Ornithine Cyclic Peptide

Creating cyclic peptides with Ornithine in CYANA
By Dan M.

aib_pic

TABLE OF CONTENTS

Introduction
Build the Initial structure
Starting sequence file
NOE tables/formats
Appendix

Introduction

This tutorial aims to build a cyclic peptide having Ornithine connections thru the NE of its chain. The cyclic geometry is driven by synthetic NOE restraints as discrete bonds are unfortunately difficult with torsional angle dynamics TAD (CYANA). Using NOEs we are able to get a very close structure regardless and take advantage of CYANAs rapid folding and NOE assignment algorithm. We will take the ensemble of structures solved here with their derived NOE/ACO restraints and refine them further in AMBER MD in a later tutorial. Some may choose to stop at the CYANA structure however which is fine as well.

Reference pages:

The CYANA/CYLIB general links are available here:

CYANA_wiki - Wiki site with many links, tutorials, formats etc
CYLIB_manuscript - The manuscript to read
CYLIB_tut - Build custom residue amino acid for CYANA

On our 8 CPU workstation we have the following settings:

  1. CentOS 7 - The Linux OS which is free
  2. Cyana3.98 - The latest version of CYANA as of 9/2020.
  3. Avogadro2 - Molecular viewing software. Avogadro2 installs OpenBabel as well for data format conversion.
  4. Molmol - Molecular viewing software which is a bit older but very useful. Molmol is another program from the Wuthrich as is DYANA/CYANA. consequently the 2 programs work well together and even use the same library files for defining the residue types and ligands. Molmol also has a number of functions which are friendly to NMR users (NOE viewing etc)
Here we'll assume the user is largely familiar with using command lines in shells within the Linux environment. If you are just starting out please see the tutorial linked here:

Code_academy - Learn line commands and BASH scripting.

The first part of this tutorial is directed towards people with access to our PC at UIC. Outside users can find the files at the bottom of this page however Appendix

Login

ssh (secure shell)

1. Use the ssh command to remote login to our workstation. -Y means portforward -l means username
Note that the "$" is the command line prompt in the terminal. 

$ ssh -Y -l username 131.193.196.29

Login in as if you are sitting at the computer. You might need to run VPN client to access UIC as well before using ssh. Most of the work for this tutorial will be done mainly through the terminal.

List files and make a directory (folder) to store your files in

When you first log in and start a terminal, your current working directory (or folder) is your home directory. It has the same name as your username, and it is where your files and directories are stored. In most cases, this is /home/username

ls (list)

2. Use the ls command to list what is in your current directory.
Note that the "$" is the command line prompt in the terminal. 

$ ls
Desktop  Documents  Downloads  Music  Pictures  Public  Templates  Videos  articles  bin
molmol  orig  people

At this point there will probably be some files and directories in your home directory that are created automatically with your account. Above is for our system. As a quick sidenote I store all the software for users in the /opt/ directory as seen here: 

$ ls /opt/
amber14  bin  cyana-3.98.13 molmol pdbstat

cd (change directory)

At this point, you'll want to move into your Tutorial directory so that you can save all of your working files there.

3. Use the cd command to change to different directory. 

$ cd people 
$ ls
andy_cyclic  francis  molmol_library  peptide_bond  sabari  sherrie  thuc  tutorials 
$ cd thuc 
$ ls
sample1 test
$ rm -rf test 
$ ls
sample1
$ cp -r sample1 test 
$ ls
sample1 test
$ cd test 
$ ls
pdb_build  refine

OK where are we now? Use:

pwd (print working directory)

Pathnames describe what directory you are relative to the entire computer's filesystem. We started from /home/username/

4. Print the working directory pathname of your current directory with pwd 

$ pwd
/home/andyn/people/thuc/test

And the directories and files located here are: 

$ ls
pdb_build  refine

There are 2 directories pdb_build and refine. In pdb_build we generate:

  1. Sequence/structure pdb demo.pdb
  2. start.prot file/table to fill in with chemical shift assignments

Build the initial structure and assignment table file

$ cd pdb_build 
$ ls
CALC.cya  clean  clean_1.pdb  demo.ovw  demo.pdb  demo.seq  go_cyana  go_pdbclean  link.lol  link.upl  orig  start.prot  start.seq

lets clean the directory using the clean script. 

$ ./clean
$ ls
clean  go_cyana  go_pdbclean  orig  start.seq

The important files left behind:

The clean script is trivial and just removes everything we will regenerate anyway.
Lets have a closer look at the start.seq. I call it start.seq as it will end up named demo.seq after it is modified somewhat with the NOE links. See below:

Sequence file "start.seq"

Peptide Sequence including linker atoms

The peptide sequence requires "linker" atoms between broken parts of the chain. This essentially keeps the entire sequence in a continuous fashion which is required by the CYANA program. The special linker atoms do not have charge or vdW values and move right thru other atoms. They only serve for book keeping purposes basically.

The linker atoms have type:

Another important aspect of the linker atoms is you need enough of them included in a run so the attached ligand/residue has enough freedom to move in space and bond/coordinate as needed. Such as having a long enough leash...

Here is the starting file start.seq with annotations:

cif1

Build initial structure and generate "start.prot" file

The many files used and created in this script are here: