A tutorial for molecular dynamics simulations using Amber package

: In this paper we present a tutorial for performing molecular dynamics calculations utilizing Amber package. Through it we explain the function of each step in a theoretical work of MD, thus enabling the application of acquired knowledge to other systems without many difficulties. For this tutorial, we simulated by MD and analyzed various geometrical and structual parameters for doxycycline, a potent antimicrobial agent


Introduction
AMBER is a package of computer programs whose function is to allow users to perform simulations of molecular dynamics (MD) with chemical structures of many types, such as: organic and inorganic molecules and biomolecules [1].The term "AMBER" is also sometimes used to refer to empirical force fields [2].
Neither individual program of the AMBER package performs all steps of the MD, but together they offer a complete procedure, being a powerful framework for many common calculations.223 mechanics (QM).
The advantage of the QM/MM simulation is in fact supporting the gas phase and buffer (with hundreds of atoms) using semi-empirical methods.The AMBER package has tools that allow the correction of the system volume by multiplication of the box solvation with explicit solvent.Moreover it has the extensive chemical structures can be used as solvent of the system.This package consists of several programs, including: • Leap: used to prepare the system for the simulation programs; • Antechamber: automates the process of parametrization; • Sander: central simulation program and has facilities for energy minimization and molecular dynamics with a wide variety of options; • Ptraj: used to perform analysis of simulation results.
In this tutorial, we use the doxycycline (Fig. 1), derived from the oxytetracycline, because presents high power antibiotic and high degree of lipossolubility.It is highly stable in normal human serum.Basically bacteriostatic, has antimicrobial action activated by inhibiting protein synthesis.The compound is used against a wide variety of Grampositive and Gram-negative bacteria and other microorganisms [3].

PREPARATION OF THE FILES TO MOLECULAR DYNAMIC SIMULATION
We use two auxiliary programs Ghemical [4] and Gaussian03 [5]) to prepare the solute to start the procedures by AMBER package.

Creating a directory
Firstly is necessary creating a directory on the machine where the calculations will Garcia et al.

Technical Note
Orbital Elec.J. Chem., Campo Grande, 4(3): 222-234, 2012 224 224 be performed.We will do all the steps remotely (using the tool for remote user), this requires access to the central directory to be used by the terminal command: ssh user@<remote machine name> After accessing the machine where the calculations are performed, we will create and enter into the directory that stores data about the calculation.To create it, the command is mkdir, and how the name is "doxycycline", we have: To open the execution command we use the command cd cd doxycycline Into the directory, having the file of the optimized coordinates of the drug by classical mechanics, the process of preparation and simulation of the chosen system can be done.The procedure was divided in stages or steps in order to facilitate understanding.

Optimization the geometry of the structure
We perform the optimization of the geometry using quantum calculations at the HF/6-31G(d,p) theory level, in order to obtain the geometry of lowest energy.
In this theory, the base set used for the calculations is divided generally for "a-bcG(d,y)", and each letter corresponds to a variable used in each part of the model, where "a" represents the number of Slater primitive (STO) necessary to describe the valence shell, "b" and "c" represents the number of Gaussians primitive (GTO) required for each STO involved and the "x" and "y" means the polarization [5].
To prepare the compound for this stage is necessary converts the format file of the coordinates (.gpr to .gau), in order to start the calculations by Gaussian03.The command that contains this function runs as follows "babel": [6] babel -igpr doxycycline.gpr-ogau doxycycline.gauThen the input file for calculating the Gaussian03 (doxycycline.gau) is edited by adding a header that indicates the type of calculation used and the theory will add the following data, which indicates a geometric optimization:

%chk=doxiciclina.chk #P HF/6-31G(d,p) opt test
With the file ready, we start the calculations in the Gaussian03 using a simple command, as below: Garcia et al.

Technical Note
Orbital Elec.J. Chem., Campo Grande, 4( 3 Where doxycycline.gaurepresents the input file with initials coordinates and doxycycline.log is the output file that will contains geometric data and physical conformation of the best energy obtained.The "&" permits the machine to be used while the calculations are performed.
We can follow the calculations from the command less, thus the calculation can be followed in real time.At the end of the doxycycline.logoutput file, we need to have the following term: "Normal termination of Gaussian 03".
With this result, begin to prepare our system for the main stage of this tutorial, the molecular dynamics simulation.To continue is necessary the conversion of doxycyline.logfile to pdb format: the starting point for AMBER program.The command will be, as follows: babel -ig03 doxycycline.log-o pdb doxycycline.pdb After conversion, our next step is to edit the file doxycycline.pdbusing a text editor.We need to withdraw all lines beginning with the term "CONNECT".

nd Step
To start the simulation for MD, the system is prepared with the compound in aqueous solution.
Using the AMBER package some important files are generated, for example, doxycycline.prmtopthat contains the parameters of the structure and doxycycline.inpcrd,the initial coordinates.
Into Amber Package exists a diverse set of solvents.For this simulation we use SPC/E water model [7] as a solvent and treated without interactions between the electronic density of atoms of the same structure.
The files that Amber package needs are obtained through three programs: antechamber, parmchk and tleap.
Antechamber is designed to build an archive of preparation for the topology of the solute [8], in this case, doxycycline.In combination with DivCon (a semiempirical QM program) calculate partial atomic charges.By default, the force field GAFF is used to describe the solute-solvent interactions, since this option is indicated on the solute with few atoms or represented by small molecule [9].The command used by antechamber is as follows:

antechamber -i doxycycline.pdb -fi pdb -o doxycycline.prep -fo prepi -c bcc
In this command each index means: It generates a cubic box, where are the solute and solvent molecules (Fig. 2).

Preparation of the files to molecular dynamic simulation
Simulations using the AMBER package are divided in four steps, and taking a specific function and dependent on each other.The first and second steps are optimization geometries; the third, is MD termalization phase (increase system temperature); and, the latter isMD equilibration.Each of the steps is described below: Step 1: We performed an energy minimization to relieve steric interactions that could interfere with the dynamics [5].
Step 2: Minimization of the system as a whole, without restrictions [5].Step 3: Restrict Position MD and termalization step.This calculation is carried out starting position to relax the molecules of solvent around solute and heat the system to target temperature.The movements of doxycycline are restricted, so a force is applied in order to maintain the its positions.The relaxation time of the solvent for water is about 10 ps, so should perform at least another 10 ps of dynamics to relax the solvent [5].&cntrl imin = 0, irest = 0,  0 = without effect condition to restart the calculation (default) ntx = 1,  1= to read the initial coordinates from the file "inpcrd" ntb = 1, cut = 10, ntr = 1, ntc = 2,  2=hydrogens bonds are contained ntf = 2,  2=hydrogen bonds are omitted in the calculations tempi = 0.0,  initial temperature of the system temp0 = 298.0, equilibrium temperature of the system ntt = 3,  thermostat scale (1 = Langevin Dynamics) gamma_ln = 1.0,  collision frequency in ps-1 when ntt = 3 nstlim = 12500, dt = 0.002,  simulation time:nº steps multiplied by integration time ntpr = 100, ntwx = 500, ntwr =1000  the calculated coordinates are archived / Keep solute fixed with weak restraints 10.0 RES 1 END END Scheme 3. Data founded in file md_1.in.(Step 3).

Unrestrained minimization &cntrl
Step 4: MD equilibration, without restriction, at desired temperature, should be used 2,000 ps [5].This value was taken to be only an example for the tutorial.
But it becomes necessary to create files to indicate the number of processors to be used and the type of calculation made by the cluster, the following are those along the line of command: In this case there was an interaction that lasted long enough to be noticed

Root-mean-square deviation (RMSD)
This analysis is a major tool of molecular dynamics simulations.It is possible to study the stability, balance and flexibility of the system [14,15].
There is a link between the number of atoms and the elevation and index RMSD is independent of choosing atoms participate in this analysis.To view this file (fig.4), we must create and edit file rmsd.in, as below:

Conclusion
We presented a tutorial explaining how to conduct computational simulations for solute-solvent systems by MD.In particular, we addressed the computational resources of the AMBER package.Additionally, we explained how to perform some analysis on the use of this very common computational technique.Thus, we believe that the information given in this work can assist beginners in the application of MD in their computational studies.

Figure 2 .
Figure 2. Cubic box with the solute and solvent molecules.
Garcia et al.