Praktikum Biophysikalische Chemie

Startup

Log in to the Windows XP 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 clicking the icon labeled BCC Linux blade41.nxs. The username and password for the Linux server are the same as for the Windows XP computer, and the data in the home directories are shared between the two systems.

In a terminal window, start the bash shell and execute the setup command

bash
. /usr/local/courseexchange/cyana_setup.sh

Create a new directory, vaso, 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 
library rename H atom=HN
rmsdrange:=1-6

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. The command library rename H atom=HN changes the name of the backbone hydrogen atom from "H" to "HN". The variable rmsdrange is set to the preferred residue range for RMSD calculation.

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.

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.

The overview file, vaso.ovw, has 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. Use the command

molmol -r 1-6 vaso.pdb

to start the program MOLMOL and to show a superposition of the 10 conformers whose coordinates are stored in the PDB file vaso.pdb. The option "-r 1-6" indicates MOLMOL to optimally superimpose the backbone atoms of residues 1-6.

Printing

A local printer can be accessed with the name bpc_lokal from Linux, or BPC HP Laser Jet 4250 PS from Windows XP.