Strukturrechnung mit CYANA (Dez 2018)

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 environment in Windows that allows us the access to the remote computer. To establish the connection to the server please follow the following steps:

• Copy the ZIP file in T:/Struktrurechnung/MobaXterm_v7.4.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 additional 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 program 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 choose 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 unassigned 15N-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).

The input 15N-resolved NOESY peak list for the 'cyana2' and 'cyana3' peak list is called 'n15.peaks'. This may be the same peak list as for 'cyana1' but CYANA will not use the assignments, only the peak positions and volumes.

Run the CYANA structure calculation

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

honshu> cyana
___________________________________________________________________

CYANA 3.98.5 (linux64-intel)
 
Copyright (c) 2002-18 Peter Guentert. All rights reserved.
___________________________________________________________________

    Demo license valid for specific sequences until 2019-12-31

    Library file "/home/guest/programs/cyana-3.98.5/lib/cyana.lib" read, 41 residue types.
    Sequence file "sequence.seq" read, 53 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

Look at the structure with a molecular viewer such as PyMol.

Structure calculation with automated NOESY peak assignment

The 'cyana1' structure calculation relies entirely on manual NOESY cross peak assignments. Alternatively, the NOESY cross peaks can be assigned automatically. In the structure calculations 'cyana2' and 'cyana3' you calculate a structure from partially or copmpletely unassigned input NOESY peak lists. The input data are :

sequence.seq

amino acid sequence

n15.peaks

peak list from 3D ¹³C-resolved NOESY spectrum

c13.peaks

peak list from 3D ¹⁵N-resolved NOESY spectrum (only in cyana3)

shifts.prot

¹H, ¹³C, and ¹⁵N chemical shift list

angles.aco

dihedral angle restraints

init.cya

initialization macro

AUTO.cya

macro for the structure calculation

Combined automated NOESY cross peak assignment and structure calculation are performed with the macro file AUTO.cya:

peaks       := n15.peaks,c13.peaks         # NOESY peak lists in XEASY format
prot        := shifts.prot                 # names of chemical shift lists
restraints  := angles.aco                  # additional (non-NOE) restraints
tolerance   := 0.040,0.030,0.45            # shift tolerances: H, H', C/N', C/N
#calibration_dref := 4.2
#calibration_constant:=6.7E5,8.2E5,8.0E4   # calibration constants, automatic if empty
structures  := 50,10                       # number of initial, final structures
steps       := 6000                        # number of torsion angle dynamics steps
randomseed  := 3206

noeassign peaks=$peaks prot=$prot autoaco  # perform NOESY assignment/structure calculation

First several variables are set: The variable peaks gives the names of the input peak lists, separated by commas without intervening blanks. The variable prot gives the name(s) of the input chemical shift list(s). If a single name is given as in the example, it specifies that a single chemical shift list file with this name will be used for all peak lists. Alternatively, it is possible to specify a separate chemical shift list for each peak list as a comma-separated list of file names. The variable restraints specifies the names of input files with additional conformational restraints that will be used together with the upper distance bounds that will be derived from the NOESY peaks. If there are several file names, they must be separated by commas without intervening blanks. The variable tolerance specifies the tolerances for the matching of chemical shifts. It is used for a consistency check of the peaks that have assignments in the input peak lists, and for the automated NOESY cross peak assignment. The calibration constants for the peak lists can be given by the variable calibration as a comma-separated list of values in the order of the peak list names given by the variable peaks. If the variable calibration is not given, the calibration parameters are determined automatically such that the median of the upper distance limits for each peak list equals the value of the variable calibration_dref. The variable calibration_dref can have a single value that applies to all peak lists, or separate values for each peak list. This variable is not used when the calibration constants are given explicitly by the variable calibration_constant.

Seven cycles of combined automated NOESY assignment and structure calculation are performed, followed by a final structure calculation. In each cycle and in the final structure calculation 100 conformers are calculated using the standard simulated annealing schedule with 10000 torsion angle dynamics steps per conformer. The 20 conformers with the lowest final target function values are analyzed. An overview table of these 20 best conformers is saved in the file final.ovw, and their coordinates are written to the PDB file final.pdb. The corresponding files from the intermediate cycles 1-7 are called cycle1.*, cycle2.*, etc.

An overview table of the complete calculation can be obtained with the command cyanatable (at the Unix prompt) during or after the completion of the calculation.