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:
- First residue number
- First residue name
- First atom name
- Second residue number
- Second residue name
- Second atom name
- 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.