Strukturrechnung mit CYANA (2015)

Startup

Log in to the Windows system with the username and password of your HRZ account.

The structure calculation will be performed on a Linux server that can be accessed by a remote connection to our computer cluster. For the remote connection we use the program MobaXterm. This program provides a shell evironment in Windows that allows us the access to the remote computer. To etablish the connection to the server please follow the following steps:

• Copy the ZIP file in T:/Struktrurechnung/MobaXterm@honshu.zip to you personal folder U: and unpack it. (Keep in mind that only files in U: are kept after this day. All other files and directories are deleted automatically.)
• The folder contains two files, MobaXterm_Personal_7.4.exe (the program executable) and MobaXterm.ini an adittional file containing all passwords and data necessary to automaticall access the server
• Start the program by klicking the execuatble MobaXterm_Personal_7.4.exe.
• The program starts with a default shell console (black area). Additionally you see on the left side a field named "Saved session". There, please click on the preconfigured connection 141.2.222.9 to connect. A new tab should open showing the prompt "honshu>". This is the name of our server.

The programm shows the list of the files in the active directory on the left side. This enables you to open files in Windows by clicking them and in addition this allows you to copy files between Windows and the active directory on the server by drag and drop.

All students use the same account. As an first step, please create a directory for your group to work in. For that the following steps are necessary:

• Use the command 'mkdir' command to create a directory. Please chose the last name of one of the group members. Use only the standard letters A-Z and a-z. Every group name has to be unique. E.g. if your name is Müller, use 'Mueller' as the groupname, type

mkdir mueller

• Now change the directory using the command 'cd'. For our example:

cd mueller

The data for the practical is in the file Data.tgz in the home directory (/home/guest) on the server. (Alternatively, it can be downloaded from Data.tgz.)

In your group directory, unpack the data

tar zxf /home/guest/Data.tgz

Three subdirectories will be created:

• cyana1: structure calculation using only the assigned 15N-resolved NOESY peak list
• cyana2: structure calculation using the assigned 15N-resolved NOESY peak list and the unassigned 13C-resolved NOESY peak list
• cyana3: structure calculation using the unassigned 15N-resolved and 13C-resolved NOESY peak lists

Enter the corresponding subdirectory. For example

cd cyana1

and copy your data to this directory. For example, for the 'cyana1' calculation, copy your chemical shift list (shifts.prot) and your assigned peak list from the 15N-resolved NOESY spectrum (n15assigned.peaks).

Run the CYANA structure calculation

Start CYANA, and execute the CYANA script CALC.cya (in 'cyana1') or AUTO.cya (in 'cyana2' and 'cyana3'):

> cyana
___________________________________________________________________

CYANA 3.0 (intel)
 
Copyright (c) 2002-10 Peter Guntert. All rights reserved.
___________________________________________________________________

    Library file "/usr/local/soft/cyana-3.0/lib/cyana.lib" read, 38 residue types.
    Sequence file "sequence.seq" read, 114 residues.
cyana> CALC

Analyze the results of the structure calculation

The results of the structure calculation are the structural statistics in the overview file, cyana.ovw ('cyana1') or final.ovw ('cyana2' and 'cyana3'), and the structure itself, which is represented by a bundle of 10 conformers whose coordinates are stored in the PDB file, cyana.pdb ('cyana1') or final.pdb ('cyana2' and 'cyana3').

The overview file has at least three parts. The file starts with a table of the target function values and restraint violation statistics. For example:

Structural statistics:

str   target     upper limits    van der Waals   torsion angles
    function   #    rms   max   #    sum   max   #    rms   max
  1     1.69   2 0.0076  0.36   4    5.6  0.34   0 0.3302  3.23
  2     1.74   2 0.0077  0.36   5    5.9  0.34   0 0.3272  3.30
  3     1.75   1 0.0075  0.36   5    5.7  0.34   0 0.3695  3.45
  4     1.87   1 0.0075  0.37   7    6.3  0.34   0 0.3159  2.69
  5     1.95   1 0.0075  0.37   5    6.7  0.37   0 0.3185  3.00
  6     2.12   2 0.0084  0.36   6    6.6  0.34   0 0.3745  3.56
  7     2.19   2 0.0100  0.50   7    6.8  0.34   0 0.3257  3.29
  8     2.35   2 0.0096  0.36   8    7.0  0.34   0 0.3748  3.50
  9     2.40   2 0.0088  0.36   5    8.4  0.35   0 0.4152  3.44
 10     2.49   2 0.0090  0.36   9    7.6  0.33   0 0.3494  3.20

Ave     2.06   2 0.0084  0.38   6    6.7  0.34   0 0.3501  3.26
+/-     0.28   0 0.0009  0.04   2    0.8  0.01   0 0.0308  0.25
Min     1.69   1 0.0075  0.36   4    5.6  0.33   0 0.3159  2.69
Max     2.49   2 0.0100  0.50   9    8.4  0.37   0 0.4152  3.56
Cut                      0.20             0.20             5.00

This table has one row for each structure, containing

• the rank of the structure sorted by target function value
• the target function value
• three columns for each type of conformational restraints:
  • the number of restraints that are violated by more than the cutoff value given in the last row (“Cut”)
  • the root-mean-square (RMS) violation calculated over all, violated and fulfilled, restraints of this type
  • the maximal violation

The five bottom lines of the Table give the average value, the standard deviation, the minimum value, and the maximum value of the corresponding quantity over the individual structures, as well as the cutoff value for significant violations.

Restraints that are violated in a significant number of structures by more than the corresponding cutoff value are reported in the second part of the overview file:

Constraints violated in 3 or more structures:
                                               #   mean   max.  1   5   10
Upper QB    LEU   17 - QB    PRO  108   3.69   3   0.10   0.50  ++    *     peak 1009
Upper HB    ILE   85 - H     ASP   86   3.80  10   0.36   0.37  +++*++++++  peak 803
VdW   N     ILE   81 - HD2   PRO   82   2.45  10   0.34   0.37  ++++*+++++
VdW   CG2   ILE   81 - C     ILE   81   2.90   6   0.20   0.21   + + ++* +
2 violated distance restraints.
0 violated angle restraints.

Each line identifies a violated restraint, and gives the number of structures in which the restraint is violated by more than the aforementioned cutoff value (column labeled “#”), its maximal violation (column “max.”), and the structures in which the violations occur (a one-character column for each structure that is analyzed). Structures in which the restraint is violated by more than the cutoff are marked with “+”, or with a “*” for the structure in which the maximal violation occurs. If available, the number of the cross peak from which the restraint originated is given at the end of the line.

At the end of the overview file, root-mean-square deviation (RMSD) values for the atom positions after optimal superposition of the individual conformers onto the mean coordinates are given:

RMSDs for residues 10..100:
Average backbone RMSD to mean   :    0.58 +/- 0.11 A (0.45..0.82 A; 10 structures)
Average heavy atom RMSD to mean :    1.08 +/- 0.11 A (0.93..1.25 A; 10 structures)

The residue range used for the superposition is indicated. RMSD values are computed for the backbone and heavy atoms of the given residues. The average value, the standard deviation, and the minimal and maximal values of the RMSDs between the analyzed structures and their mean coordinates are calculated.

Visualize the structure

The program MOLMOL can visualize the bundle of conformers that represents the solution structure of the peptide with the command

molmol -r 7-37 cyana.pdb

to start the program MOLMOL and to show a superposition of the 10 conformers whose coordinates are stored in the PDB file cyana.pdb. The option "-r 7-37" indicates MOLMOL to optimally superimpose the backbone atoms of residues 8-21.

Alternatively, other molecular viewers such as 'pymol' can be used.