Computational Chemistry

L-tert-Leucine

This is a tutorial for intermediate level users. To comprehend it, you will need to know how to:

• Build a system to run molecular dynamics (MD) with Amber.
• Submit calculations using Gaussian.

This tutorial was designed as an alternative (or as a supplement) to the official Amber tutorials for parameterizing a custom residue. It summarizes information from three Amber tutorials:

I invested a considerable amount of time following Amber’s recipes to parameterize custom residues. I achieved my goal by combining parts of them to create my own protocol. I hope it also helps you. As I mentioned, this is a recipe; there is no technical or theoretical information included. That part is well explained in the Amber tutorials.

This is not the only or the best method, but it is one that works.

This tutorial can be viewed from a bird’s eye perspective by advanced users who are just looking for a few lines or commands to remember (or to tweak something in their regular workflow). Alternatively, beginners can follow along at a slower pace by reading the basics of what is being done. If additional information is needed, links to explanations will be included.

#### I) Residue Structure Selection

To calculate the partial atomic charges, we will need a 3D representation of our non-standard residue. For this, we can:

1. Build it using, Gaussview, Maestro or any other software you are familiar with.
2. Obtain the atomic coordinates from a PDB file or any other source that includes 3D information about the molecule.

In this case, the residue to be parameterized is L-tert-Leucine, which I refer to as LTL. However, the side chain can be anything you desire.

Keep in mind:

In a amino acid C=O is the tail and the NH is the head. (Fig. 1)

When naming your custom residues, use three or four capital letters, ensuring that the name hasn’t already been used in the names of Amber force field residues.

The residue is capped using NME and ACE groups, which has a purpose beyond maintaining the unit charge of the amino acid. When a CH3 is bonded to the pre-tail (C=O) or post-head (NH) atoms, Antechamber will recognize the C atom as an SP3 alpha-carbon and the Hs as alpha hydrogens. So, by using these capping groups, Antechamber will create the parameters for the backbone of our L-tert-Leucine (angle and dihedral values). This way, the residue will automatically bond to the previous and next residues in the chain. Please continue reading for further details.

#### II) Geometry Optimization

To acquire the parameters for our custom residue, its geometry needs to be optimized. This optimized geometry will be used to compute the electrostatic potential of the system. This procedure will be conducted in vacuo using B3LYP and a 6-31G* basis set.

B3LYP geometry optimization input

LTL_opt.com

Submit the Calculation as usual:

`g16  LTL_opt.com`

Examine the log file (LTL_opt.log) in GaussView, or your preferred software, to confirm that everything is in order..

#### III) Electrostatic potential Charges calculation (ESP-charges)

Now, with the system in its optimized geometry, a Hartree-Fock (HF) calculation must be performed to obtain the Electrostatic Potential.

The procedure used in the development of the Amber force field to acquire the partial atom charges for every residue will be employed here. In other words, the electrostatic potential will be obtained in the gas phase using a HF/6-31G* QM calculation because “Conveniently the error in the HF/6-31G* calculation is close to the difference between the charge distribution in gas phase and that in solution “

Remember, do not change the QM level of theory when deriving charges. Use the same QM level that was used to derive the charges in the Amber forcefield.

LTL_hf.com

Submit:

`g16  LTL_hf.com`

Output: LTL_hf.log

#### IV) Restrained Fitting of the Partial Charges to the Electrostatic Potentials (RESP Calculation)

The electrostatic potential must be in a format that the resp program can understand. Run the following command to extract the electrostatic potential from the Gaussian output using espgen:

submit:

`espgen -i LTL_hf.log -o esp.dat`

Output: esp.dat

Now, we need to derive the partial charges for every atom in our residue, fitted to the ESP. Additionally, we must fix the values of the partial charges of the atoms in the ACE and NME residues to their corresponding values in the Amber force field. (Fig. 2)

Once you have the esp.dat file, you will need two additional files to instruct the RESP program which atoms will maintain a given partial charge value, and for which ones the charge will be calculated.

The first is the resp.in file. This serves as the input for this calculation (the file here contains some comments that will help you to understand it better). The description of this file isn’t included in the Amber manual, but you can read about it here: : http://ambermd.org/tutorials/advanced/tutorial1/section1.htm

resp.in (with comments, delete them before using the file)

CAUTION: Pay attention to the spaces, and blank lines at the end of the resp.in file, as it is sensitive to these.

The Second file is the resp.qin file. This is where the charges of the NME and ACE are provided to the RESP calculation.

resp.qin

This file has the following characteristics: each value is a partial charge, each line contains 8 entries (first line corresponds to atoms 1 to 8, the second line to atoms 9 to 16, and so on), with 10 characters for each entry. A new value is read subsequently. Each value here corresponds to an atom in the resp.in file. Thus, if a value here is different from 0.0000000, a -1 should be written in the second column for that atom in the resp.in file. Comparing the resp.in and the resp.qin files can enhance your understanding.

submit:

`resp -O -i resp.in -o resp.out -p resp.pch -t resp.chg\ -q resp.qin -e esp.dat`

Output: resp.out, resp.pch, resp.chg

If you encounter the “At line 403 of file resp.F (unit = 5, file = ‘resp.in’)” error, you may need to update your AmberTools. This issue was resolved in Amber 18 but not in Amber 16. Another possible error is related to the end-of-file during read. In such a case, include a blank line at the end of the resp.in file.

Another common error eported in the comments of this tutorial is:

At line 386 of file resp.F (unit = 5, file = ‘resp.in’)

As Adrian Garcia pointed out in his response, the “number of the molecule” in the resp.in file is read as a floating point, while it’s written as an integer in the file. For him, changing 1 to 1.0000 solved the problem. Thanks for sharing your solutions to this issue.

In the resp.chg file are now the partial charges for each atom in the custom residue.

#### VI) .ac File Generation or Starting to Speak Amber Language

Here, we’ll associate the calculated partial charges with each atom in the previously optimized structure. We’ll also assign a name to the residue using Amber’s nomenclature. This is all accomplished in a single file, LTL.ac.

`antechamber -fi gout -i LTL_hf.log -bk LTL -fo ac\ -o LTL.ac -c rc -cf resp.chg -at amber`

output : LTL.ac

#### VII) Creating the Residue for Amber Recognition

Now, we have to remove the capping groups from the custom residue and turn it into a new residue using prepgen. The main chain file contains the information needed for this. LTL.mc

The names used in the main chain file (.mc) are the same as those in the .ac file.

`prepgen -i LTL.ac -o LTL.prepin -m LTL.mc -rn LTL`

The final step is renaming the atom names in the .prepin file to match the atom names in the PDB file, if for some reason they differ.

output: LTL.prepin

In the 4-Hydroxyl-Proline (PR4) tutorial, the final step simply involves loading the .prepin file to obtain a system ready for simulation. However, if you’ve modified the backbone or are attempting to model a highly modified N or C terminal, prepgen may not generate the appropriate atom types for these unique atoms. Thus, you’ll need to generate a file with the atom types in the GAFF AND another with the atom types in the ff14SB. The next section demonstrates how to accomplish this when necessary. For a “standard custom residue”, we’re done.

#### VII.2) Generating the Parameters Files for Special Cases

By “special cases”, I refer to residues that possess a bond, angle, dihedral, or functional group not present in the protein Amber force field. As such, these parameters must be obtained using the General Amber Force Field (GAFF). To obtain them, execute:

`parmchk2 -i LTL.ac -f ac -o LTL_gaff.frcmod`

This command yields the file: LTL_gaff.frcmod, which needs to be included in the next step before loading the “LTL_ff14SB.frcmod” parameters. This ensures that the ff14SB parameters take precedence over GAFF whenever applicable..

You’ll know if you need to perform this step if, after completing the following step, you see lines with the message “ATTN: needs revision”:

`parmchk2 -i LTL.ac -f ac -o LTL_ff14SB.frcmod -a Y -p \$AMBERHOME/dat/leap/parm/parm10.dat`

#### VIII) Finally, Putting It All Together

open tleap by typing: tleap

once inside, build a test system typing the following lines

```loadamberprep LTL.prepin
source leaprc.protein.ff14SBloadamberparams LTL_gaff.frcmod   !if you obtained lines with the ATTN message in the ff14SB fileloadamberparams LTL_ff14SB.frcmod !after having deleted the lines with the ATTN message
aa = sequence {ACE LTL NME}
saveAmberParm aa aa.prmtop aa.rst7```

If you need help with a specific topic, let me know in the comments.

1. Anderson

Dear,
I’m trying your tutorial and got stuck in the following error:

At line 386 of file resp.F (unit = 5, file = ‘resp.in’)

Thank you!

• CarlosRamosG

Hi Anderson.

Did you delete the text after the exclamation signs in the resp.in file? ah! and don’t forget to include a blank line after the last line in the text.

try again and let me know if that was the problem.

Regards.

I had a similar problem and by looking at the code in resp.F I noticed that the variable reading “number of the molecule” in the resp.in file is defined as floating point when read, whereas in the file is written as an integer. Changuing 1 by 1.0000 solved the problem for me, so try that.

2. Johanna

Did this tutorial without having ever worked with amber before. It was easy to follow and everything worked out perfectly fine. Great tutorial- Thank you for your work!

3. Zahra A.

Thank you so much for preparing such a great tutorial on “Amber Custom Residue Parameterization”. I got an issue at RESP step calculation for my model which is ligand covalently bonded to Cys residue of protein. (resp -O -i resp.in -o resp.out -p resp.pch -t resp.chg -q resp.qin -e esp.dat). It will be great to have an email for sharing results with you which is a big help to me fixing the issue. I appreciate your consideration.
Bests
Zahra

• CarlosRamosG

Hello Zahra,
Thank you for your interest in this tutorial. If you wish to parameterize a residue bonded to a Cys residue, likely through the sulfur, the recommended approach is to begin with an initial structure that includes both residues bonded to each other. Additionally, ensure that each residue has its corresponding cap termini residues ACE and NME.

your initial structure should looks like this:

If you follow this approach, you can parameterize your custom Cysteine (CYA) and your custom residue (RES). To obtain charges for each atom in the RESP program, ensure the preparation of corresponding resp.in and resp.qin files, incorporating charge values for atoms in the ACE and NME residues, as demonstrated in the tutorial. If you choose not to parameterise the new Cys bonded to your RES, include the charges of the CYX amber residue in the resp.qin file. This way, only the charges of RES will be fitted based on the values provided in the resp.qin file.

The main distinction arises when creating the unit without additional atoms, particularly when creating the residue for Amber recognition. Specify the exclusion of atoms by indicating their numbers in the main chain file. Your RES.mc file should resemble the following:

TAIL_NAME C8
MAIN_CHAIN C3
OMIT_NAME C1
.
.
.
OMIT_NAME H18
POST_TAIL_TYPE N
CHARGE 0.0 (the charge of your residue)

Adapt it accordingly to your system. List each atom you wish to exclude from your residue by employing the OMIT_NAME keyword, as exemplified in the LTL.mc example file.

Proceed with the rest of the tutorial to generate the frcmod and prepin files.

When preparing your system using leap, remember to bond the two atoms between CYS and RES with the following line:

bond SYSTEM.XX.AA SYSTEM.YY.BB

Here, SYSTEM is the name you assign to your system in leap, XX denotes the number of the Cys atom named AA bonded to the atom named BB with number YY in your “RES.”

I hope this information helps you with the parameterization of your custom residue.

4. Zahra A.

Great! Thank you so much Carlos for the prompt response and sharing technical details. I appreciate that. I will follow all details you mentioned. Yes, there is a covalent bond between S-Cys and C-ligand so I am wondering if I need to CAP ligand or not, and the ligand has charge of -2 at its terminal group. I have considered this coordination model to start optimization with Gaussian, If it looks fine then I can move on with other steps.
Bests
Zahra

C -2.69100000 -17.32800000 23.67100000
C -3.97800000 -16.74700000 24.26500000
O -4.15700000 -16.81200000 25.49900000
H -2.69500000 -17.18900000 22.59000000
H -2.63300000 -18.39200000 23.90000000
H -1.82900000 -16.81600000 24.10000000
N -4.84000000 -16.19000000 23.41000000
C -6.12000000 -15.51000000 23.79000000
C -6.86000000 -15.13000000 22.50000000
S -8.23000000 -13.97000000 22.87000000
C -6.96000000 -16.39000000 24.74000000
O -7.67000000 -15.81000000 25.59000000
C -9.79000000 -14.58000000 21.84000000
C -10.90000000 -14.33000000 22.87000000
N -10.02000000 -13.80000000 20.68000000
P -8.47000000 -23.96000000 22.39000000
O -9.80000000 -24.68000000 22.12000000
O -7.17000000 -24.74000000 22.02000000
O -8.36000000 -23.29000000 23.80000000
C -9.55000000 -22.01000000 20.83000000
O -8.37000000 -22.66000000 21.27000000
C -9.43000000 -20.49000000 20.94000000
O -9.67000000 -20.08000000 22.29000000
C -8.05000000 -19.99000000 20.49000000
O -7.88000000 -20.28000000 19.10000000
C -7.84000000 -18.48000000 20.71000000
O -6.53000000 -18.11000000 20.28000000
C -8.85000000 -17.68000000 19.90000000
N -10.06000000 -17.02000000 22.24000000
C -10.81000000 -16.77000000 23.36000000
O -11.16000000 -17.65000000 24.15000000
N -11.25000000 -15.44000000 23.59000000
O -11.38000000 -13.22000000 23.03000000
C -9.06000000 -13.94000000 19.68000000
C -8.67000000 -12.90000000 18.82000000
C -7.69000000 -13.09000000 17.84000000
C -7.29000000 -11.95000000 16.94000000
C -7.06000000 -14.36000000 17.72000000
C -5.97000000 -14.59000000 16.71000000
C -7.46000000 -15.40000000 18.57000000
C -8.47000000 -15.21000000 19.53000000
C -9.63000000 -16.02000000 21.49000000
N -8.96000000 -16.27000000 20.34000000
H -4.61000000 -16.19000000 22.42000000
H -5.88000000 -14.60000000 24.37000000
H -7.23000000 -16.03000000 21.99000000
H -6.16000000 -14.62000000 21.81000000
H -10.43000000 -22.34000000 21.40000000
H -9.72000000 -22.26000000 19.76000000
H -10.21000000 -20.07000000 20.26000000
H -9.84000000 -19.10000000 22.29000000
H -7.30000000 -20.53000000 21.09000000
H -7.05000000 -19.82000000 18.85000000
H -7.96000000 -18.26000000 21.78000000
H -5.92000000 -18.42000000 21.00000000
H -9.86000000 -18.11000000 20.00000000
H -8.58000000 -17.68000000 18.84000000
H -11.90000000 -15.31000000 24.37000000
H -10.38000000 -12.86000000 20.87000000
H -9.14000000 -11.91000000 18.94000000
H -6.21000000 -11.71000000 17.05000000
H -7.44000000 -12.21000000 15.87000000
H -7.87000000 -11.04000000 17.16000000
H -5.11000000 -13.91000000 16.87000000
H -5.59000000 -15.63000000 16.76000000
H -6.33000000 -14.40000000 15.68000000
H -6.96000000 -16.38000000 18.51000000
N -6.85500000 -17.72100000 24.64600000
C -7.80200000 -18.61800000 25.35500000
H -6.11900000 -18.12200000 24.08300000
H -8.52400000 -18.01900000 25.90900000
H -7.25100000 -19.25500000 26.04700000
H -8.32700000 -19.23900000 24.62900000

• CarlosRamosG

Hi Zahra,
Having visualised your structure, it’s clear to me that you don’t need to cap your “RES” residue as it’s unlikely to be attached to a backbone chain. If this is the case, you can start optimising the geometry of this structure and then follow the tutorial.

Let me know if this is clear enough.

• Zahra A.

Hi Carlos. I have done geom optimization, RESP (-2 charge, 73 atoms and setting -1 to ACE and NME residues and other atoms equal to zero). There were two issues at RESP steps (FORTRAN runtime error: Bad value during floating point read, Fortran runtime error: End of file) and have been fixed based on reported commands from other users. Then, antechamber -fi gout -i FMN-hf.log -bk FMN -fo ac -o FMN.ac -c rc -cf resp.chg -at amber, here is the FMN.ac file:
(base) cat FMN.ac
CHARGE -2.00 ( -2 )
Formula: H31 C23 N6 O11 P1 S1
ATOM 1 N1 MOL 1 -1.402 -1.545 3.066 -0.591590 N
ATOM 2 N2 MOL 1 -0.334 -3.052 0.932 -0.342128 N
ATOM 3 C1 MOL 1 -2.278 -2.023 2.002 -0.025870 CT
ATOM 4 C2 MOL 1 -0.100 -1.751 5.125 -0.612845 CT
ATOM 5 C3 MOL 1 0.415 -4.051 0.182 -0.334400 CT
ATOM 6 C4 MOL 1 -1.652 -3.191 1.205 0.660925 C
ATOM 7 C5 MOL 1 -1.043 -2.359 4.104 0.783892 C
ATOM 8 O1 MOL 1 -2.337 -4.141 0.808 -0.612160 O
ATOM 9 O2 MOL 1 -1.451 -3.526 4.174 -0.611120 O
ATOM 10 C6 MOL 1 -2.576 -0.818 1.089 -0.543266 CT
ATOM 11 S1 MOL 1 -3.612 -1.308 -0.343 0.000000 S
ATOM 12 P1 MOL 1 6.748 0.192 -0.040 0.000000 P
ATOM 13 O3 MOL 1 7.293 -0.106 -1.453 -0.544005 O2
ATOM 14 O4 MOL 1 7.606 1.139 0.833 -0.610434 O2
ATOM 15 O5 MOL 1 6.171 -1.023 0.735 -0.574457 O2
ATOM 16 C7 MOL 1 4.666 1.277 -1.581 0.044452 CT
ATOM 17 O6 MOL 1 5.386 1.204 -0.401 -0.175829 OS
ATOM 18 C8 MOL 1 3.337 0.530 -1.462 -0.022976 CT
ATOM 19 O7 MOL 1 3.659 -0.851 -1.651 -0.496317 OH
ATOM 20 C9 MOL 1 2.675 0.736 -0.100 0.131863 CT
ATOM 21 O8 MOL 1 2.771 2.117 0.218 -0.656784 OH
ATOM 22 C10 MOL 1 1.208 0.295 -0.082 0.045337 CT
ATOM 23 O9 MOL 1 0.658 0.441 1.204 -0.608289 OH
ATOM 24 C11 MOL 1 0.466 1.144 -1.081 -0.072047 CT
ATOM 25 N3 MOL 1 -0.881 -0.765 -2.454 -0.492301 DU
ATOM 26 C12 MOL 1 -1.475 -1.698 -3.268 0.595808 C
ATOM 27 O10 MOL 1 -0.863 -2.626 -3.792 -0.539308 O
ATOM 28 N4 MOL 1 -2.858 -1.540 -3.563 -0.378562 N
ATOM 29 C13 MOL 1 -3.714 -0.672 -2.946 0.540779 C
ATOM 30 C14 MOL 1 -3.119 0.021 -1.716 -0.347934 CT
ATOM 31 O11 MOL 1 -4.879 -0.481 -3.279 -0.517951 O
ATOM 32 C15 MOL 1 -3.144 2.153 -0.608 0.007567 CA
ATOM 33 N5 MOL 1 -3.692 1.299 -1.557 -0.296909 NT
ATOM 34 C16 MOL 1 -3.898 3.148 0.037 -0.271554 CA
ATOM 35 C17 MOL 1 -3.317 4.014 0.974 0.070769 CA
ATOM 36 C18 MOL 1 -4.155 5.071 1.653 -0.241331 CT
ATOM 37 C19 MOL 1 -1.937 3.871 1.286 0.072834 CA
ATOM 38 C20 MOL 1 -1.277 4.766 2.309 -0.240244 CT
ATOM 39 C21 MOL 1 -1.185 2.881 0.636 -0.217269 CA
ATOM 40 C22 MOL 1 -1.765 2.027 -0.318 0.017070 CA
ATOM 41 C23 MOL 1 -1.616 0.084 -1.767 0.693489 CM
ATOM 42 N6 MOL 1 -1.011 1.053 -1.027 -0.074009 NT
ATOM 43 H1 MOL 1 0.338 -5.041 0.668 0.140227 H1
ATOM 44 H2 MOL 1 1.470 -3.742 0.145 0.153090 H1
ATOM 45 H3 MOL 1 -0.496 -1.942 6.136 0.174640 HC
ATOM 46 H4 MOL 1 0.876 -2.262 5.047 0.190410 HC
ATOM 47 H5 MOL 1 -1.058 -0.584 3.031 0.331452 H
ATOM 48 H6 MOL 1 0.165 -2.275 1.363 0.292543 H
ATOM 49 H7 MOL 1 0.049 -0.670 4.981 0.154416 HC
ATOM 50 H8 MOL 1 -3.208 -2.429 2.434 0.178143 H1
ATOM 51 H9 MOL 1 0.028 -4.134 -0.849 0.146485 H1
ATOM 52 H10 MOL 1 -1.614 -0.420 0.728 0.173219 H1
ATOM 53 H11 MOL 1 -3.091 -0.033 1.668 0.263073 H1
ATOM 54 H12 MOL 1 5.229 0.827 -2.426 0.063424 H1
ATOM 55 H13 MOL 1 4.453 2.339 -1.827 0.016626 H1
ATOM 56 H14 MOL 1 2.679 0.882 -2.282 0.111883 H1
ATOM 57 H15 MOL 1 2.826 -1.352 -1.742 0.318709 HO
ATOM 58 H16 MOL 1 3.254 0.121 0.620 0.124128 H1
ATOM 59 H17 MOL 1 2.341 2.236 1.088 0.422025 HO
ATOM 60 H18 MOL 1 1.149 -0.766 -0.401 0.090879 H1
ATOM 61 H19 MOL 1 1.198 -0.083 1.828 0.412515 HO
ATOM 62 H20 MOL 1 0.747 0.830 -2.093 0.009937 H1
ATOM 63 H21 MOL 1 0.730 2.201 -0.947 0.148513 H1
ATOM 64 H22 MOL 1 -3.235 -2.133 -4.307 0.309656 H
ATOM 65 H23 MOL 1 -4.699 1.339 -1.727 0.331129 H
ATOM 66 H24 MOL 1 -4.963 3.234 -0.205 0.180659 HA
ATOM 67 H25 MOL 1 -4.164 4.930 2.750 0.076636 HC
ATOM 68 H26 MOL 1 -3.749 6.082 1.467 0.084836 HC
ATOM 69 H27 MOL 1 -5.196 5.044 1.293 0.065213 HC
ATOM 70 H28 MOL 1 -1.762 4.673 3.298 0.066900 HC
ATOM 71 H29 MOL 1 -0.210 4.515 2.423 0.084432 HC
ATOM 72 H30 MOL 1 -1.352 5.831 2.019 0.076821 HC
ATOM 73 H31 MOL 1 -0.134 2.758 0.897 0.194484 HA
BOND 1 1 3 1 N1 C1
BOND 2 1 7 1 N1 C5
BOND 3 1 47 1 N1 H5
BOND 4 2 5 1 N2 C3
BOND 5 2 6 1 N2 C4
BOND 6 2 48 1 N2 H6
BOND 7 3 6 1 C1 C4
BOND 8 3 10 1 C1 C6
BOND 9 3 50 1 C1 H8
BOND 10 4 7 1 C2 C5
BOND 11 4 45 1 C2 H3
BOND 12 4 46 1 C2 H4
BOND 13 4 49 1 C2 H7
BOND 14 5 43 1 C3 H1
BOND 15 5 44 1 C3 H2
BOND 16 5 51 1 C3 H9
BOND 17 6 8 2 C4 O1
BOND 18 7 9 2 C5 O2
BOND 19 10 11 1 C6 S1
BOND 20 10 52 1 C6 H10
BOND 21 10 53 1 C6 H11
BOND 22 11 30 1 S1 C14
BOND 23 12 13 9 P1 O3
BOND 24 12 14 9 P1 O4
BOND 25 12 15 9 P1 O5
BOND 26 12 17 1 P1 O6
BOND 27 16 17 1 C7 O6
BOND 28 16 18 1 C7 C8
BOND 29 16 54 1 C7 H12
BOND 30 16 55 1 C7 H13
BOND 31 18 19 1 C8 O7
BOND 32 18 20 1 C8 C9
BOND 33 18 56 1 C8 H14
BOND 34 19 57 1 O7 H15
BOND 35 20 21 1 C9 O8
BOND 36 20 22 1 C9 C10
BOND 37 20 58 1 C9 H16
BOND 38 21 59 1 O8 H17
BOND 39 22 23 1 C10 O9
BOND 40 22 24 1 C10 C11
BOND 41 22 60 1 C10 H18
BOND 42 23 61 1 O9 H19
BOND 43 24 42 1 C11 N6
BOND 44 24 62 1 C11 H20
BOND 45 24 63 1 C11 H21
BOND 46 25 26 1 N3 C12
BOND 47 25 41 2 N3 C23
BOND 48 26 27 2 C12 O10
BOND 49 26 28 1 C12 N4
BOND 50 28 29 1 N4 C13
BOND 51 28 64 1 N4 H22
BOND 52 29 30 1 C13 C14
BOND 53 29 31 2 C13 O11
BOND 54 30 33 1 C14 N5
BOND 55 30 41 1 C14 C23
BOND 56 32 33 1 C15 N5
BOND 57 32 34 7 C15 C16
BOND 58 32 40 8 C15 C22
BOND 59 33 65 1 N5 H23
BOND 60 34 35 8 C16 C17
BOND 61 34 66 1 C16 H24
BOND 62 35 36 1 C17 C18
BOND 63 35 37 7 C17 C19
BOND 64 36 67 1 C18 H25
BOND 65 36 68 1 C18 H26
BOND 66 36 69 1 C18 H27
BOND 67 37 38 1 C19 C20
BOND 68 37 39 8 C19 C21
BOND 69 38 70 1 C20 H28
BOND 70 38 71 1 C20 H29
BOND 71 38 72 1 C20 H30
BOND 72 39 40 7 C21 C22
BOND 73 39 73 1 C21 H31
BOND 74 40 42 1 C22 N6
BOND 75 41 42 1 C23 N6

• CarlosRamosG

Ok, this is good. But remember that if you do it this way, you will end up with one large residue containing both your “RES” and the CYS to which it is attached. This means that your “FMN” unit is actually two residues and that they need to be included in your PDB file in this way. Before continuing with the parameterisation process, let me know if this is what you want to do, because if it is not, the approach to solving the problem will be different.

• Zahra A.

Hi Carlos. Thank you ! yes, the residue is a bit big and I need to consider them as a merged residue with name of FMN and should also rename it on final PDB file. I think you have seen my message out of order that I posted 🙂 but it should be fine. There was an issue with parmchk2 for preparing FMN_gaff.frcmod file and it has been fixed. Now I have prepared FMN_gaff.frcmod and FMN_ff14SB.frcmod but both have couple of records tagged with “ATTN, need revision”. Can you please let me know how to modify them for using in tleap and writing parm files for MD. Bests/Zahra

5. Zahra A.

Then, prepgen -i FMN.ac -o FMN.prepin -m FMN.mc -rn FMN as:
POST_TAIL_TYPE is N
Net charge of truncated molecule is -2.00
TAIL_ATOM 6 C4
MAIN_CHAIN 1 1 N1
MAIN_CHAIN 2 3 C1
MAIN_CHAIN 3 6 C4
OMIT_ATOM 1 4 C2
OMIT_ATOM 2 45 H3
OMIT_ATOM 3 46 H4
OMIT_ATOM 4 49 H7
OMIT_ATOM 5 7 C5
OMIT_ATOM 6 9 O2
OMIT_ATOM 7 2 N2
OMIT_ATOM 8 48 H6
OMIT_ATOM 9 5 C3
OMIT_ATOM 10 43 H1
OMIT_ATOM 11 44 H2
OMIT_ATOM 12 51 H9
Number of mainchain atoms (including head and tail atom): 3
Number of omited atoms: 12
Warning: ATOM N3 has unfilled valence, assuming the linked atom name (in other residue) is “M”
change “M” to “-M” if ATOM N3 is linked to the immediate previous residue
change “M” to “+M” if ATOM N3 is linked to the immediate after residue(base)
then I stopped at the following step with Segmentation fault error.
parmchk2 -i FMN.ac -f ac -o FMN_gaff.frcmod
Warning: Atom type (DU) is not in PARMCHK.DAT; using default values
for improper_flag [0], group_id [0], and equivalent_flag [0].
Warning: No mass information for atom type (DU); set to 0.0.
It is recommended to add the new atom type (DU) to PARMCHK.DAT.
/home/jirka/amber18/bin/parmchk2: line 4: 65155 Segmentation fault (core dumped) \$AMBERHOME/bin/to_be_dispatched/parmchk2 “\$@”

• CarlosRamosG

Hi Zahra, could you please repeat the generation of the .ac file with antechamber but this time use ‘-at gaff2’ instead of ‘-at amber’. As your molecule is very different from a protein or a nucleic acid it is likely that this is the reason why amber is not recognising your N3 nitrogen and is assigning the DU atom type.
Please let me know if this solves the problem.

• Zahra A.

Sure! Actually it is combination of amino acid + organic molecule, here is the .ac file using -at gaff2 :
(base) cat FMN.ac
CHARGE -2.00 ( -2 )
Formula: H31 C23 N6 O11 P1 S1
ATOM 1 N1 MOL 1 -1.402 -1.545 3.066 -0.591590 ns
ATOM 2 N2 MOL 1 -0.334 -3.052 0.932 -0.342128 ns
ATOM 3 C1 MOL 1 -2.278 -2.023 2.002 -0.025870 c3
ATOM 4 C2 MOL 1 -0.100 -1.751 5.125 -0.612845 c3
ATOM 5 C3 MOL 1 0.415 -4.051 0.182 -0.334400 c3
ATOM 6 C4 MOL 1 -1.652 -3.191 1.205 0.660925 c
ATOM 7 C5 MOL 1 -1.043 -2.359 4.104 0.783892 c
ATOM 8 O1 MOL 1 -2.337 -4.141 0.808 -0.612160 o
ATOM 9 O2 MOL 1 -1.451 -3.526 4.174 -0.611120 o
ATOM 10 C6 MOL 1 -2.576 -0.818 1.089 -0.543266 c3
ATOM 11 S1 MOL 1 -3.612 -1.308 -0.343 0.000000 ss
ATOM 12 P1 MOL 1 6.748 0.192 -0.040 0.000000 p5
ATOM 13 O3 MOL 1 7.293 -0.106 -1.453 -0.544005 o
ATOM 14 O4 MOL 1 7.606 1.139 0.833 -0.610434 o
ATOM 15 O5 MOL 1 6.171 -1.023 0.735 -0.574457 o
ATOM 16 C7 MOL 1 4.666 1.277 -1.581 0.044452 c3
ATOM 17 O6 MOL 1 5.386 1.204 -0.401 -0.175829 os
ATOM 18 C8 MOL 1 3.337 0.530 -1.462 -0.022976 c3
ATOM 19 O7 MOL 1 3.659 -0.851 -1.651 -0.496317 oh
ATOM 20 C9 MOL 1 2.675 0.736 -0.100 0.131863 c3
ATOM 21 O8 MOL 1 2.771 2.117 0.218 -0.656784 oh
ATOM 22 C10 MOL 1 1.208 0.295 -0.082 0.045337 c3
ATOM 23 O9 MOL 1 0.658 0.441 1.204 -0.608289 oh
ATOM 24 C11 MOL 1 0.466 1.144 -1.081 -0.072047 c3
ATOM 25 N3 MOL 1 -0.881 -0.765 -2.454 -0.492301 ne
ATOM 26 C12 MOL 1 -1.475 -1.698 -3.268 0.595808 c
ATOM 27 O10 MOL 1 -0.863 -2.626 -3.792 -0.539308 o
ATOM 28 N4 MOL 1 -2.858 -1.540 -3.563 -0.378562 ns
ATOM 29 C13 MOL 1 -3.714 -0.672 -2.946 0.540779 c
ATOM 30 C14 MOL 1 -3.119 0.021 -1.716 -0.347934 c3
ATOM 31 O11 MOL 1 -4.879 -0.481 -3.279 -0.517951 o
ATOM 32 C15 MOL 1 -3.144 2.153 -0.608 0.007567 ca
ATOM 33 N5 MOL 1 -3.692 1.299 -1.557 -0.296909 nu
ATOM 34 C16 MOL 1 -3.898 3.148 0.037 -0.271554 ca
ATOM 35 C17 MOL 1 -3.317 4.014 0.974 0.070769 ca
ATOM 36 C18 MOL 1 -4.155 5.071 1.653 -0.241331 c3
ATOM 37 C19 MOL 1 -1.937 3.871 1.286 0.072834 ca
ATOM 38 C20 MOL 1 -1.277 4.766 2.309 -0.240244 c3
ATOM 39 C21 MOL 1 -1.185 2.881 0.636 -0.217269 ca
ATOM 40 C22 MOL 1 -1.765 2.027 -0.318 0.017070 ca
ATOM 41 C23 MOL 1 -1.616 0.084 -1.767 0.693489 c2
ATOM 42 N6 MOL 1 -1.011 1.053 -1.027 -0.074009 nh
ATOM 43 H1 MOL 1 0.338 -5.041 0.668 0.140227 h1
ATOM 44 H2 MOL 1 1.470 -3.742 0.145 0.153090 h1
ATOM 45 H3 MOL 1 -0.496 -1.942 6.136 0.174640 hc
ATOM 46 H4 MOL 1 0.876 -2.262 5.047 0.190410 hc
ATOM 47 H5 MOL 1 -1.058 -0.584 3.031 0.331452 hn
ATOM 48 H6 MOL 1 0.165 -2.275 1.363 0.292543 hn
ATOM 49 H7 MOL 1 0.049 -0.670 4.981 0.154416 hc
ATOM 50 H8 MOL 1 -3.208 -2.429 2.434 0.178143 h1
ATOM 51 H9 MOL 1 0.028 -4.134 -0.849 0.146485 h1
ATOM 52 H10 MOL 1 -1.614 -0.420 0.728 0.173219 h1
ATOM 53 H11 MOL 1 -3.091 -0.033 1.668 0.263073 h1
ATOM 54 H12 MOL 1 5.229 0.827 -2.426 0.063424 h1
ATOM 55 H13 MOL 1 4.453 2.339 -1.827 0.016626 h1
ATOM 56 H14 MOL 1 2.679 0.882 -2.282 0.111883 h1
ATOM 57 H15 MOL 1 2.826 -1.352 -1.742 0.318709 ho
ATOM 58 H16 MOL 1 3.254 0.121 0.620 0.124128 h1
ATOM 59 H17 MOL 1 2.341 2.236 1.088 0.422025 ho
ATOM 60 H18 MOL 1 1.149 -0.766 -0.401 0.090879 h1
ATOM 61 H19 MOL 1 1.198 -0.083 1.828 0.412515 ho
ATOM 62 H20 MOL 1 0.747 0.830 -2.093 0.009937 h1
ATOM 63 H21 MOL 1 0.730 2.201 -0.947 0.148513 h1
ATOM 64 H22 MOL 1 -3.235 -2.133 -4.307 0.309656 hn
ATOM 65 H23 MOL 1 -4.699 1.339 -1.727 0.331129 hn
ATOM 66 H24 MOL 1 -4.963 3.234 -0.205 0.180659 ha
ATOM 67 H25 MOL 1 -4.164 4.930 2.750 0.076636 hc
ATOM 68 H26 MOL 1 -3.749 6.082 1.467 0.084836 hc
ATOM 69 H27 MOL 1 -5.196 5.044 1.293 0.065213 hc
ATOM 70 H28 MOL 1 -1.762 4.673 3.298 0.066900 hc
ATOM 71 H29 MOL 1 -0.210 4.515 2.423 0.084432 hc
ATOM 72 H30 MOL 1 -1.352 5.831 2.019 0.076821 hc
ATOM 73 H31 MOL 1 -0.134 2.758 0.897 0.194484 ha
BOND 1 1 3 1 N1 C1
BOND 2 1 7 1 N1 C5
BOND 3 1 47 1 N1 H5
BOND 4 2 5 1 N2 C3
BOND 5 2 6 1 N2 C4
BOND 6 2 48 1 N2 H6
BOND 7 3 6 1 C1 C4
BOND 8 3 10 1 C1 C6
BOND 9 3 50 1 C1 H8
BOND 10 4 7 1 C2 C5
BOND 11 4 45 1 C2 H3
BOND 12 4 46 1 C2 H4
BOND 13 4 49 1 C2 H7
BOND 14 5 43 1 C3 H1
BOND 15 5 44 1 C3 H2
BOND 16 5 51 1 C3 H9
BOND 17 6 8 2 C4 O1
BOND 18 7 9 2 C5 O2
BOND 19 10 11 1 C6 S1
BOND 20 10 52 1 C6 H10
BOND 21 10 53 1 C6 H11
BOND 22 11 30 1 S1 C14
BOND 23 12 13 9 P1 O3
BOND 24 12 14 9 P1 O4
BOND 25 12 15 9 P1 O5
BOND 26 12 17 1 P1 O6
BOND 27 16 17 1 C7 O6
BOND 28 16 18 1 C7 C8
BOND 29 16 54 1 C7 H12
BOND 30 16 55 1 C7 H13
BOND 31 18 19 1 C8 O7
BOND 32 18 20 1 C8 C9
BOND 33 18 56 1 C8 H14
BOND 34 19 57 1 O7 H15
BOND 35 20 21 1 C9 O8
BOND 36 20 22 1 C9 C10
BOND 37 20 58 1 C9 H16
BOND 38 21 59 1 O8 H17
BOND 39 22 23 1 C10 O9
BOND 40 22 24 1 C10 C11
BOND 41 22 60 1 C10 H18
BOND 42 23 61 1 O9 H19
BOND 43 24 42 1 C11 N6
BOND 44 24 62 1 C11 H20
BOND 45 24 63 1 C11 H21
BOND 46 25 26 1 N3 C12
BOND 47 25 41 2 N3 C23
BOND 48 26 27 2 C12 O10
BOND 49 26 28 1 C12 N4
BOND 50 28 29 1 N4 C13
BOND 51 28 64 1 N4 H22
BOND 52 29 30 1 C13 C14
BOND 53 29 31 2 C13 O11
BOND 54 30 33 1 C14 N5
BOND 55 30 41 1 C14 C23
BOND 56 32 33 1 C15 N5
BOND 57 32 34 7 C15 C16
BOND 58 32 40 8 C15 C22
BOND 59 33 65 1 N5 H23
BOND 60 34 35 8 C16 C17
BOND 61 34 66 1 C16 H24
BOND 62 35 36 1 C17 C18
BOND 63 35 37 7 C17 C19
BOND 64 36 67 1 C18 H25
BOND 65 36 68 1 C18 H26
BOND 66 36 69 1 C18 H27
BOND 67 37 38 1 C19 C20
BOND 68 37 39 8 C19 C21
BOND 69 38 70 1 C20 H28
BOND 70 38 71 1 C20 H29
BOND 71 38 72 1 C20 H30
BOND 72 39 40 7 C21 C22
BOND 73 39 73 1 C21 H31
BOND 74 40 42 1 C22 N6
BOND 75 41 42 1 C23 N6
(base)

• fyymls

I also encountered this problem “Atom type of DU does not shown up in PARMCHK.DAT”, and understood the reason and solution based on your conversation, thank you very much

6. Dillon

I capped my modified residue, and added the capped atoms to the OMIT_NAME to my mainchain file. During tleap the omitted/capped residues are being added again and I can’t bond my residue to the backbone residues. Do you know how I can prevent the residues from being added?

• CarlosRamosG

Dear Dillon,

I’m glad you found the tutorial useful.
What happens if you create a prmtop and inpcrd files for your residue only?
This can be done in the following way:
aa = sequence {LTL}
saveAmberParm aa LTLT.prmtop LTL.rst7

If you see the capped atoms here, something may be missing in the .mc file
If you don’t see the capped atoms here, you may have extra atoms in the PDB file you are using to build your system.

7. Gerardo

Hi Carlos, thank you for this helpful and insightful tutorial!
As I was going through I got a question. What would it happen if we use a higher level of theory to calculate the partial charges than the the one that was used to derive the partial charges of the forcefield?
Would it be safe to say that the ESP of the molecules would not be properly described since the partial charges of that force field are not compatible with that level of theory?

Thank you!

• CarlosRamosG

Hi Gerardo,

Thanks for your message. According to the Amber recommendations, using a higher level of theory to calculate partial charges than that used to derive the force field partial charges could lead to discrepancies in the description of the electrostatic potential (ESP) of molecules. This is because the electrostatic potential is sensitive to the distribution of partial charges on atoms.

If the partial charges derived from a higher level of theory differ significantly from those of the force field, the ESP may not be correctly described. This could potentially lead to inaccurate predictions of properties that depend on electrostatic interactions, such as molecular conformations, binding energies or solvation energies.

My recommendation is that it is advisable to follow the methodology outlined in the documentation to ensure consistency and reliability in simulations. If there are compelling reasons to explore a higher level of theory for partial charge calculations, a thorough validation and recalibration of the force field parameters would be necessary to maintain accuracy and compatibility.

8. Mrfath

Dear Carlos,
I have downloaded the 8CIX structure from the PDB and I want to run molecular dynamics (MD) simulations using AMBER. The peptide in the 8CIX PDB contains four non-standard amino acids: MP8, MVA, MLU, and NZC. I have successfully adjusted the parameters for MP8, MVA, and MLU.
However, I am facing issues with the NZC amino acid. NZC forms peptide bonds with neighboring residues and additionally forms a bond with a glycine residue via its oxygen atom. Specifically, NZC makes two peptide bonds and an additional bond with the carbonyl carbon (C=O) of the glycine backbone.
I am having trouble obtaining the parameters for this additional bond between NZC and glycine. Could you please advise on how to introduce this bond into the AMBER24 program?
Best regards,

• CarlosRamosG

Hi, I’m glad you managed parameterize the other three residues. For the case of NZC I imagine the problem is related with the fact that this is bound to three residues instead of two as the example in the tutorial. So in these cases what I should do is the geometry optimisation of the structure of the NZC bound to the MP8, both residues with their corresponding capping groups. Perform the calculation of the electrostatic potential charges and the RESP calculation, since at this point you have already obtained the charges for MP8, you can include them in the respq.in file in the same way as the charges of the ACE and the NME. Follow the steps VI and VII of the tutorial but in this last step modify the .mc file to delete not only the atoms of the ACE and NME cap groups but the MP8 as well.
Now you have the .prepin file for your residue, the .frcmod should be generated with the .ac file obtained in the step VI.
Now, the bond between the NZC nitrogen atom and the MP8 Carbon is created during the preparation step using tleap. use the command bond SYSTEM.4.N SYSTEM.3.C. Here, SYSTEM is the name you assign to your system in leap, the 4 and 3 are the number of your residues in the PDB file and N and C are the names of the atoms to be bonded. it’s the same procedure used to create a disulfide bond.

Check my reply to Zhara on “January 11, 2024 at 10:42 am” this can also helps you.
Hope it helps!

• Mrfath

Dear Carlos,
Thank you for your prompt response.
The issue we’re encountering is not between NZC and MP8 but between NZC and GLY. The bond between NZC and GLY is an ester bond rather than a peptide bond. Specifically, NZC has two peptide bonds with MP8 and L, and one ester bond with GLY. In the PDB file, the ester bond is represented with the oxygen atom from NZC binding to the carbon atom of the C=O group in GLY.
When I omit the atoms of GLY in the MC file, the prepin file results in multiple ‘X’ signs and lacks all the necessary residues due to identical atom names. Additionally, when I run TLEaP, it adds an OXT atom to GLY. This leads to the modified PDB file showing the same bond twice, appearing aligned but not as a double bond. It also gives me a “one atom does not have a type” error.
Could you please advise on how to properly define that the oxygen atom of NZC binds to the C=O group of GLY? Alternatively, could you please advise on how to run MD simulations for depsipeptides? Is there any example or website you can suggest?
Best

• CarlosRamosG

Oh I see, in this case the approach will remain the same as previously mentioned. However, since this is not a regular GLY, you won’t be able to bond it normally to your NZC residue. You’ll need to parameterize it as a new residue, let’s call it GLZ. This residue should be bond to your NZC later you will cutting it using the .mc file and you’ll need to create the bond between it and the NZC in tleap later. The bond with the preceding MLU residue should be created automatically as you include the N and CA atoms in the main chain description in the .mc file.

Hope this helps, please let me know.

9. Peng

Thanks for this great tutorial! Very useful!!

I got an issue at the step “resp -O -i resp.in -o resp.out -p resp.pch -t resp.chg -q resp.qin -e esp.dat”. The error message looks like “forrtl: severe (64): input conversion error, unit 5, file /home/pz19/Work/Morgan/test/PPP_02/resp.in”

I have deleted the comment lines in resp.in and I am using Amber24. Any insight? Thanks!

• CarlosRamosG

Hi Peng,

I’m glad you find this useful.

I understand that working with this file can be a bit tricky. To help, try generating a template for this file using the following antechamber command:
antechamber -fi gout -i LTL_hf.log -bk LTL -fo ac -o LTL.ac -c rc -c resp -at amber
This command is similar to the one you’ll use later to generate the .ac file. However, the key here is to obtain the ANTECHAMBER_RESP1.IN outfile. Rename this file to resp.in, make the necessary modifications, and continue with the fitting charges procedure as outlined in the tutorial, “Restrained Fitting of the Partial Charges to the Electrostatic Potentials (RESP Calculation)”. Compare your file with the generated file and you will see the error you had.

Use the modified resp.in file later with the following command:
resp -O -i resp.in -o resp.out -p resp.pch -t resp.chg -q resp.qin -e esp.dat
Best wishes!

• Peng

Thank you so much!

• Peng

Hi Carlos

Following your suggestions, I was able to proceed successfully. Thanks again!

I have one additional question regarding the ” MAIN_CHAIN C3″ in LTL.mc. I am not sure I understand this. Does this mean that C3 connect the head N1 and tail C8? In a general case, should I look for the shorted path that connects the head and tail? Thanks!

Best wishes

Peng