52PheCa, as suggested by the synthetic peaks (and the fact that these are connections to adjacent residues), so that we don't get any improper combinations. To specify the resonance pairing, in the Assignment:Assignment Panel check Assignment Groups so that the "G" columns (which shows group numbers) appear to the left. Then for a pair of correlated resonances, e.g. 53ValCb and 52PheCa, double click in the "G" column and select <New> for the first resonance, which will cause the number 2 to appear, and then "2" for the second resonance of the pair (i.e. in the other peak dimension). This pair of resonances is now in group 2 and thus distinct from group 1, so the two will not get mixed up when making distance restraints etc.

Note that when dealing with multiple peak lists, only the active peak list can be selected and assigned in a spectrum window. The active peak list for a spectrum is simply set in the M:Peak:Peak Lists {Peak Lists} table.

Making Synthetic Peaks Using Structure Information

Next we will look at how we can make better synthetic peak lists for this kind of spectrum if we have 3D structure information, albeit from crystallography, NMR or modelling. For the following part we will be working with the PDB structure entry 1U06 which relates to the following reference:

• • Detection of dynamic water molecules in a microcrystalline sample of the SH3 domain ofalpha-spectrin by MAS solid-state NMR. Chevelkov, V., Faelber, K.  Diehl, A.  Heinemann, U.  Oschkinat, H.  Reif, B. Journal: (2005) J. Biomol. Nmr 31: 295-310

Load the coordinates of the PDB structure by going to M:Structure:Structures. And in the first {Ensembles} tab click [Import]. Select the file 1U06.pdb (which can be downloaded at http://www.rcsb.org/pdb/explore/explore.do?structureId=1U06) and click [Ok]. A new row should now appear in the Ensembles table. This structure only contains one coordinate model, but this system can load a file with multiple multiple models in a structure ensemble in the same way. To see the structure in a 3-dimensional representation for whichever is selected in the table click the [Viewer] at the top.

The controls for the structural viewer are as follows:

• • Rotate with middle-click & drag.

  • Zoom with the mouse wheel, or middle-click, <Shift> & drag.

  • Move with middle-click, <Ctrl> & drag.

The mouse right-click brings up a menu that allows you to change the display mode, spin, and print the structure. The left-click is used for atom selection. Try the atom selection by first ensuring that the PDSD500_2C:sh3 peak list is selected at the top, click on the 23ValCA atom on the structure vie (i.e. the grey atom nearest to the 23Val label) and then click [Show Peaks]. This will show a table containing all of the peaks that relate to connections from the selected atom in the structure as well as display those connections in the 3D structure view. The numbers on the dashed lines represent the distances between the atoms. This should show the peak we assigned above (and is actually the only assignment so far for the peak list)

The same sort of functionality is present in the assignment popup (M:Assignment:Assignment Panel). - If you look at the  PDSD500_2C:sh3 spectrum in PDSD, for the ambiguously assigned peak (by pressing <a> with the cursor over a peak), you can see assignment possibilities via the [Show On Structure] button. Also note that because we now have a structure the Assignment Panel will show distances between one peak assigned 13C resonance and the 13C possibilities in another peak dimension (choosing the closest distance in case of ambiguity).

Next we will make a synthetic peak list using this structure as a guide, together with the chemical shift and labelling pattern data. Hence go to: M:Peak:PeakLists and select the {Synthetic Lists} tab once again. In the From Shifts and Structure panel set the Spectrum to PDSD500_2C:sh3, the Min Isotope Fraction to  0.1 and the Max Dist to 6.5. Then press the big blue [Predict from Structure] button to the left. Once the calculation has finished you will see a red synthetic peak list displayed on the spectrum and the predicted peak locations listed in the peak table. Observe how the red peak list explains much more of the PDSD spectrum than the blue peak lists, which do not use the structure information. As before we can use these predictions to give a good guide for assigning the real peak list, although we should naturally be cautious if the structure, or parts of the structure, may be inaccurate, e.g. from modelling or a preliminary NMR structure calculation.

EXERCISE: Repeat the synthetic peak list prediction using a structure and a shorter distance threshold (equivalent to using a shorter PDSD mixing time) or repeat for the 1,3-13C glycerol labelling pattern (overriding the automated, sample based default) to see the effect of the isotope labelling.

9. Setup for Structure Calculations

The next part of the exercise is to look at how we can use a CCPN project to get peaks lists and restraints (both dihedral and distance) that can be used in an ARIA (or CYANA) structure calculation, and how we can use structural information within Analysis to help with violation analysis and NOE peak assignment.

To make a list of distance restraints from the PDSD peak lists first go to M:Structure:Make Distance Restraints. At the top of the popup, change the peak list to PDSD500_13Cali:sh3:3 and set the Restraint Set pulldown to "4" . Note how that the Isotope Labelling pulldown menu is set to "Automatic from sample", which is exactly what we want in this situation. The Restraint Distance Params section allows us to specify how the NOE peak intensities relate to the distance bounds of any generated distance restraints. In this case we will be using fixed distance bonds for our data, in which case change the Distance function pulldown to "Distance bins" - and the settings for this will already be setup.

Note that the default method (i.e. common for solution NMR) is to calculate a target distance as peak volume raised to the power of -1/6 multiplied by some scaling factor, such that the reference intensity (which defaults to the peak list's average volume) exactly corresponds to the reference distance (in this case 3.2 Angstroms). The upper and lower bounds of the distance restraint are calculated as fractional changes from the calculated target distance (default is 20% above and below) while observing absolute minimum and maximum values for the bounds (1.72 & 8.00 Angstroms respectively by default). The {Residue Ranges} and {Chem Shift Ranges} tabs would allow you to make only restraints for specific assigned regions of your molecule or for specific shift ranges.

Making Distance Restraints From Through-Space Peaks

A common way to generate distance restraints is to match the chemical shifts of resonances to though-space peak positions, thus generating potentially highly ambiguous distance restraints. Naturally, if we had a more refined, assigned peak list with structure information then we could used that too.

Shift-matched restraints, with significant ambiguity, would typically be used as input for an iterative structure generation program like ARIA, where they would eventually be filtered to select only the correct contributing resonance pairs. Firstly, we could leave the matching of chemical shifts to the ARIA program by sending the program peak lists rather than restraint lists. However, it is also possible to make such restraints in CCPN. Accordingly, the {Shift Match Tolerances} and {Network Anchoring} tabs in the M:Structure:Make Distance Restraints popup allow you to generate such distance restraints for peaks which do not have assignments. To generate distance restraints by shift matching for the PDSD500_13Cali:sh3:3 peak list click [Make Shift Match Restraints].

In the case of the shift-matching method potentially ambiguous distance restraints are generated by matching peak positions to all close chemical shifts (within a tolerance). These matches can then be further refined by excluding any atom pairs that do not have sufficient spin active label, considering the 1,3-13C glycerol labelling scheme that was used for this sample. In the case of network anchoring method, chemical shifts are also matched to peaks, but the ambiguous possibilities are refined by selecting only through-space assignments from amongst the possibilities that are supported by other, assigned peaks or covalent structure.

The restraints popup will appear and in its table you will see the restraints listed. Each restraint has one green-coloured row. Note some restraints also have following grey rows. These grey rows are alternative distance pairs for restraints that are ambiguous, i.e. a possible connection between two different pairs of 13C resonances. Note that such ambiguous restraints can represent logical uncertainty (before ambiguity is resolved) or real physical ambiguity where a peak is caused by two or more overlapping pairs of resonances.

After a short pause you will see the Restraints and Violations popup appear. This shows that you have one restraint set (a way of grouping related restraints and violations) containing several restraint lists. One of these lists is the one we just made and the others were already in the project. Click on the row of the restraint list in the central table and then click on the {Restraints} tab. Note that you can also get to this point via the M:Structure:Restraints and Violations option.

EXERCISE: If you have spare moment try generating distance restraints for the PDSD500_13Cali:sh3:3 peak list with the  Isotope Labelling set to <None>, but do not use them for the structure calculation later. Note how the amount of ambiguity is somewhat larger than the one obtained by sensibly employing the1,3-13C glycerol labelling pattern.

Making Dihedral Restraints from Chemical Shifts

Next we will generate structural restraints in a different manner; dihedral restraints from backbone chemical shifts. We will be using a program called DANGLE (Dihedral ANgles from Global Likelihood Estimates) which is embedded within Analysis. DANGLE estimates dihedral angles from chemical shifts in a similar manner to TALOS; i.e. it matches a chemical shift & sequence query to a structural database of known PHI/PSI angles and chemical shifts. However, DANGLE uses a different (Bayesian) method to produce an angle estimate and tolerance, compared to TALOS. The idea is to use Bayesian inference to infer what the range of likely PHI/PSI angles might be (using the chemical shifts) by checking all PHI/PSI combinations in 10 degree square bins to see how well such angles can be used to explain the data. Such an analysis allows for the user to see uncertainties in the angle predictions, including where the chemical shift to structure mapping is redundant and there are multiple regions in the Ramachandran plot which could explain the chemical shift data.

To run DANGLE select M:Structure:DANGLE: Predict Dihedrals. Note that at the top that the Chain should already be set as "MS1:A", the Shift List as "ShiftList 1:1" and Max No. of Islands as 2. This last setting simply specifies which data to use and how strict the analysis should be. Using two islands means that we will reject predictions that result in more than two discrete regions of the Ramachandran plot. To start the analysis press [Run Prediction] and accept "Run1" as the name for the job by pressing [OK] at the opportune moment. Please be aware that DANGLE will take several minutes to finish the calculation.

Once the calculation is over you will see the main table filled in with PHI and PSI backbone dihedral angle predictions and their associated error ranges. Further, if you select a row in the main table you will see a plot in Ramachandran (PHI/PSI) space of where the likely angles are deemed to be. Click on the "40 Asp" row and note that there is red colour in different regions of the chart, indicating that DANGLE was not able to make a distinct choice of PHI/PSI: you should not use such a prediction in a structure calculation. Click on the [Next] button to get to "41 Trp". The prediction for this residue is somewhat better, and you could use this in a structure calculation (it has one discrete region) although the error bounds for such a dihedral restraint would be suitably large. Click on the  "27 Lys" row. This residue has a very precise range of predicted PHI/PSI angles. Such a residue could be used in a structure calculation with a high degree of confidence and proportionately narrow error margins.

Note that DANGLE also predicts the secondary structure of the residues, but that this calculation is not made from the angles, but directly from the measured secondary structures in the shift-structure database. To make the restraints themselves set the Restraint Set to "4", which will place the PHI/PSI dihedral restraints with our existing distance restraints and press [Commit Constraints].  View the generated restraints by going to M:Structure:Restraints & Violations:{Restraints}. Note that if you have a structural model for your protein you can see how the model's angles match with the DANGLE prediction.

Starting an ARIA Calculation with CCPN Grid

If for any reason you are not confident about the state of your CCPN project at this stage of the demonstration before we do a structure calculation you may like to open a pre-prepared CCPN project, which has all of the expected restraints present: In the Analysis menu bar select M:Project:Open Project then [Yes] to close the current project and [No] to not save.  Navigate to find and select the CcpnCourseMas2c project, then click [Open].

To start the structure calculation using the restraints we have created we will launch a panel that is dedicated to setting up an ARIA job, which will be run remotely on the CcpnGrid service. The ARIA Setup panel is available at M:Structure:ARIA: Structure Calculation. This allows us to control which data goes to the ARIA calculation from the CCPN project. For more fine-grained control you need to use the ARIA GUI; for example to change the annealing protocol.

Create a new job for ARIA to work on by pressing the green "New Run" button. This "Run" object links together all the data that goes into the same calculation. Ensuring that the {Input Data} tab is selected (they ought to be by default) select the inner {Restraint Lists} tab and add restraint lists to the ARIA run: Select the list name from the pulldown menu and click [Add Restraint List]. Do this for all of the restraint lists, i.e. the four distance and dihedral restraint lists. Now that all of the input data is set we have to tell ARIA how to run on the data.

Move to the {Run Settings} tab, found at the top. Here you can see some of the settings to control the ARIA run. In the lower table make sure that the "Ambiguous protocol?" column set to  "No" for the NCOCX and NCACX derived distance restraint lists. If we wanted to run the ARIA structure calculation locally, noting you will have to have a CNS executable and ARIA itself available, we can [Launch ARIA GUI], make relevant changes, save the ARIA project and then [Setup Project], which will put the ARIA data in a state that it can be run from the command line in the usual way.

For this demonstration you may a the ARIA calculation remotely using a test account on the prototype CCPN Grid service. Note that if you are doing this practical as part of a large class only the demonstrator should submit the calculation; at present only one person can use a CCPN Grid account at a time from Analysis. Under normal circumstances you would use you own personal account to do calculations; the CCPN team will be happy to give academic and non-profit users a login.

To start a calculation first create a compressed archive of the CCPN project at M:Project:Archive and press [Save]. Next the CCPN project archive is uploaded at  http://webapps.ccpn.ac.uk/ccpngrid/; select Submit a Project at the left of the web page and, to try the demonstration yourself use the user identification ''test" and the password "test123". The File is set to the name of the CCPN project CcpnCourseMas2<id>.tgz archive and an identifying Title. For this demo data select the option to use the log-harmonic potential and [Submit]. We will not wait for the final structure calculation, which will take some time (although this protein will take less than an hour on an unloaded server), but instead we will look at a structure which has been deposited in the PDB.

Via a web browser go to the page http://webapps.ccpn.ac.uk/ccpngrid/status: if you do start a grid calculation this is the page that you get to via the [Show web page] button. If asked, log on with the same username and password as before. Here you will see various ARIA calculations, one of which will have status "Finished" and be entitled ''CcpnCourseMas...". Click on the [Results] button for this job and look at the available data. Note that we have the option to download an updated CCPN project which contains the newly calculated structures and a violation analysis, all of which remained linked to the NMR data in the CCPN project; so that for example we can easily jump from a violation to the offending point in the spectrum.