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ASDP_public/Tutorial.md

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1. Getting Started 1.1 Tools in Bin Directory 1.2 Input Files 1.3 Defining Project 2. ASDP Run 2.1 ASDP Protocol used in CASD-NMR (Rosato et al, 2009 625-626) 2.2 Using Cyana in ASDP 2.3 Using Xplor in ASDP 2.4 CreateProc 2.5 Useful ASDP Options 3. Analyzing the Output 4. Data in the Examples Directory (contributed by the CASD-NMR) 4.1 CtR69A ASDP run with cyana/cns: ASDP run with xplor/cns: 4.2 Oxidized _ET109 4.3 TAF3 5. Compile C/C++ source code
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1. Getting Started

1.1 Tools in Bin Directory

For most of the ASDP programs, type the command name for help.

ASDP -> Run in command-line. It will execute "noesyassign" to assign NOEs and generate constraints, and then execute "CreateProc" and "Refinement".

ASDP-GUI -> Run with GUI interface

CreateProc -> Setup structure calculations with Cyana or Xplor on a cluster

Other tools:

bmrb2lacs.pl -> Convert bmrb file to lacs input format (bija.nmrfam.wisc.edu/MANI-LACS/)

bmrb2talos.pl -> Convert bmrb file to talos input format

talos2aco.pl -> Convert talos output to dihedral angle file in cyana format

The program talos2aco.pl convert talos output into cyana dihedral angle constraints with score = 10, with error range +/- max(20 degree, 2 * standard deviation).

1.2 Input Files

The input data for ASDP are:

  • The resonance assignment table (in bmrb 2.x or 3.x format) (see data in examples directory). ASDP does interpret the ambiguity code column. This is important for denoting stereospecific assignments.

  • 2D, 3D, and/or 4D NOESY peak lists (see data in examples directory). In principle, the program can read any column formatted peak list file. We typically use either Sparky or Xeasy formats.

  • Other data: RDC (see data in examples directory)

1.3 Defining Project

You need to create a control file for each project, which specifies the protein name, inputs and how to run structure calculations.

For first time users, you may start with the GUI interface to prepare the control file (see for wiki site). If you are familiar with the ASDP control-file format, you can also use the control-files in the examples directory as templates to prepare your new control file.

2. ASDP Run

2.1 ASDP Protocol used in CASD-NMR (Rosato et al, 2009 625-626)

  1. Use LACS to refine C13 referencing (use bmrb2lacs.pl to prepare LACS input, http://bija.nmrfam.wisc.edu/MANI-LACS/)
  2. Generate Cyana dihedral angle constraints with Talos+ results (bmrb2talos.pl and talos2aco.pl).
  3. Run ASDP for 5 or 6 cycles. Type command 'asdp -c control-file -o output' (see data in examples/) Each cycle calculates 100 models and selects the top 20 (See control-file in examples). Cyana is only used for structure calculation. No NOE assignment is made from Cyana. At cycle 5 and 6, Cyana models can be refined with CNSw refinements. CNSw refinement protocol in ASDP does not handle special residues and RDCs. Projects using special residues and RDC data shall not use CNSw refinement in the ASDP protocol.
  4. Remove violated talos+ constraints if any and Rerun Step 3.

2.2 Using Cyana in ASDP

If you want to use Cyana for structure calculation, please specify it in the control-file.

Special residues: 1. Define special residues in the bmrb file (see examples directory) 2. Create the corresponding special cyana lib and add the library file into the Control-file

RDC data: Specify the ORI residues and add the file name into the control-file.

See examples directory for preparation

Manual constraints: * UPL= * ACO=

Advanced Users: ASDP uses the standard CYANA simulated annealing schedule with 10000 torsion angle dynamics steps (15000 with RDC data). The Cyana protocols are defined in lib/ASDP/Cyana.pm.

The structures generated by Cyana require further energy refinement.

At the last cycle, ASDP will generate constraints in CNS/Xplor format (*_noe.tbl, *_dihe.tbl and *_hbond.tbl), which can be used for energy refinement. ASDP will convert the dihedral angel constraint input file from cyana format into CNS/Xplor format, but not the distance constraint input file.

2.3 Using Xplor in ASDP

The current Xplor protocol supports only disulfide bonds and does not support RDCs and special residues. We will support these features in the next version.

Manual constraints: * XPL= * DIHE=

Advanced Users: The Xplor protocols are defined in lib/ASDP/Xplor.pm.

2.4 CreateProc

CreateProc manages the structure calculation runs using Cyana or Xplor.

It can be used by ASDP or stand-alone.

Advanced Users: The CreateProc protocols are defined in lib/ASDP/CreateProc.pm.

2.5 Useful ASDP Options

-c Control_file Required

-o Output_dir Required

-h Help

-m Exclude PCT-filter of Cycle1 for symmetry analysis

-n Exclude CSI-based secondary structure analysis

-i Structure_file Initial fold for bootstrapping

-k Float_number Calibration distance (default 3.4)

-q Structure_file Calculate the F and DP scores of the input structure

-r Out Restore from an old ASDP run

-v Calculate the M score and average chemical shifts

Calibration (-k)

The default average calibrated distance is set to 3.4 in each peak list. You can change this value with -k.

Restore a previous run (-r)

The program will read the pdb models from a previous run specified by -r and skip the modeling generation and selection steps. It only calculates structures when no model is available from the older run.

Start with an initial fold (-i)

You can give a model as input. The program will interpret the NOE peaks solely using the input model. Not recommended unless the model is highly accurate.

Verify input (-v)

It will calculate an M score and a global average chemical shifts, a measure to estimate the consistency between the peak lists and resonance assignments (ref).

3. Analyzing the Output

In the output directory (-o) , you will see a list of CYCLE* directories. In each cycle, you will find intermediate NOE assignments, constraints, and structures. The final structures are in the last cycle of the ASDP output (-o). In the last cycle, constraints in CNS/Xplor format are generated, which can then be used for standalone CNSw refinement.

If CNSw refinement is used, looking for <protein_name>_cns.pdb file.

The constraints are <protein_name>_noe.tbl, <protein_name>_dihe.tbl and <protein_name>_hbond.tbl.

The structures generated by Cyana or Xplor will be named <protein_name>.pdb.

The constraints used by Cyana are <protein_name>.upl, <protein_name>.aco and hbond.upl and hbond.lol.

The constraints used by Xplor are <protein_name>_noe.tbl, <protein_name>_dihe.tbl and <protein_name>_hbond.tbl.

NOE assignments can be found in each cycle:

In Sparky format: <peaklist_name>_assSparky Order by residue-residue distance: <peaklist_name>_assSparky

In the main output directory, files are better to be reviewed with the GUI interface:

  • Main report: <protein_name>_AS.ovw -> summary of AutoStructure runs

    <protein_name>_DP.ovw -> summary of the RPF/DP scores

    <protein_name>_DP.QM/QP/QR -> summary of the RPF/DP analysis

  • Others: <protein_name>_AS.sec -> summary of secondary structure analysis

    <protein_name>_AS.unassign -> list of peaks violated and unassigned in ASDP runs.

    <protein_name>.exm -> calculated with the M score?QM?

    <protein_name>.note -> Print distance calculation errors.

    <peaklist_name>_match.gz -> The match files list matches between peak list and chemical shifts.

    <peaklist_name>_noise -> Peaks with no chemical shift matches.

4. Data in the Examples Directory (contributed by the CASD-NMR)

4.1 CtR69A

ASDP run with cyana/cns:

Type command 'asdp -c control_fileCyana -o runCyana'

result: runCyana/

DP score: 0.822 (CtR69A_DP.ovw)

In the final cycle directory, you will find:

Structures: CtR69A.pdb (from cyana)

Constraints: *.upl, *.aco, hbond.lol/upl (cyana) *.tbl (xplor format)

We recommend to run Rosetta refinement for CYANA models.

ASDP run with xplor/cns:

Type command 'asdp -c control_fileXplor -o runXplor'

result: runXplor/

DP score: 0.82 (CtR69A_DP.ovw)

In the final cycle directory, you will find:

Structures: CtR69A.pdb (from xplor)

CtR69A_cns.pdb (from cns refinement, not released)

Constraints: *.tbl (xplor/cns)

4.2 Oxidized _ET109

It has a disulfide bond 141-177, and a HIS159 in a tautomeric state (HIST) with RDC data. The current version of ASDP supports RDC with Cyana. The Xplor and CNS calculation scripts included in ASDP don't support RDCs.

To use Cyana without CNSw refinement:

HIST is defined in the final.bmrb (sequence section) and special.lib

Disulfide bond and RDC are defined in the control_fileCyana file.

Step 1: Run asdp: Type command 'asdp -c control_fileCyana -o runCyana'

Step 2: Run CNSw Refinement (under development, not released)

Copy all constraints and ET109A.pdb files from CYCLE6-0_final into cns_refine. Type command 'Refinement -na ET109A -ss 141-177 -hise 159’

To run Xplor without RDC:

See controlfileXpor as an example. Please note HIST is not used in this example.

4.3 TAF3

It has two ZNs. one binds to 21C,24C,44H,47C, and another one binds to 36C,39C,62C,65C

To use Cyana without CNSw refinement:

Zn binding residue modification - defined in the chemical shift file (final.bmrb) and special.lib

Zn geometry constraints are zn_dist2.lol and zn_dist2.upl

Run asdp: Type command 'asdp -c control_fileCyana -o runCyana'

Refinement:

Copy all constraints and taf3.pdb files from CYCLE6-0_final into a new directory. Need to 1) prepare patches for zn binding residues (not provided), 2) Add zn geometry constraints into TAF3_noe.tbl (not provided)

5. Compile C/C++ source code

Two programs noesyassign and pdbstat are written in C/C++ programming languages. Other components are written in Perl programming languages.

Programs noesyassign and pdbstat are compiled for Linux system. You need to recompile them for other operating systems.

noesyassign -> Go to noesyassign/Release/ directory and type make noesyassign. Copy the executable noesyassign to bin/

pdbstat -> Go to PDBSTAT/src directory and type make to follow the instruction. Copy the executable pdbstat to bin/