ENORA and multi-state structure calculations: Difference between revisions

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  print "Displacement of the LIG (to ref xray): ${s/n} ($n atoms)"
  print "Displacement of the LIG (to ref xray): ${s/n} ($n atoms)"


=== Exercise xx: Analysis of multi-state calculations ===
=== Exercise: Analysis of multi-state calculations ===
 


=== On improving the final structure ===
=== On improving the final structure ===

Revision as of 11:33, 5 April 2019

In this tutorial we will provide you with a guided example for calculating eNOEs and multi-state structure calculations.

To this end we will first run the modules of eNORA and then use the obtained eNOEs to calculate a single state and a two-state structure model using automated sorting to group the states. Along the way you will learn some additional CYANA skills useful for other purposes as well.

The eNORA module offers in principle two methods to calculate spin diffusion, FRM and TSS. FRM is the generally applicable way to do these calculations and we will set a main focus on that method, will however have one section which explains the principles at play for TSS and how to set it up.

To finalize you will .... And ultimately you can try to improve ....

CYANA setup

Obtaining and installing the CYANA demo version and data

Please follow the following steps carefully (exact Linux commands are given below; you may copy them to a terminal):

  1. Go to your home directory (or data directory).
  2. Get the demo data from the server.
  3. Unpack the input data for the practical.
  4. Get the demo version of CYANA.
  5. Unpack CYANA.
  6. Setup the CYANA environment variables.
  7. Change into the newly created directory 'eNORA'.
  8. Copy the demo_data directory to 'enoe'.
  9. Change into the subdirectory 'enoe'.
  10. Test whether CYANA can be started by typing its name, 'cyana'.
  11. Exit from CYANA by typing 'q' or 'quit'.
cd ~
wget 'http://www.cyana.org/wiki/images/6/64/eNORA_multiState.tar.gz'

on mac OS X (use curl instead of wget): 

curl http://www.cyana.org/wiki/images/6/64/eNORA_multiState.tar.gz -o eNORA_multiState.tar.gz

then 

tar zxf eNORA_multiState.tar.gz 

wget 'http://www.cyana.org/wiki/images/6/64/Cyana-3.98.9_Demo.tgz'

again, on mac OS X: 

curl http://www.cyana.org/wiki/images/6/64/Cyana-3.98.9_Demo.tgz -o Cyana-3.98.9_Demo.tgz

then 

tar zxf Cyana-3.98.9_Demo.tgz
cd cyana-3.98.9/
./setup

cd ~

cd eNORA
cp -r demo_data enoe 
cd enoe

Try to run CYANA by entering 'cyana' at the command prompt of your terminal (q to quit cyana): 

cyana
___________________________________________________________________

CYANA 3.98 (mac-intel)

Copyright (c) 2002-17 Peter Guentert. All rights reserved.
___________________________________________________________________

    Demo license valid for specific sequences until 2018-12-31
cyana> q

If all worked, you are ready to go in terms of everything related to CYANA!

Execution scripts or "macros" in CYANA

For more complex task within CYANA, rather than to enter the execution commands line by line at the CYANA prompt, the necessary commands are collected in a file named '*.cya'. Collecting the commands in macros has the added advantage, that the macros serve as a record allowing to reconstruct previous calculations.

Hint: For comprehensive information on the CYANA commands etc. is in the CYANA 3.0 Reference Manual.

Preparing input data

Structure input for spin-diffusion calculations

Preparing an xray structure to use within CYANA

Deposited structures many times lack specific features, i.e. Xray structures often lack proton coordinates or contain sequence mutations and ligands. Using the regularize command one can get a structure recalculated within CYANA that has these issues fixed but is still very close to the input structure.

In the data directory you find the 'regualbb' directory. 

cd regulabb

In this directory you find the 'CALC_reg.cya' macro and an 'init.cya' macro.

The initialization macro has the fixed name 'init.cya' and is executed automatically each time CYANA is started. It can also be called any time one wants to reinitialize the program by typing 'init'. It contains normally at least two commands. However for reasons that will become clear in a minute, this time there is only one command. 

cyanalib

The command 'cyanalib' reads the standard CYANA library.

After reading the library file, one normally reads a sequence file before reading pdb file or a peak list.

Inspection of the 'CALC_reg.cya': 

read 1PIN.pdb unknown=warn hetatm new
write 1PIN.seq 
write 1PIN_2.pdb

Where the option 'hetatm' allows for reading of coordinate labeled HETATM, rather than ATOM in the pdb. The parameter 'new' directs CYANA to read the sequence from the pdb.

We require two mutations to the sequence (S18N and W34F), furthermore we do not have a ligand and the expressed protein for NMR is truncated. Therefore we use cyana truncate the Xray structure and build in the mutations. We read the structure again, but this time provide a sequence, containing the two mutations and use the options unknown=warn or unknown=skip to skip the parts of the Xray structure not specified in the sequence file and later reconstruct those during regularization: 

read demo.seq
read 1PIN_2.pdb rigid unknown=warn
write 1PIN_ed.pdb

./init

read pdb 1PIN_ed.pdb
regularize steps=20000 link=LL keep

Download the Xray structure used for this exercise: 

wget 'https://files.rcsb.org/view/1PIN.pdb'

or for mac os x: 

curl https://files.rcsb.org/view/1PIN.pdb -o 1PIN.pdb

Inspect the pdb using chimera: There are several issues besides HETATM, that we are fixing within CYANA before using the structure.

Execute the 'CALC_reg.cya' macro: 

cyana CALC_reg.cya

The resulting structure is 'regula.pdb'.

Calculating a structure from an experimental peak list

In the data directory you find the 'noecalib' directory.

The 'CALC_noecalib.cya' contains the following commands: 

peaks:=5.peaks
calibration peaks=$peaks
peaks calibrate simple
write upl noesimple.upl

The following block of commands, reads the restraints 'noesimple.upl', an angle file called 'demo.aco', calculates a structure: 

read upl noesimple.upl     
read aco talos.aco			 
calc_all 100 steps=50000		        

overview demo.ovw structures=20 pdb

The statistics are in demo.ovw file and the 20 conformers with the lowest target function in the 'demo.pdb'.

Estimating spin-diffusion in NOESY experiments

Recording NOESY experiments with 13C/15N simultaneous evolution

Preparing experimental peak lists

Peak lists in XEASY format are prepared by automatic peak picking with a visualization program such as CcpNmr Analysis, NMRdraw or NMRview and saved as XXX.peaks, where XXX denotes the name of the xeasy peak list file. Since NMRdraw peak lists are of different file type, cyana provides the command read tab to convert the files to XEASY format. 

# Number of dimensions 3
#FORMAT xeasy3D
#INAME 1 H
#INAME 2 HN
#INAME 3 N
#SPECTRUM N15NOESY H HN N  ??? change to 13C, procerssed as 13C!!!!
   17086    4.098    4.099   57.441 1 U   6.990943E+08  0.000000E+00 e 0  HA.5      HA.5      CA.5
   89532    4.355    1.829   33.507 1 U   1.720779E+06  0.000000E+00 e 0  HA.6      HB2.6     CB.6
   89544    4.353    1.757   33.513 1 U   2.939628E+06  0.000000E+00 e 0  HA.6      HB3.6     CB.6

The first line specifies the number of dimensions (3 in this case). The '#SPECTRUM' (no space between characters) lines gives the experiment type (N15NOESY, which refers to the corresponding experiment definition in the CYANA library), followed by an identifier for each dimension of the peak list (H HN N) that specifies which chemical shift is stored in the corresponding dimension of the peak list. The experiment type and identifiers must correspond to an experiment definition in the general CYANA library (see below) in most uses of the definition, here however we cheat slightly and get away with it. We are cheating, because for eNOE calculations we record our NOESY spectra with simultanous evolution of 13C and 15N dimensions, since we require 15N and 13C bound spins within the same spectrum for purposes of normalization (see...).

After the '#SPECTRUM' line follows one line for every peak. For example, the first peak in the 'HNCA.peaks' list has

  • Peak number 17086
  • H chemical shift 4.098 ppm
  • ("HN") chemical shift 4.099 ppm (in this case 13C bound)
  • Heavy atom chemical shift 57.441 ppm (in this case 13C labeled)

The other data are relevant entry for the eNOE mudules is the peak volume or intensity (6.990943E+08).

Assignment dimensions have to be arranged with the flow of magnetization, first row spin i, second row spin j


The protein sequence is supplied by three-letter code in the demo.seq file.

SPECTRUM definitions in the CYANA library

When you start CYANA, the program reads the library and displays the full path name of the library file. You can open the standard library file to inspect, for example, the NMR experiment definitions . For instance, the definition for the N15NOESY spectrum (search for 'N15NOESY' in the library file 'cyana.lib') is 

SPECTRUM N15NOESY H HN N
 0.900 N:N_AM* HN:H_AMI ~4.0 H:H_*
 0.800 N:N_AM* HN:H_AMI ~4.5 H:H_*
 0.700 N:N_AM* HN:H_AMI ~5.0 H:H_*
 0.600 N:N_AM* HN:H_AMI ~5.5 H:H_*
 0.500 N:N_AM* HN:H_AMI ~6.0 H:H_*

The first line corresponds to the '#SPECTRUM' line in the peak list. It specifies the experiment name and identifies the atoms that are detected in each dimension of the spectrum. The number of identifiers defines the dimensionality of the experiment (3 in case of N15NOESY).

Each line below defines a (formal) magnetization transfer pathway that gives rise to an expected peak. in the case of N15NOESY there are five lines, corresponding to the through space magnetization transfer by dipol-dipol mechanism. The peak definition starts with the probability to observe the peak (0.900), followed by a series of atom types, e.g. H_AMI for amide proton etc. The atoms whose chemical shifts appear in the spectrum are identified by their labels followed by ':', e.g. for N15NOESY 'H:', 'HN:', and 'N:'. If you were to use the CYANA functions to simulate peaks, expected peaks are generated for each molecular fragment in which these atom types occur.

You may have realized that our peak list contains peaks that are 13C bound, therefore the spectrum definition is wrong, since we are only reading the peak lists and not generating any, this is not a problem.

From nmrDraw to XEASY

In the 'nmrDrawX' directory you find the 'nmrDrawX.cya' macro, it does the following:

  • Conversion of the nmrDraw peak file to XEASY format with the atom assignments in the file.
  • Sorting of the peaks in each peaks list per mixing time.
  • Writing out the peaks in XEASY format with atom names contained in the file, one peak list for each mixing time.
# read nmrDraw tab file
read tab demo.tab

# sort the peaks
peaks sort

# write out peak lists
do i 1 npkl
  peaks select "** list=${i}"
  write peaks $i.peaks names
end do

Using Talos to generate torsion angle restraints

Torsion angle restraints from the backbone chemical shifts help restrict angular conformation space. We wish to use only "strong assignments" to generate these restraints.

If you do not have TALOS installed get it from here. It is part of the nmrpipe software package.

In the 'acoPREP' directory, inspect the 'CALC_talos.cya' file with the commands to calculate the talos angle restraints: 

# convert
read 5.peaks
shifts initialize
shifts adapt
atom set "* shift=990.0.." shift=none
write demo.prot

read prot demo.prot unknown=skip

talos talos=talos+                
talosaco pred.tab

write aco talos.aco

This will call the program TALOS+ and store the resulting torsion angle restraints in the file 'talos.aco'.

Since this is not a calculation suited for the MPI scheduler, start CYANA first, then call the 'CALC_talos.cya' macro from the prompt.


Hint: change to a cshell before running cyana (since talos needs a cshell to run): 

csh

eNOE calculations from a single mixing time

All eNOE related calculations within cyana are carried out using the eNORA modules.

NOESY experiment measured at different mixing times (keeping the mixing times as much as possible within the linear regime of NOE buildup) supply very precise distance restraints used for a structure calculation. In addition other restraints such as backbone angles from chemical shifts and scalar couplings for backbone and aromatic side chains are also used.


eNORA

In the 'enoe1pt' directory you find the relevant 'init.cya' and 'CALC_enoe1pt.cya' macro's.

The init macro

The initialization macro file has the fixed name 'init.cya' and is executed automatically each time CYANA is started. It can also be called any time one wants to reinitialize the program by typing 'init'. It contains normally at least two commands that read the CYANA library and the protein sequence: 

rmsdrange:=8-33
cyanalib
read seq demo.seq
swap=0
expand=1

The first line sets the appropriate rmsdrange, and the command 'cyanalib' reads the standard CYANA library. The next command reads the protein sequence.

The protein sequence is stored in three-letter code in the file 'demo.seq'.

Stereo-specificity of dia-stereocenters
atoms stereo "HA? 10"
atoms stereo "HB? 7 8 11 13 14 21 23 24 25 26 27 33 34 35 37 38"
atoms stereo "HG? 8 12 14 17 36 37"
atoms stereo "HD2? 18 26 30"
atoms stereo "QG? 22"
atoms stereo "QD? 7"
atoms stereo "HE2? 33"
atoms stereo "HD? 8 14 21 37"
atoms stereo "HG1? 28"

However, one may do the following to supply all atoms as stereo specific: 

atoms select
atoms stereo

or to supply all atoms as non stereo specific, use: 

atoms select
atoms stereo delete

To get a feedback of the supplied stereo specific assignment add to your 'init.cya' the command: 

atoms stereo list
D20 exchange

With 3% D2O in the nmr buffer for exchange of backbone amide atoms 

atoms set "H" protlev=0.97

The eNORA CALC macro

The 'CALC_enoe1pt.cya': 

# ----------------------------------          eNORA routine         ---------------------------------- 
# ----------------------------------   basic parameter definitions  ---------------------------------- 

echo:= on
mixingtimes = 0.06
avgrho      = 5.3
tauc        = 4.25
b0field     = 700
maxdistance = 6.5
rhofile     = 'none'
normed      = 0
normspin    = 2
mode        = 1

# ----------------------------------         structure input        ---------------------------------- 

# specify the conformers for calculations
read pdb demo rigid
structure select 1

# ----------------------------------        peak file reading       ----------------------------------

# read in the peak lists
read peaks 5.peaks
  
# ----------------------------------   initializing the routine     ----------------------------------
# initialize the routine, fit experimental decays and buildups
enoe init normalize=normspin normed=normed rhoavg=avgrho time=$mixingtimes

# write the auto-relaxation values to file
write rhoOut.rho
 
# ----------------------------------        spin-diffusion          ----------------------------------

# calculate the spin-diffusion correction
enoe init normalize=normspin normed=normed rhoavg=avgrho time=$mixingtimes
 
# ----------------------------------     apply spin-diffusion       ---------------------------------- 

# apply spin-diffusion correction to experimental buildups (if calculated) and calculate reff
enoe reff b0field=b0field tauc=tauc

# prepare the cyana restraints (scaling, error margins)
enoe restraint errStereoFlag=1 errStereo=-1 chiN=-1 b0field=b0field tauc=tauc
 
# ----------------------------------        write restraints        ----------------------------------

# delete fixed distances from upl/lol output
distance delete fixed

# write the distance restraints to file
write upl enoe.upl
write lol enoe.lol
 
# ----------------------------------            overview            ----------------------------------

enoe overview

Run the eNOE calculation such as: 

cyana CALC_enoe1pt.cya

eNORA output files

The FLYA algorithm will produce the following output files:

  • missRhoIzero: List of spins that lack a diagonal decay, and do not provide a rho or I(0) value (if you set normed=0).
  • nonNormalizableNOEs.out: List of NOEs that lack a diagonal decay and are not normalizable (if you set the parameter normed=0 to normed=1, all non normalizable NOES are left out).
  • rhoOut.rho: Experimentally fitted rho and I(0) values values form diagonal decays.
  • enoe.upl and enoe.lol: Upper limit and lower limit restraint files with tolerances applied.
  • enoe.ovw: Collated results file.

The missRhoIzero file

List of spins that lack a diagonal decay and therefore have no experimentally determined rho and I(0) values. The file is formatted to be used as basis for a rhoIn.rho file derived from average values. The file may also be used to setup generic normalized eNOE calcuations, see .....

The rhoOut.rho file

The enoe.upl/lol distance restrains

Tolerances Comments


The enoe.ovw file

Collated file that may be generated at any time during the routine and will be populated with the values available at the momentary progress of calculations.

  • ASSIGNMENT(i->j): Assignment arranged with the flow of magnetization.
  • REFFixEXP: The experimental sigma of spin x, where x=i,j
  • REFFxCORR: The reff of spin x, where x=i,j
  • SIGxEXP: The experimental sigma of spin x, where x=i,j
  • SIGxCORR: The spin-diffusion corrected experimental sigma of spin x, where x=i,j
  • SDCx: The spin-diffusion correction of spin x, where x=i,j
  • SDPx: The number of other partner spins involved in spin-diffusion for spin x, where x=i,j
  • IZEROx: The back calculated I(0) value of spin x, where x=i,j
  • exp_chiNx: The goodness of fit of the experimental data, where x=i,j
  • corr_chiN x: The goodness of fit of the spin-diffusion corrected experimental data, where x=i,j

Considerations regarding the obtained eNOE restraints

Mapping calculated eNOE restraints onto a known structure

One can map the calculated restraints, such as distance restraints (upl/lol) onto a known structure (in the example here the modified xray structure). This is an approach to analyze restraints and their influence on the results.

Below you find the commands to accomplish this. You see by studying the commands, which files are needed to execute the macro. Therefore, create a new directory ('mkdir') or copy a directory containing the respective files. Delete what you do not need. Use the regularized xray structure from exercise xxxx.

You need an init file: 

rmsdrange:=8-33
cyanalib
read seq demo.seq


And the main macro (name it 'CALC_xraymap.cya'): 

read upl enoe.upl
read lol enoe.lol

read regula.pdb unknown=warn

weight_vdw=0
overview enoe_xray.ovw
  • If the restraints do not match with the xray structure, does it mean they are wrong?
  • If you tried the two options, what is (are) the difference(s)?

eNOE calculations from buildup data

All eNOE related calculations within cyana are carried out using the eNORA modules.

NOESY experiment measured at different mixing times (keeping the mixing times as much as possible within the linear regime of NOE buildup) supply very precise distance restraints used for a structure calculation. In addition other restraints such as backbone angles from chemical shifts and scalar couplings for backbone and aromatic side chains are also used.


eNORA

You will find the relevant macro's in the directory 'enoebup'.

The init macro

The initialization macro file has the fixed name 'init.cya' and is executed automatically each time CYANA is started. It can also be called any time one wants to reinitialize the program by typing 'init'. It contains normally at least two commands that read the CYANA library and the protein sequence: 

rmsdrange:=8-33
cyanalib
read seq demo.seq
swap=0
expand=1

The first line sets the appropriate rmsdrange, and the command 'cyanalib' reads the standard CYANA library. The next command reads the protein sequence.

The protein sequence is stored in three-letter code in the file 'demo.seq'.

Stereo-specificity of dia-stereocenters
atoms stereo "HA? 10"
atoms stereo "HB? 7 8 11 13 14 21 23 24 25 26 27 33 34 35 37 38"
atoms stereo "HG? 8 12 14 17 36 37"
atoms stereo "HD2? 18 26 30"
atoms stereo "QG? 22"
atoms stereo "QD? 7"
atoms stereo "HE2? 33"
atoms stereo "HD? 8 14 21 37"
atoms stereo "HG1? 28"

However, one may do the following to supply all atoms as stereo specific: 

atoms select
atoms stereo

or to supply all atoms as non stereo specific, use: 

atoms select
atoms stereo delete

To get a feedback of the supplied stereo specific assignment add to your 'init.cya' the command: 

atoms stereo list
D20 exchange

With 3% D2O in the nmr buffer for exchange of backbone amide atoms 

atoms set "H" protlev=0.97

The eNORA CALC macro

The 'CALC_enoe.cya' starts with the following:

  • The parameter definitions, their function is explained later with respect to the functions that use them.
  • The structure input and which conformers to use for spin-diffusion calculations. If multiple conformers are used to average the spin-diffusion values, the command 'structure select' may be used to select multiple conformers of the pdb.
  • Reading the XEASY peak lists in the order of increasing mixing time.
# ----------------------------------          eNORA routine         ---------------------------------- 
# ----------------------------------   basic parameter definitions  ---------------------------------- 

echo:= on
mixingtimes = '0.02,0.03,0.04,0.05,0.06'
avgrho      = 5.3
tauc        = 4.25
b0field     = 700
maxdistance = 6.5
rhofile     = 'rhoInApo.rho'
normed      = 0
normspin    = 2
mode        = 1
bname       = 'bupplots'
dname       = 'decplots'

# ----------------------------------         structure input        ---------------------------------- 

# specify the conformers for calculations
read pdb demo rigid
structure select 1

# ----------------------------------        peak file reading       ----------------------------------

# read in the peak lists
do i 1 npkl
  read peaks $i.peaks $if(i.eq.1,' ','append')
end do	

# supply averaged rho or izero values
if (existfile('$rhofile')) then
  read rho $rhofile
end if
  
# ----------------------------------   initializing the routine     ----------------------------------
# initialize the routine, fit experimental decays and buildups
enoe init normalize=normspin normed=normed rhoavg=avgrho time=$mixingtimes

# print average experimental auto-relaxation and I(0) values to screen
enoe avgExpVal

# plot the diagonal decay's  
enoe decay plot=$dname
graf $dname.pdf

# write the auto-relaxation values to file
write rhoOut.rho

exit

# ----------------------------------        spin-diffusion          ----------------------------------

# calculate the spin-diffusion correction
enoe spindiff b0field=b0field tauc=tauc maxdist=maxdistance mode=mode
 
# ----------------------------------     apply spin-diffusion       ---------------------------------- 

# apply spin-diffusion correction to experimental buildups (if calculated) and calculate reff
enoe reff b0field=b0field tauc=tauc

# prepare the cyana restraints (scaling, error margins)
enoe restraint errStereoFlag=1 errStereo=-1 chiN=-1 b0field=b0field tauc=tauc
 
# ----------------------------------        write restraints        ----------------------------------

# delete fixed distances from upl/lol output
distance delete fixed

# write the distance restraints to file
write upl enoe.upl
write lol enoe.lol

# ---------------------------------         plotting buildups         --------------------------------
enoe buildup b0field=b0field tauc=tauc plot=$bname
graf $bname.pdf
 
# ----------------------------------            overview            ----------------------------------

enoe overview

Start CYANA and execute the 'CALC_enoe.cya' macro from the CYANA prompt as such: 

cyana CALC_enoe.cya

The program if all is setup properly the program will run and display averages per spin type of autorelaxation and I (0) values and then stop as soon as it finished plotting the diagonal decay's and has written the 'rhoOut.rho' file.

The command 

exit

following the command 'write rhoOut.rho' stops the routine from running to completion. Before commenting out (#) the exit command and running the routine to its end, do the following exercise.

Exercise: Compiling the autorelaxation file

Before compiling the rhoIn.rho file, check the diagonal decays for their quality. Edit out any diagonal peaks from the tab file that give bad decays, then run the routine again the same way to get averages of rho and I(0) values. Then compile the rhoIn.rho file by copying the 'rhoOut.rho' file and adding the lines from 'missRhoIzero.out', filling in the rho values as needed.

When compiling the rhoIn.rho file see * auto-relaxation and I(0) values.

eNORA output files

The FLYA algorithm will produce the following output files:

  • missRhoIzero: List of spins that lack a diagonal decay, and do not provide a rho or I(0) value (if you set normed=0).
  • nonNormalizableNOEs.out: List of NOEs that lack a diagonal decay and are not normalizable (if you set the parameter normed=0 to normed=1, all non normalizable NOES are left out).
  • rhoOut.rho: Experimentally fitted rho and I(0) values values form diagonal decays.
  • enoe.upl and enoe.lol: Upper limit and lower limit restraint files with tolerances applied.
  • enoe.ovw: Collated results file.
  • bupplots:
  • decplots:

The missRhoIzero file

List of spins that lack a diagonal decay and therefore have no experimentally determined rho and I(0) values. The file is formatted to be used as basis for a rhoIn.rho file derived from average values. The file may also be used to setup generic normalized eNOE calcuations, see .....

The rhoOut.rho file

The enoe.upl/lol distance restrains

Tolerances Comments


The enoe.ovw file

Collated file that may be generated at any time during the routine and will be populated with the values available at the momentary progress of calculations.

  • ASSIGNMENT(i->j): Assignment arranged with the flow of magnetization.
  • REFFixEXP: The experimental sigma of spin x, where x=i,j
  • REFFxCORR: The reff of spin x, where x=i,j
  • SIGxEXP: The experimental sigma of spin x, where x=i,j
  • SIGxCORR: The spin-diffusion corrected experimental sigma of spin x, where x=i,j
  • SDCx: The spin-diffusion correction of spin x, where x=i,j
  • SDPx: The number of other partner spins involved in spin-diffusion for spin x, where x=i,j
  • IZEROx: The back calculated I(0) value of spin x, where x=i,j
  • exp_chiNx: The goodness of fit of the experimental data, where x=i,j
  • corr_chiN x: The goodness of fit of the spin-diffusion corrected experimental data, where x=i,j

eNOE calculations and the TSS approach to spin-diffusion calculations

Labeling Schemes

Deuterated labeling schemes often involve methyl labeling with 3% D2O in the nmr buffer, i.e. 

atoms set "H" protlev=0.97
# labeling scheme: VAL_G1 0% LEU_D1 0% ILE_D1 0%
atoms set "QG1 @VAL + QD1 @LEU + QD1 @ILE" protlev=0.0

Multi-state structure calculation with ensemble-averaged restraints

To facilitate the discussion of multi-state structure calculations and ensembles we use the following definitions:

  • The CYANA target function is a measure for the quality of the computed structural ensembles given in terms of the squared violation of the experimental restraints.
  • A structure is defined by a bundle (or an ensemble) of conformers fulfilling the experimental data.
  • A conformer is the result of one individual structure calculation that fulfills the experimental data and may be composed of one or more states.
  • A state is one set of coordinates for all atoms of a molecule. If there are multiple states they fulfill the experimental data on average and not individually.
  • Sub-bundles are formed by sorting the states according to structural similarity in the region of interest. There are as many sub-bundles as there are states in a conformer, and each sub-bundle comprises as many conformers as the original structure bundle. This requires for each state to belong to exactly one sub-bundle. The sub-bundle for each structural state is a measure of the precision of the individual structural states similar to the conventional bundle representation. 


Calculating a single state structure

We will perform calculations based on eNOEs by using torsion angle dynamics in order to compute the three-dimensional structure of the protein.

The 'enoe.upl' and 'enoe.lol' files will be used together with the aco based on chemical shifts of the backbone and scalar couplings from backbone, Ha-HB and aromatic residues determined by experiment.


The single-state structure calculation is in principle a regular structure calculation, using your upl/lol, aco and cco files as input. This would look something like this (we will do it differently): 

syntax inputseed=@i=3771

# ------ Structure calculation ------
read upl XXX.upl
read lol XXX.lol
read aco XXX.aco
read cco XXX.cco

anneal_weight_aco := 1.0, 1.0, 0.0, 0.0
anneal_weight_cco := 0.0, 0.5

seed=inputseed
calc_all 100 steps=50000

if (master) then
  cut_cco=1.0
  cut_rdc=3.0
  weight_aco = 0.0

  rmsdrange:=8-33,108-133
  overview sstate structures=20 pdb
end if

However, since we end up calculating also multi-state structures later on, it makes sense to setup the single-state calculation exactly the same way as the multi-state calculations, and only edit as few parameters as possible. As soon as you understand the 'PREP.cya' macro below, you will realize why this makes sense.

Exercise: Setting up a single state calculation

Copy the 'enoe' directory and give it the name 'sstate', then delete all the files and data we do not need to reduce clutter and have better oversight. 

cp -r enoe sstate
cd sstate
rm *.peaks *.out *.job
rm -rf details

From the directory 'acoPREP' copy the calculated talos restraints ('talos.aco').

The init macro

The 'init.cya' macro needs to be updated to read the multi-state sequence in order to prepare the restraints and run the structure calculation: 

cyanalib
if (existfile('bundle.seq')) then
  read seq bundle.seq
  molecules define *
  atom set * vdwgroup=bundle
else
  read seq demo.seq
end if
rmsdrange:=8-33

swap=0
expand=1

atoms stereo "HA? 10"
atoms stereo "HB? 7 8 11 13 14 21 23 24 25 26 27 33 34 35 37 38"
atoms stereo "HG? 8 12 14 17 36 37"
atoms stereo "HD2? 18 26 30"
atoms stereo "QG? 22"
atoms stereo "QD? 7"
atoms stereo "HE2? 33"
atoms stereo "HD? 8 14 21 37"
atoms stereo "HG1? 28"

atoms set "H" protlev=0.97

The PREP macro

syntax nbundle=@i=1 togetherweight=@r=0.1 multitensor

#multitensor=.true.
together=.true.
moloffset=100

# ------ Sequence file ------

read seq demo.seq
print "\# Bundle of $nbundle conformers" >bundle.seq
do j 1 nbundle
  do i 1 nr
    if (j.lt.nbundle .and. rnam(i).eq.'PL') break
    print "$rnam(i) ${$rnum(i)+moloffset*(j-1)}" >>
  end do
  if (j.lt.nbundle) print "PL LL2 LL2 LL2 LL2 LL2 LL2 LL2 LL2 LL2 LL2 LL2 LL2 LL2 LL2 LP" >>
end do
print >>.

# ------ Make bundle angle restraints ------

read aco demo.aco
write aco bundle.aco
do j 2 nbundle
  atom set * residue=residue+moloffset
  write aco bundle.aco append
end do

# ------ Make bundle coupling constant restraints ------

read demo.seq
read cco demo_backbone.cco
read cco demo_aro.cco append
read cco demo_JHaHb.cco append
 
print "\# Coupling constant restraint file" >bundle.cco
do i 1 ncco
  i1=iccoa(1,i); i2=iccoa(2,i)
  do j 1 nbundle
    m=moloffset*(j-1)
    print "${$rnum(iar(i1))+m} $rnam(iar(i1)) $anam(i1) ${$rnum(iar(i2))+m} $rnam(iar(i2)) $anam(i2) $cco(i) $tolcco(i) 1.0 $karplus(1,i) $karplus(2,i) $karplus(3,i) $if(j.ne.nbundle,'&',' ')" 
>>bundle.cco
  end do
end do 
print >>.
read seq bundle.seq
read cco bundle.cco 
write cco bundle.cco karplus

# ------ Make bundle RDC restraints ------

#read bundle.seq
#read rdc demo.rdc
#print "\# RDC restraint file" >bundle.rdc
#do i 1 orientations
#  if (multitensor) then
#    do j 1 nbundle
#      print "${i+orientations*(j-1)} $magnitude(i) $rhombicity(i) ${$rnum(iar(irtena(4,i)))+moloffset*(j-1)}" >>
#    end do
#  else
#    print "$i $magnitude(i) $rhombicity(i) $rnum(iar(irtena(4,i)))" >>
#  end if
#end do
#do i 1 nrdc
#  i1=irdca(1,i); i2=irdca(2,i)
#  do j 1 nbundle
#    m=moloffset*(j-1)
#    iten=irdct(i); if (multitensor) iten=iten+orientations*(j-1)
#    print "${$rnum(iar(i1))+m} $rnam(iar(i1)) $anam(i1) ${$rnum(iar(i2))+m} $rnam(iar(i2)) $anam(i2) $rdc(i) $tolrdc(i) $weirdc(i) $iten $rdcsca(i) $if(j.lt.nbundle,'&',' ')" >>bundle.rdc
#  end do
#end do
#print >>.
#read seq bundle.seq
#read rdc bundle.rdc
#write rdc bundle.rdc

# ------ Make ambiguous bundle distance restraints ------

subroutine PURGE	
	#distance delete "HA 9, HB2 9"
end


init

read upl enoe.upl

PURGE
distance modify info=full
molecules symmetrize
if (nbundle.gt.1) distances set "$moloffset.., $moloffset.." bound=0.0
distances set "*, *" bound=bound*(1.0*nbundle)**(-1.0/6.0)
write upl bundle.upl

init

read lol enoe.lol

PURGE
distance modify info=full
molecules symmetrize
if (nbundle.gt.1) distances set "$moloffset.., $moloffset.." bound=0.0001
distances set "*, *" bound=bound*(1.0*nbundle)**(-1.0/6.0)
write lol bundle.lol

# ------ Make restraints to keep corresponding atoms together ------

if (together .and. nbundle.gt.1 .and. togetherweight.gt.0.0) then
  read seq bundle.seq
  molecules define *
  atom set * vdwgroup=bundle
  atom select "N C*"
  do i 1 na
    if (iamol(i).ne.1) break
    if (asel(i)) then
      distance make "$atom(i)" "$anam(i) ${$rnum(iar(i))+moloffset}" upl=1.2 weight=$togetherweight info=none
    end if
  end do
  distances set "* - N CA C CB, * - N CA C CB" weight=weight*0.1
  molecules symmetrize
  distances unique
  write upl together.upl
end if

The single-state CALC macro

Inside the 'sstate' directory, use a text editor to create the 'CALC_sstate.cya' file for structure calculation as outlined below. 

syntax inputseed=@i=3771

if (master) then
  PREP
end if

# ------ Structure calculation ------
read upl bundle.upl
read lol bundle.lol
read aco bundle.aco
read cco bundle.cco
#read rdc bundle.rdc
if (existfile('together.upl')) read upl together.upl append

anneal_weight_rdc := 0.0, 0.5
anneal_weight_aco := 1.0, 1.0, 0.0, 0.0
anneal_weight_cco := 0.0, 0.5

seed=inputseed
calc_all 100 steps=50000

if (master) then
  cut_cco=1.0
  cut_rdc=3.0
  weight_aco = 0.0

  rmsdrange:=8-33,108-133
  overview bundle structures=20 pdb

  #read pdb bundle.pdb
  #rmsdrange:=11-16,21-26,31-34,111-116,121-126,131-134
  #overview bundleSec structures=20 pdb # reference=xxx.pdb

  #molecules sort "BACKBONE 26-30" base=1
  #write sortStates.pdb all
  #SPLIT
end if


These commands tell the program to calculate 100 conformers, and to analyze the best 20 of them. 50000 torsion angle dynamics steps will be applied per conformer.


When you are done preparing the macros as outlined run the calculation.

The structure calculation will be performed by running the 'CALC_sState.cya' macro: 

cyana -n 33 CALC_sState.cya

Doing this, basically means each processor will calculate 100/33=3 conformers. If you changed the setup to calculate 50 structures, you would start the calculation with 'cyana -n 25 CALC_sState.cya'.

Carefully analyze the WARNING and ERROR messages if any.


Statistics on the the structure calculation will be displayed to screen. The final structure will be 'bundle.pdb'.

Calculating Multi-state structures

The SPLIT macro

Each conformer now contains to sets of coordinates corresponding to two states, and the 

moloffset=100

read pdb sortStates.pdb
n=nstruct
write_all split

nbundle=$rnum(nr)/moloffset+1
show nbundle

do i 1 nstruct
  read seq bundle.seq
  read pdb split$i(I3.3).pdb
  do j 1 nbundle
    atoms select 1-80
    write pdb split$i(I3.3)-$j.pdb selected
    atoms set * residue=residue-moloffset
  end do
  read seq demo.seq
  read_all split$i(I3.3)-*.pdb unknown=skip
  write split$i(I3.3).pdb all
  do j 1 nbundle
    remove split$i(I3.3)-$j.pdb
  end do
end do

read seq demo.seq
do i 1 n
  read pdb split$i(I3.3).pdb append
end do
write pdb splitall.pdb all
rmsd

Grouping the coordinates of multi-state calculations

Exercise: Setting up a two-state calculation

Copy the 'sstate' directory and give it the name 'twostate', then delete all the previous, unnecessary output files to reduce clutter and have better oversight. Copy the 'CALC_sstate.cya' and rename it 'CALC_multistate.cya'. 

cp -r sstate twostate
cd twostate 
mv CALC_sstate.cya CALC_multistate.cya
rm  *.out *.job final* rama*

With a text editor, edit the 'CALC_multistate.cya' macro to activate the inactive commands (by deleting the preceeding hashtag #) necessary to perform the grouping of states and splitting of the conformers.

With a text editor, change the number of states (nbundle=@i=) from one to two in the 'PREP.cya' macro: 

syntax nbundle=@i=1 togetherweight=@r=0.1 multitensor

When you are done preparing the macros as outlined perform the calculation by running the 'CALC_multistate.cya' macro: 

cyana -n 33 CALC_multistate.cya.cya

Carefully analyze the WARNING and ERROR messages if any.

Results: analysis

Download and install the molecular viewer Chimera

  1. Download Chimera (to your personal laptop) from: Chimera

Exercise xx: Single state structure analysis

The final structure will be 'final.pdb'. You can visualize it, for example, with the command 

chimera final.pdb

Analyze the result, the bundle seems unnatuarly tight for an NMR structure bundle. Why?

Exercise: Calclulate the RMSD of NMR vs. xray structure using a CYANA macro

Using the INCLAN language of CYANA (Writing and using INCLAN macros,Using INCLAN variables,Using INCLAN control statements) it is possible to write complex macros that interact with the FORTRAN code of CYANA. Reading internal variables and manipulating them to achieves custom task.

  • save the manually edited xray structure (exercise 11) or the the regularized xray structure (containing the ligand and called 'regula.pdb') as 'reg_xray.pdb' to use the macro below (or change the name in the macro accordingly).
  • what do you think about the RMSD, does the value make sense? Does the range make sense?

Below you find the commands for a macro (call it 'CALC_RMSD.cya') that will read the regularized xray structure and the calculated nmr structure, then calculating the rmsd of both the protein and ligand parts of the complex: 

read demoLong.seq

rmsd range=8-33 structure=final.pdb reference=reg_xray.pdb

atom select "BACKBONE 8-33"
t=rmsdmean
j=rindex('333')
n=0
s=0.0
do i ifira(j) ifira(j+1)-1
  if (element(i).gt.1) then
    n=n+1
    s=s+displacement(i)
   end if
end do
print "RMSD of the LIG: ${s/n} ($n atoms)"

read pdb final.pdb
structure mean
write pdb mean.pdb

read pdb mean.pdb
read pdb reg_xray.pdb append

atom select "BACKBONE 15-111"
t=rmsdmean
atom select "WITHCOORDALL"
j=rindex('333')
n=0
s=0.0
do i ifira(j) ifira(j+1)-1
  if (element(i).gt.1.and.asel(i)) then
    n=n+1
    s=s+displacement(i)*2
  end if
end do
print "Displacement of the LIG (to ref xray): ${s/n} ($n atoms)"

Exercise: Analysis of multi-state calculations

On improving the final structure

Using what you have learned so far, employing some of the options


General questions to answer regarding this task:

  • Name additional experimental restraints (or inputs) you could use for structure calculation.
  • Name additional NMR experiments you could measure, to acquire experimental data that are not supplied with the demo_data.

eNORA extensions and options

There are a variety of commands to modify eNORA runs to accommodate experimental labeling schemes or etc...


Averaging Of Spin-Diffusion Over Multiple Conformers

Generating XEASY peak list with expected FRM or two spin intensities

# --------------------------   get the shifts from a XEASY peaks list    ---------------------------

./init

# convert
read 1.peaks
shifts adapt contribution=0.0
shifts renumber
atoms set "* shift=900.0.." shift=none
write demo.prot

# --------------------------------    basic parameter definitions    --------------------------------
./init
echo:= on
mixingtimes:= 0.02,0.03,0.04,0.05,0.06
tauc        = 4.25
b0field     = 700
maxdistance = 6.5

# ----------------------------------          structure input           ----------------------------------- 

# specify the conformers for calculations
read pdb demo rigid
structure select 1

# ----------------------------------             peak input             ----------------------------------  

loadspectra structure=demo.pdb peaks=N15NOESY,C13NOESY prot=demo.prot simulate

# ---------------------------------- run eNORA elements and write peaks ----------------------------------

do n 1 length('mixingtimes')
	# FM
	enoe spindiff b0field=b0field tauc=tauc time=$mixingtimes(n) mode=3 labilatom='NONE'
	read peaks N15NOESY_exp.peaks
	enoe intensities
	write peaks N15NOESY_FM_$n.peaks names
	read peaks C13NOESY_exp.peaks
	enoe intensities
	write peaks C13NOESY_FM_$n.peaks names

	# 2 spin
	enoe twospin  b0field=b0field tauc=tauc time=$mixingtimes(n) mode=3 labilatom='NONE'
	read peaks N15NOESY_exp.peaks
	enoe intensities mode=2
	write peaks N15NOESY_2spin_$n.peaks names
	read peaks C13NOESY_exp.peaks
	enoe intensities mode=2
	write peaks C13NOESY_2spin_$n.peaks names
end do

read peaks C13NOESY_FM_1.peaks
peaks2dplot dimensions=12

read peaks C13NOESY_FM_1.peaks
read peaks N15NOESY_FM_1.peaks append

shifts initialize
shifts adapt
atom set "* shift=990.0.." shift=none
write prot NOESY_1.prot
write peaks NOESY_1.peaks
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