Praktikum Biophysikalische Chemie

Download and install the course material

Store the file PBCPraktikumCyana.tgz in your home directory and execute the setup command:

 tar zxf PBCPraktikumCyana.tgz
 setup_cyana

Make a new directory for the structure calculation, and change into it:

 mkdir vaso
 cd vaso

Write the sequence file

Use a text editor to write a new file called vaso.seq that contains the peptide sequence, one upper-case residue name per line, given in the standard three-letter code for amino acids (except for cysteine residues that are involved in a disulfide bond, which are denoted by "CYSS"), e.g.

 CYSS
 TYR
 PHE
 GLN
 ASN
 CYSS
 PRO
 ARG
 GLY

Write the initialization script

Use a text editor to write a new initialization script, init.cya, for the program CYANA with the following content:

 cyanalib
 read seq vaso.seq 

These two commands will be executed automatically whenever the program CYANA is started. The cyanalib command reads the standard residue library of CYANA, and the command read seq vaso.seq reads the polypeptide sequence.

Write the NOE distance restraint file

Use a text editor to write a new file, vaso.upl, that contains the upper distance bounds derived from NOESY cross peaks, using the standard CYANA nomenclature for atoms in proteins and the same format as in the following example:

91 THR  HB     93 GLN  QB      5.50
80 SER  HB2    81 ILE  H       4.22
80 SER  HB3    81 ILE  H       4.22
81 ILE  HA     84 LEU  H       4.01
81 ILE  HA     84 LEU  HB2     4.47
81 ILE  HA     81 ILE  QG2     3.46
81 ILE  HA     81 ILE  HG12    3.77
28 VAL  HA     39 LEU  HG      3.97
52 SER  H      52 SER  HB2     3.96
52 SER  H      52 SER  HB3     3.96
99 SER  QB    101 VAL  H       5.50
43 SER  H      43 SER  QB      3.12
43 SER  QB     48 GLU  H       4.07
42 GLU  HA     43 SER  QB      5.50
43 SER  QB     48 GLU  HB2     3.95

Each line specifies an upper bound on the distance between two hydrogen atoms. The data in the 7 columns are:

1. First residue number
2. First residue name
3. First atom name
4. Second residue number
5. Second residue name
6. Second atom name
7. Upper distance bound in Å

Residue and atom names are given in upper case letters. The exact number of spaces between different items is irrelevant, but the "TAB" key should not be used.

Note that the backbone amide hydrogen is called "H", not "HN".

Degenerate groups of atoms, e.g. methyl groups, and diastereotopic pairs of hydrogen atoms, e.g. HB2/HB3 in serine, are referred to by "pseudoatoms" whose names are derived from the names of the hydrogen atoms that they represent by changing the first letter from "H" to "Q" and omitting the last digit. For instance, "HB2" and "HB3" are represented by a pseudoatom called "QB".

Write the CYANA script to execute the structure calculation

Use a text editor to write a new CYANA script, CALC.cya, with the following content:

 read upl vaso.upl
 ssbond 1-6
 calc_all 50 steps=3000
 overview vaso.ovw structures=10 pdb

The read upl command reads the input file with upper distance limits, vaso.upl.

The ssbond command adds restraints for the disulfide bond between residues Cys1 and Cys6.

The calc_all command performs a structure calculation starting from 50 conformers with random torsion angle values. Simulated annealing with 3000 torsion angle dynamics steps per conformer is used.

The overview command sorts the resulting structures by ascending target function value, analyzes the 10 best conformers for violations of the conformational restraints, and saves the results of the analysis in an overview file, vaso.ovw, and the coordinates of the 10 best conformers in a PDB file, vaso.pdb.

Run the CYANA structure calculation

Start CYANA, and execute the CYANA script CALC.cya:

 > cyana
 ___________________________________________________________________

 CYANA 3.0 (intel)
  
 Copyright (c) 2002-08 Peter Guntert. All rights reserved.
 ___________________________________________________________________

     Library file "/usr/local/soft/cyana-3.0/lib/cyana.lib" read, 38 residue types.
     Sequence file "demo.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, vaso.ovw, and the structure itself, which is represented by a bundle of 10 conformers whose coordinates are stored in the PDB file, vaso.pdb.

   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

   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     LEU   39 - CD1   LEU   39   3.05  10   0.23   0.24  ++++*+++++
   VdW   N     ILE   81 - CD    PRO   82   3.05  10   0.26   0.29  ++++*+++++
   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   + + ++* +
   VdW   CB    THR   91 - H     GLN   92   2.55   7   0.22   0.28  ++*+ +++
   VdW   O     THR   91 - CB    GLN   92   2.90   3   0.16   0.20     *+   +
   VdW   HA    VAL  107 - CD    PRO  108   2.60   5   0.19   0.27     +* ++ +
   2 violated distance restraints.
   0 violated angle restraints.

   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)