Objective and Overview

This tutorial illustrates the use of Normal Mode Flexible Fitting (NMFF) algorithm and MMTSB Tool Set for generating atomically detailed pathways connecting the known initial and final conformations of the system. Here, we demonstrate the scope of the method by investigating conformational transition pathway of Adenylate Kinase (ADK). Adenylate Kinase catalyzes the phosphate transfer reaction and undergoes large-scale motions from "open" to "closed" form upon binding its substrates.

Specifically, in this tutorial we will:

1. Generate initial, low-resolution pathway between the "open" and "closed" conformations of ADK with the use of NMFF program. Step-by-step instructions for doing this exercise can be found under following link (NMFF tutorial).
2. Visualize the conformational transition pathway of ADK using VMD.
3. Extract the high-frequency motions from the low-resolution pathway generated using NMFF in step 1.
4. Visualize the refined pathway from step 3 and compare it with the low-resolution pathway generated in step 1.

Generate Low-resolution Conformational Closing Pathway of Adenylate Kinase Using NMFF

Provided the "open" and "closed" structures of ADK we can obtain intermediate states between the two forms employing NMFF. To work on this section, refer to the NMFF tutorial for the detailed instructions and obtaining the required input files. Briefly, the NMFF tutorial illustrates the use of NMFF software to create the electron density map corresponding to the closed state of ADK and subsequently refine the atomic coordinates of the open conformation into this density. After the completion of the NMFF tutorial you will have about 30 intermediate states connecting open and closed form of Adenylate Kinase. It takes about an hour to complete the NMFF tutorial. If you are not able to complete the entire exercsie in the stipulated time-period, you may obtain all the conformations along the closing pathway from the files directory and proceed to the next step.

Extracting High-Frequency Motions From the NMFF Pathway

Integrating the low-resolution models with atomically detailed simulations and subsequently determining thermodynamic quantities (e.g., free energy) along the pathway is of extreme interest in the field of computational biophysics. Here, we present a way to integrate information from low-resolution models with the atomically detailed models.

Specifically, we will perform minimization with restraints starting from each structure along the pathway generated using NMFF. We will use CHARMM for this purpose. The example CHARMM input file (kinase_bead1.inp) that acomplish's this task is as follows:

* This tutorial example illustrates how to perform minimization with restraints
* in CHARMM.
* Note first we will build the missing atoms in the starting structure.
* In an accompanying example we will do all this with mmtsb command.
*

! Read in the parameter and topology files.
! We'll use the $CHARMMDATADIR as our source for top/param files

open unit 1 read form name "$CHARMMDATA/top_all22_prot_cmap.inp"
read rtf card unit 1
close unit 1

open unit 1 read form name "$CHARMMDATA/par_all22_prot_cmap.inp"
read param card unit 1
close unit 1

! Read the sequence explicitly
!read sequ card unit 5
!* Sequence for 214 residue ADK
!*
!*
read sequ card
* Adenylate Kinase
*
214
MET ARG ILE ILE LEU LEU GLY ALA PRO GLY ALA GLY LYS
GLY THR GLN ALA GLN PHE ILE MET GLU LYS TYR GLY ILE
PRO GLN ILE SER THR GLY ASP MET LEU ARG ALA ALA VAL
LYS SER GLY SER GLU LEU GLY LYS GLN ALA LYS ASP ILE
MET ASP ALA GLY LYS LEU VAL THR ASP GLU LEU VAL ILE
ALA LEU VAL LYS GLU ARG ILE ALA GLN GLU ASP CYS ARG
ASN GLY PHE LEU LEU ASP GLY PHE PRO ARG THR ILE PRO
GLN ALA ASP ALA MET LYS GLU ALA GLY ILE ASN VAL ASP
TYR VAL LEU GLU PHE ASP VAL PRO ASP GLU LEU ILE VAL
ASP ARG ILE VAL GLY ARG ARG VAL HSD ALA PRO SER GLY
ARG VAL TYR HSD VAL LYS PHE ASN PRO PRO LYS VAL GLU
GLY LYS ASP ASP VAL THR GLY GLU GLU LEU THR THR ARG
LYS ASP ASP GLN GLU GLU THR VAL ARG LYS ARG LEU VAL
GLU TYR HSD GLN MET THR ALA PRO LEU ILE GLY TYR TYR
SER LYS GLU ALA GLU ALA GLY ASN THR LYS TYR ALA LYS
VAL ASP GLY THR LYS PRO VAL ALA GLU VAL ARG ALA ASP
LEU GLU LYS ILE LEU GLY

generate PRO0 first NTER last CTER setup

open unit 9 read card name adk_mov1.pdb ! Read coordinates of the first structure
read coor pdb unit 9
close unit 9

! Since the structures from the NMFF path do not have hydrogens atoms
! we add them here. Use ic build and then hbuild.
ic param
ic build
coor init select hydrogen end

! For consistency with defaults in the mmtsb tool minCHARMM.pl, we use
! this hbuild set-up
hbuild atom cdie eps 80.0 cutnb 10.0 ctofnb 7.5 ctonnb 6.5 shift vshift bygr
update atom rdie eps 1 cutnb 12 ctofnb 11 ctonnb 8 switch vswitch bygr

! Now we will do a short minimization with harmonic restraints on CA atoms
!Restraint CA atoms with high force constant

! Copy current coordinates to comparison set
coor copy compare

cons harm force 100 mass select ( ( resid 1:214 .and. segid PRO0 ) ) .and. ( atom * * ca ) end

mini sd nprint 10 step 0.02 nstep 500 ! Steepest-Descent minimization
mini abnr nprint 10 step 0.005 nstep 1000 tolenr 1e-05 ! ABNR minimization scheme

open unit 10 write form name "movmin1.pdb" ! minimized coordinates
write coor pdb unit 10
*
close unit 10

STOP

Use the MMTSB tool convpdb.pl to "convert" from generic resiude naming convention to "charmm22" convention for residues and atoms.

convpdb.pl -out charm22 mov1.pdb > adk_mov1.pdb

The ADK conformation is minimized using CHARMM with the command:
$CHARMMEXEC < kinase_bead1.inp > kinase_bead1.out

Preferably, use minCHARMM.pl routine in the MMTSB Tool Set to perform the same task.

minCHARMM.pl -par minsteps=0,sdsteps=500,sdstepsz=0.02 \
-par minsteps=1000,minmode=abnr \
-par trunc=switch,dielec=rdie,cutnb=12,cuton=8,cutoff=11 \
-par param=22x,cmap\
-par xtop=top_all22_prot_cmap.inp \
-par xpar=par_all22_prot_cmap.inp \
-cons ca self 1:214_100 -cmd mmtsbmin.inp -log mmtsbmin$i.log \
mov$i.pdb > movmin$i.pdb

Now minimize other intermediate states along the pathway using the same protocol. You have a choice of either submitting jobs manually after making changes to CHARMM input file (kinase_bead1.inp) to read the next structure along the pathway or execute this simple shell script (runcharmm.sh) to perform the task automatically (it will take approximately 1 hour to minimize all the intermediate conformations). If more than one CPU is available this task can also be performed in parallel. This is particularly attractive feature of the method and makes it computationally tractable to investigate conformational transitions of large biomolecules.

After minimization jobs are finished, load in VMD all the minimized intermediate conformations along the ADK pathway. You can do this by issuing the following commands in the "tkcon" console of VMD:

source animatepbs.tcl
animatepdbs 1 30 "movmin%0d.pdb"

Turn on the simultaneously the representation of the molecule to "Cartoon" for the entire protein and to "Licorice" for only few selected residues, numbered 36, 88, 123, 156, 16 and 13 in the PDB files. Now play the trajectory and visualize the atomically detailed residue motions that were missing in your initial NMFF pathway.

In principle, it is also feasible to extract thermodynamically relevant information from the NMFF pathways. Although, to put this into practice much more computationally intensive calculations are required and is beyond the scope of this workshop. If time permits, interested participants may work on the suggested exercise.

Suggested exercise: To obtain more thermodynamically meanigful pathway perform dynamics with harmonic restraints on backbone atoms instead of minimization. Run a short dynamics simulation starting from intermediate structures along the Adenlylate Kinase pathway using mdCHARMM.pl routine in MMTSB Tool Set as explained in RNAse C-peptide tutorial.