{"id":7006,"date":"2026-04-20T12:42:14","date_gmt":"2026-04-20T03:42:14","guid":{"rendered":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/?p=7006"},"modified":"2026-04-20T12:42:15","modified_gmt":"2026-04-20T03:42:15","slug":"qm-mm-md-simulation-of-ntmgam-in-complex-with-maltose","status":"publish","type":"post","link":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/en\/2026\/04\/20\/qm-mm-md-simulation-of-ntmgam-in-complex-with-maltose\/","title":{"rendered":"QM\/MM MD Simulation of NtMGAM in Complex with Maltose"},"content":{"rendered":"\n<p>This note details the reproduction of the QM\/MM modeling protocols described by <strong>Br\u00e1s et al. (2018)<\/strong> using <strong>GROMACS<\/strong> (interfaced with <strong>CP2K<\/strong>).<\/p>\n\n\n\n<p><strong>Reference:<\/strong> Br\u00e1s, N. F., et al., <em>J. Phys. Chem. B<\/em> 2018, 122, 3889\u20133899.<a target=\"_blank\" rel=\"noreferrer noopener\" href=\"https:\/\/www.google.com\/search?q=https:\/\/doi.org\/10.1021\/acs.jpcb.8b01321\">doi: 10.1021\/acs.jpcb.8b01321<\/a>.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\">1. System Preparation<\/h3>\n\n\n\n<h4 class=\"wp-block-heading\">Structural Assembly<\/h4>\n\n\n\n<p><strong>Residue Recovery:<\/strong> The missing <strong>Q837<\/strong> residue was extracted from the 3L4U.pdb structure and inserted into 2QMJ.pdb to generate 2QMJ_plus_Q837.pdb.<\/p>\n\n\n\n<p><strong>Ligand Alignment:<\/strong> A maltose structure was extracted from 8YBE.pdb and fitted to the acarbose molecule in 2QMJ.pdb to produce maltose_fitted.pdb. This file was processed via <strong>CHARMM-GUI\u2019s Glycan Reader &amp; Modeler<\/strong> to generate the GROMACS topology file (CARA.itp).<\/p>\n\n\n\n<p><strong>Assembly:<\/strong> After removing acarbose coordinates, the NEWPDB.PDB (generated by Antechamber) was inserted following the NAG glycan coordinates in 2QMJ.pdb, resulting in 2QMJ_maltose_input.pdb.<\/p>\n\n\n\n<p><strong>Q837_from_3L4U_aligned.pdb<\/strong><\/p>\n\n\n\n<pre class=\"prettyprint\">ATOM      1  N   GLN A 837     -56.319  -1.918 -54.152  1.00 52.24           N  \nATOM      2  CA  GLN A 837     -56.489  -3.228 -53.471  1.00 51.57           C  \nATOM      3  C   GLN A 837     -57.877  -3.508 -52.879  1.00 50.76           C  \nATOM      4  O   GLN A 837     -58.136  -3.210 -51.705  1.00 51.36           O  \nATOM      5  CB  GLN A 837     -55.377  -3.492 -52.436  1.00 51.89           C  \nATOM      6  CG  GLN A 837     -55.042  -2.327 -51.488  1.00 52.50           C  \nATOM      7  CD  GLN A 837     -55.174  -2.711 -50.028  1.00 52.02           C  \nATOM      8  OE1 GLN A 837     -54.177  -2.786 -49.310  1.00 49.07           O  \nATOM      9  NE2 GLN A 837     -56.420  -2.967 -49.583  1.00 50.20           N  \n<\/pre>\n\n\n\n<p><strong>maltose_fitted.pdb<\/strong><\/p>\n\n\n\n<pre class=\"prettyprint\">HETATM    1  C1  GLC B   1     -18.637  -6.555  -5.479  1.00 20.00           C  \nHETATM    2  C2  GLC B   1     -18.080  -5.440  -6.357  1.00 20.00           C  \nHETATM    3  C3  GLC B   1     -18.902  -5.280  -7.625  1.00 20.00           C  \nHETATM    4  C4  GLC B   1     -19.046  -6.614  -8.329  1.00 20.00           C  \nHETATM    5  C5  GLC B   1     -19.495  -7.694  -7.358  1.00 20.00           C  \nHETATM    6  C6  GLC B   1     -19.515  -9.049  -8.044  1.00 20.00           C  \nHETATM    7  O1  GLC B   1     -19.972  -6.247  -5.064  1.00 20.00           O  \nHETATM    8  O2  GLC B   1     -18.077  -4.209  -5.634  1.00 20.00           O  \nHETATM    9  O3  GLC B   1     -18.244  -4.343  -8.478  1.00 20.00           O  \nHETATM   10  O4  GLC B   1     -20.058  -6.525  -9.322  1.00 20.00           O  \nHETATM   11  O5  GLC B   1     -18.621  -7.761  -6.237  1.00 20.00           O  \nHETATM   12  O6  GLC B   1     -20.151  -9.974  -7.172  1.00 20.00           O  \nHETATM   13  C1  GLC B   2     -19.555  -5.932 -10.525  1.00 19.82           C  \nHETATM   14  C2  GLC B   2     -20.762  -5.492 -11.333  1.00 18.22           C  \nHETATM   15  C3  GLC B   2     -21.625  -6.709 -11.603  1.00 19.03           C  \nHETATM   16  C4  GLC B   2     -20.799  -7.724 -12.372  1.00 19.44           C  \nHETATM   17  C5  GLC B   2     -19.558  -8.067 -11.566  1.00 24.91           C  \nHETATM   18  C6  GLC B   2     -18.675  -9.055 -12.311  1.00 20.27           C  \nHETATM   19  O2  GLC B   2     -21.513  -4.523 -10.609  1.00 26.01           O  \nHETATM   20  O3  GLC B   2     -22.777  -6.339 -12.355  1.00 24.65           O  \nHETATM   21  O4  GLC B   2     -21.561  -8.906 -12.618  1.00 28.65           O  \nHETATM   22  O5  GLC B   2     -18.825  -6.875 -11.301  1.00 25.46           O  \nHETATM   23  O6  GLC B   2     -17.756  -9.627 -11.384  1.00 20.51           O  \n<\/pre>\n\n\n\n<p><strong>CARA.itp<\/strong><\/p>\n\n\n\n<pre class=\"prettyprint\">;;\n;; Generated by CHARMM-GUI FF-Converter\n;;\n;; Correspondance:\n;; jul316@lehigh.edu or wonpil@lehigh.edu\n;;\n;; GROMACS topology file for CARA\n;;\n\n\n&#91; moleculetype &#93;\n; name\tnrexcl\nCARA\t     3\n\n&#91; atoms &#93;\n; nr\ttype\tresnr\tresidu\tatom\tcgnr\tcharge\tmass\n     1     CC3162      1     AGLC     C1      1     0.340000    12.0110   ; qtot  0.340\n     2       HCA1      1     AGLC     H1      2     0.090000     1.0080   ; qtot  0.430\n     3      OC311      1     AGLC     O1      3    -0.650000    15.9994   ; qtot -0.220\n     4       HCP1      1     AGLC    HO1      4     0.420000     1.0080   ; qtot  0.200\n     5     CC3163      1     AGLC     C5      5     0.110000    12.0110   ; qtot  0.310\n     6       HCA1      1     AGLC     H5      6     0.090000     1.0080   ; qtot  0.400\n     7     OC3C61      1     AGLC     O5      7    -0.400000    15.9994   ; qtot  0.000\n     8     CC3161      1     AGLC     C2      8     0.140000    12.0110   ; qtot  0.140\n     9       HCA1      1     AGLC     H2      9     0.090000     1.0080   ; qtot  0.230\n    10      OC311      1     AGLC     O2     10    -0.650000    15.9994   ; qtot -0.420\n    11       HCP1      1     AGLC    HO2     11     0.420000     1.0080   ; qtot -0.000\n    12     CC3161      1     AGLC     C3     12     0.140000    12.0110   ; qtot  0.140\n    13       HCA1      1     AGLC     H3     13     0.090000     1.0080   ; qtot  0.230\n    14      OC311      1     AGLC     O3     14    -0.650000    15.9994   ; qtot -0.420\n    15       HCP1      1     AGLC    HO3     15     0.420000     1.0080   ; qtot -0.000\n    16     CC3161      1     AGLC     C4     16     0.090000    12.0110   ; qtot  0.090\n    17       HCA1      1     AGLC     H4     17     0.090000     1.0080   ; qtot  0.180\n    18      OC301      1     AGLC     O4     18    -0.360000    15.9994   ; qtot -0.180\n    19      CC321      1     AGLC     C6     19     0.050000    12.0110   ; qtot -0.130\n    20       HCA2      1     AGLC    H61     20     0.090000     1.0080   ; qtot -0.040\n    21       HCA2      1     AGLC    H62     21     0.090000     1.0080   ; qtot  0.050\n    22      OC311      1     AGLC     O6     22    -0.650000    15.9994   ; qtot -0.600\n    23       HCP1      1     AGLC    HO6     23     0.420000     1.0080   ; qtot -0.180\n    24     CC3162      2     AGLC     C1     24     0.290000    12.0110   ; qtot  0.110\n    25       HCA1      2     AGLC     H1     25     0.090000     1.0080   ; qtot  0.200\n    26     CC3163      2     AGLC     C5     26     0.110000    12.0110   ; qtot  0.310\n    27       HCA1      2     AGLC     H5     27     0.090000     1.0080   ; qtot  0.400\n    28     OC3C61      2     AGLC     O5     28    -0.400000    15.9994   ; qtot -0.000\n    29     CC3161      2     AGLC     C2     29     0.140000    12.0110   ; qtot  0.140\n    30       HCA1      2     AGLC     H2     30     0.090000     1.0080   ; qtot  0.230\n    31      OC311      2     AGLC     O2     31    -0.650000    15.9994   ; qtot -0.420\n    32       HCP1      2     AGLC    HO2     32     0.420000     1.0080   ; qtot -0.000\n    33     CC3161      2     AGLC     C3     33     0.140000    12.0110   ; qtot  0.140\n    34       HCA1      2     AGLC     H3     34     0.090000     1.0080   ; qtot  0.230\n    35      OC311      2     AGLC     O3     35    -0.650000    15.9994   ; qtot -0.420\n    36       HCP1      2     AGLC    HO3     36     0.420000     1.0080   ; qtot -0.000\n    37     CC3161      2     AGLC     C4     37     0.140000    12.0110   ; qtot  0.140\n    38       HCA1      2     AGLC     H4     38     0.090000     1.0080   ; qtot  0.230\n    39      OC311      2     AGLC     O4     39    -0.650000    15.9994   ; qtot -0.420\n    40       HCP1      2     AGLC    HO4     40     0.420000     1.0080   ; qtot -0.000\n    41      CC321      2     AGLC     C6     41     0.050000    12.0110   ; qtot  0.050\n    42       HCA2      2     AGLC    H61     42     0.090000     1.0080   ; qtot  0.140\n    43       HCA2      2     AGLC    H62     43     0.090000     1.0080   ; qtot  0.230\n    44      OC311      2     AGLC     O6     44    -0.650000    15.9994   ; qtot -0.420\n    45       HCP1      2     AGLC    HO6     45     0.420000     1.0080   ; qtot -0.000\n\n&#91; bonds &#93;\n; ai\taj\tfunct\tb0\tKb\n    1     2     1\n    1     3     1\n    1     7     1\n    1     8     1\n    3     4     1\n    5     6     1\n    5     7     1\n   16     5     1\n    5    19     1\n    8     9     1\n    8    10     1\n    8    12     1\n   10    11     1\n   12    13     1\n   12    14     1\n   12    16     1\n   14    15     1\n   16    17     1\n   16    18     1\n   18    24     1\n   19    20     1\n   19    21     1\n   19    22     1\n   22    23     1\n   24    25     1\n   24    28     1\n   24    29     1\n   26    27     1\n   26    28     1\n   37    26     1\n   26    41     1\n   29    30     1\n   29    31     1\n   29    33     1\n   31    32     1\n   33    34     1\n   33    35     1\n   33    37     1\n   35    36     1\n   37    38     1\n   37    39     1\n   39    40     1\n   41    42     1\n   41    43     1\n   41    44     1\n   44    45     1\n\n&#91; pairs &#93;\n; ai\taj\tfunct\tc6\tc12 or\n; ai\taj\tfunct\tfudgeQQ\tq1\tq2\tc6\tc12\n    1     6     1\n    1    11     1\n    1    13     1\n    1    14     1\n    1    16     1\n    1    19     1\n    2     4     1\n    2     5     1\n    2     9     1\n    2    10     1\n    2    12     1\n    3     5     1\n    3     9     1\n    3    10     1\n    3    12     1\n    4     7     1\n    4     8     1\n    5     8     1\n    5    13     1\n    5    14     1\n    5    23     1\n    5    24     1\n    6    12     1\n    6    17     1\n    6    18     1\n    6    20     1\n    6    21     1\n    6    22     1\n    7     9     1\n    7    10     1\n    7    12     1\n    7    17     1\n    7    18     1\n    7    20     1\n    7    21     1\n    7    22     1\n    8    15     1\n    8    17     1\n    8    18     1\n    9    11     1\n    9    13     1\n    9    14     1\n    9    16     1\n   10    13     1\n   10    14     1\n   10    16     1\n   11    12     1\n   12    19     1\n   12    24     1\n   13    15     1\n   13    17     1\n   13    18     1\n   14    17     1\n   14    18     1\n   15    16     1\n   16    20     1\n   16    21     1\n   16    22     1\n   16    25     1\n   16    28     1\n   16    29     1\n   17    19     1\n   17    24     1\n   18    19     1\n   18    26     1\n   18    30     1\n   18    31     1\n   18    33     1\n   20    23     1\n   21    23     1\n   24    27     1\n   24    32     1\n   24    34     1\n   24    35     1\n   24    37     1\n   24    41     1\n   25    26     1\n   25    30     1\n   25    31     1\n   25    33     1\n   26    29     1\n   26    34     1\n   26    35     1\n   26    40     1\n   26    45     1\n   27    33     1\n   27    38     1\n   27    39     1\n   27    42     1\n   27    43     1\n   27    44     1\n   28    30     1\n   28    31     1\n   28    33     1\n   28    38     1\n   28    39     1\n   28    42     1\n   28    43     1\n   28    44     1\n   29    36     1\n   29    38     1\n   29    39     1\n   30    32     1\n   30    34     1\n   30    35     1\n   30    37     1\n   31    34     1\n   31    35     1\n   31    37     1\n   32    33     1\n   33    40     1\n   33    41     1\n   34    36     1\n   34    38     1\n   34    39     1\n   35    38     1\n   35    39     1\n   36    37     1\n   37    42     1\n   37    43     1\n   37    44     1\n   38    40     1\n   38    41     1\n   39    41     1\n   42    45     1\n   43    45     1\n\n&#91; angles &#93; \n; ai\taj\tak\tfunct\tth0\tcth\tS0\tKub\n    2     1     3     5\n    2     1     7     5\n    2     1     8     5\n    3     1     7     5\n    3     1     8     5\n    7     1     8     5\n    1     3     4     5\n    6     5     7     5\n    6     5    16     5\n    6     5    19     5\n    7     5    16     5\n    7     5    19     5\n   16     5    19     5\n    1     7     5     5\n    1     8     9     5\n    1     8    10     5\n    1     8    12     5\n    9     8    10     5\n    9     8    12     5\n   10     8    12     5\n    8    10    11     5\n    8    12    13     5\n    8    12    14     5\n    8    12    16     5\n   13    12    14     5\n   13    12    16     5\n   14    12    16     5\n   12    14    15     5\n    5    16    12     5\n    5    16    17     5\n    5    16    18     5\n   12    16    17     5\n   12    16    18     5\n   17    16    18     5\n   16    18    24     5\n    5    19    20     5\n    5    19    21     5\n    5    19    22     5\n   20    19    21     5\n   20    19    22     5\n   21    19    22     5\n   19    22    23     5\n   18    24    25     5\n   18    24    28     5\n   18    24    29     5\n   25    24    28     5\n   25    24    29     5\n   28    24    29     5\n   27    26    28     5\n   27    26    37     5\n   27    26    41     5\n   28    26    37     5\n   28    26    41     5\n   37    26    41     5\n   24    28    26     5\n   24    29    30     5\n   24    29    31     5\n   24    29    33     5\n   30    29    31     5\n   30    29    33     5\n   31    29    33     5\n   29    31    32     5\n   29    33    34     5\n   29    33    35     5\n   29    33    37     5\n   34    33    35     5\n   34    33    37     5\n   35    33    37     5\n   33    35    36     5\n   26    37    33     5\n   26    37    38     5\n   26    37    39     5\n   33    37    38     5\n   33    37    39     5\n   38    37    39     5\n   37    39    40     5\n   26    41    42     5\n   26    41    43     5\n   26    41    44     5\n   42    41    43     5\n   42    41    44     5\n   43    41    44     5\n   41    44    45     5\n\n&#91; dihedrals &#93;\n; ai\taj\tak\tal\tfunct\tphi0\tcp\tmult\n    2     1     3     4     9\n    2     1     7     5     9\n    3     1     7     5     9\n    2     1     8     9     9\n    2     1     8    10     9\n    2     1     8    12     9\n    3     1     8     9     9\n    3     1     8    10     9\n    3     1     8    12     9\n    7     1     8     9     9\n    7     1     8    10     9\n    7     1     8    12     9\n    4     3     1     7     9\n    4     3     1     8     9\n    6     5    16    12     9\n    6     5    16    17     9\n    6     5    16    18     9\n    7     5    16    12     9\n    7     5    16    17     9\n    7     5    16    18     9\n    6     5    19    20     9\n    6     5    19    21     9\n    6     5    19    22     9\n    7     5    19    20     9\n    7     5    19    21     9\n    7     5    19    22     9\n   16     5    19    20     9\n   16     5    19    21     9\n   16     5    19    22     9\n    5     7     1     8     9\n    1     7     5     6     9\n    1     7     5    16     9\n    1     7     5    19     9\n    1     8    10    11     9\n    9     8    10    11     9\n    1     8    12    13     9\n    1     8    12    14     9\n    1     8    12    16     9\n    9     8    12    13     9\n    9     8    12    14     9\n    9     8    12    16     9\n   10     8    12    13     9\n   10     8    12    14     9\n   10     8    12    16     9\n   11    10     8    12     9\n    8    12    14    15     9\n   13    12    14    15     9\n    8    12    16    17     9\n    8    12    16    18     9\n   13    12    16    17     9\n   13    12    16    18     9\n   14    12    16    17     9\n   14    12    16    18     9\n   15    14    12    16     9\n   12    16     5    19     9\n   17    16     5    19     9\n   18    16     5    19     9\n    5    16    12     8     9\n    5    16    12    13     9\n    5    16    12    14     9\n    5    16    18    24     9\n   12    16    18    24     9\n   17    16    18    24     9\n   16    18    24    25     9\n   16    18    24    28     9\n   16    18    24    29     9\n    5    19    22    23     9\n   20    19    22    23     9\n   21    19    22    23     9\n   18    24    28    26     9\n   25    24    28    26     9\n   18    24    29    30     9\n   18    24    29    31     9\n   18    24    29    33     9\n   25    24    29    30     9\n   25    24    29    31     9\n   25    24    29    33     9\n   28    24    29    30     9\n   28    24    29    31     9\n   28    24    29    33     9\n   27    26    37    33     9\n   27    26    37    38     9\n   27    26    37    39     9\n   28    26    37    33     9\n   28    26    37    38     9\n   28    26    37    39     9\n   27    26    41    42     9\n   27    26    41    43     9\n   27    26    41    44     9\n   28    26    41    42     9\n   28    26    41    43     9\n   28    26    41    44     9\n   37    26    41    42     9\n   37    26    41    43     9\n   37    26    41    44     9\n   26    28    24    29     9\n   24    28    26    27     9\n   24    28    26    37     9\n   24    28    26    41     9\n   24    29    31    32     9\n   30    29    31    32     9\n   24    29    33    34     9\n   24    29    33    35     9\n   24    29    33    37     9\n   30    29    33    34     9\n   30    29    33    35     9\n   30    29    33    37     9\n   31    29    33    34     9\n   31    29    33    35     9\n   31    29    33    37     9\n   32    31    29    33     9\n   29    33    35    36     9\n   34    33    35    36     9\n   29    33    37    38     9\n   29    33    37    39     9\n   34    33    37    38     9\n   34    33    37    39     9\n   35    33    37    38     9\n   35    33    37    39     9\n   36    35    33    37     9\n   33    37    26    41     9\n   38    37    26    41     9\n   39    37    26    41     9\n   26    37    33    29     9\n   26    37    33    34     9\n   26    37    33    35     9\n   26    37    39    40     9\n   33    37    39    40     9\n   38    37    39    40     9\n   26    41    44    45     9\n   42    41    44    45     9\n   43    41    44    45     9\n\n#ifdef POSRES\n&#91; position_restraints &#93;\n    1     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   \n    3     1    POSRES_FC_SC    POSRES_FC_SC    POSRES_FC_SC   \n    5     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   \n    7     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   \n    8     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   \n   10     1    POSRES_FC_SC    POSRES_FC_SC    POSRES_FC_SC   \n   12     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   \n   14     1    POSRES_FC_SC    POSRES_FC_SC    POSRES_FC_SC   \n   16     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   \n   18     1    POSRES_FC_SC    POSRES_FC_SC    POSRES_FC_SC   \n   19     1    POSRES_FC_SC    POSRES_FC_SC    POSRES_FC_SC   \n   22     1    POSRES_FC_SC    POSRES_FC_SC    POSRES_FC_SC   \n   24     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   \n   26     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   \n   28     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   \n   29     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   \n   31     1    POSRES_FC_SC    POSRES_FC_SC    POSRES_FC_SC   \n   33     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   \n   35     1    POSRES_FC_SC    POSRES_FC_SC    POSRES_FC_SC   \n   37     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   \n   39     1    POSRES_FC_SC    POSRES_FC_SC    POSRES_FC_SC   \n   41     1    POSRES_FC_SC    POSRES_FC_SC    POSRES_FC_SC   \n   44     1    POSRES_FC_SC    POSRES_FC_SC    POSRES_FC_SC   \n#endif\n\n#ifdef DIHRES\n&#91; dihedral_restraints &#93;\n    1     7     5    16     1     60.0      2.5       DIHRES_FC\n    7     5    16    12     1    -60.0      2.5       DIHRES_FC\n    5    16    12     8     1     60.0      2.5       DIHRES_FC\n   16    12     8     1     1    -60.0      2.5       DIHRES_FC\n   12     8     1     7     1     60.0      2.5       DIHRES_FC\n    8     1     7     5     1    -60.0      2.5       DIHRES_FC\n   24    28    26    37     1     60.0      2.5       DIHRES_FC\n   28    26    37    33     1    -60.0      2.5       DIHRES_FC\n   26    37    33    29     1     60.0      2.5       DIHRES_FC\n   37    33    29    24     1    -60.0      2.5       DIHRES_FC\n   33    29    24    28     1     60.0      2.5       DIHRES_FC\n   29    24    28    26     1    -60.0      2.5       DIHRES_FC\n#endif\n<\/pre>\n\n\n\n<pre class=\"prettyprint\">obabel -ipdb maltose_fitted.pdb -h -opdb -O maltose_fitted_h.pdb \nantechamber -fi pdb -i maltose_fitted_h.pdb -fo prepi -o maltose.prep\nobabel -ipdb NEWPDB.PDB -omol2 -O maltose_antechamber.mol2\nsed -i &#34;&#34; &#34;s&#47;GLC  &#47;GLC B&#47;g&#34; NEWPDB.PDB\n<\/pre>\n\n\n\n<h4 class=\"wp-block-heading\">CHARMM-GUI Workflow<\/h4>\n\n\n\n<p>The 2QMJ_maltose_input.pdb was processed through the following steps:<\/p>\n\n\n\n<ol start=\"1\" class=\"wp-block-list\">\n<li><strong>Input Generator &gt; Glycan Reader &amp; Modeler:<\/strong> Uploaded the PDB file.<\/li>\n\n\n\n<li><strong>Component Selection:<\/strong> Selected <strong>Protein<\/strong>, <strong>HETA<\/strong>, and <strong>Water<\/strong>.<\/li>\n\n\n\n<li><strong>Ligand Parameters:<\/strong> Set &#8220;Reading Hetero Chain Residues&#8221; to use <strong>CHARMM General Force Field (CGenFF)<\/strong>.\n<ul class=\"wp-block-list\">\n<li>Uploaded maltose_antechamber.mol.<\/li>\n\n\n\n<li>Unchecked &#8220;Model missing residues.&#8221;<\/li>\n\n\n\n<li>Manually set protonation states for: <strong>HIS831, ASP261, ASP336, ASP366, ASP542, GLU559,<\/strong> and <strong>GLU788<\/strong>.<\/li>\n<\/ul>\n<\/li>\n\n\n\n<li><strong>Solvation &amp; Ion addition:<\/strong> Set the edge distance to <strong>12.0 \u00c5<\/strong>. Added <strong>150 mM NaCl<\/strong> (standardizing the ionic strength rather than only adding 23 Na\u207a ions to neutralize).<\/li>\n<\/ol>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-large is-resized\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"806\" src=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-3.56.35-PM-1024x806.png\" alt=\"\" class=\"wp-image-6946\" style=\"aspect-ratio:1.270500798084597;width:250px;height:auto\" srcset=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-3.56.35-PM-1024x806.png 1024w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-3.56.35-PM-300x236.png 300w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-3.56.35-PM-768x604.png 768w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-3.56.35-PM.png 1032w\" sizes=\"auto, (max-width: 1024px) 100vw, 1024px\" \/><figcaption class=\"wp-element-caption\">Step 2<\/figcaption><\/figure>\n<\/div>\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-large is-resized\"><img loading=\"lazy\" decoding=\"async\" width=\"515\" height=\"1024\" src=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-4.33.42-PM-515x1024.png\" alt=\"\" class=\"wp-image-6947\" style=\"aspect-ratio:0.502937361715114;width:248px;height:auto\" srcset=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-4.33.42-PM-515x1024.png 515w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-4.33.42-PM-151x300.png 151w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-4.33.42-PM-768x1527.png 768w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-4.33.42-PM-772x1536.png 772w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-4.33.42-PM-1030x2048.png 1030w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-4.33.42-PM.png 1076w\" sizes=\"auto, (max-width: 515px) 100vw, 515px\" \/><figcaption class=\"wp-element-caption\">Step 3<\/figcaption><\/figure>\n<\/div>\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-large is-resized\"><img loading=\"lazy\" decoding=\"async\" width=\"881\" height=\"1024\" src=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-17-at-10.51.31-AM-881x1024.png\" alt=\"\" class=\"wp-image-6951\" style=\"aspect-ratio:0.8603664879571097;width:236px;height:auto\" srcset=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-17-at-10.51.31-AM-881x1024.png 881w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-17-at-10.51.31-AM-258x300.png 258w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-17-at-10.51.31-AM-768x893.png 768w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-17-at-10.51.31-AM.png 996w\" sizes=\"auto, (max-width: 881px) 100vw, 881px\" \/><figcaption class=\"wp-element-caption\">Step 4<\/figcaption><\/figure>\n<\/div>\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-large is-resized\"><img loading=\"lazy\" decoding=\"async\" width=\"649\" height=\"1024\" src=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-5.43.50-PM-649x1024.png\" alt=\"\" class=\"wp-image-6949\" style=\"aspect-ratio:0.6337834744792858;width:193px;height:auto\" srcset=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-5.43.50-PM-649x1024.png 649w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-5.43.50-PM-190x300.png 190w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-5.43.50-PM-768x1212.png 768w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/Screenshot-2026-04-16-at-5.43.50-PM.png 788w\" sizes=\"auto, (max-width: 649px) 100vw, 649px\" \/><figcaption class=\"wp-element-caption\">Step 6<\/figcaption><\/figure>\n<\/div>\n\n\n<h4 class=\"wp-block-heading\">Topology Finalization<\/h4>\n\n\n\n<p>The CGenFF-generated maltose topology and coordinates were manually replaced with the outputs from the Glycan Reader &amp; Modeler to create step3_input_modified.pdb. In topol.top, the downloaded CHARMM36 force field was included, and the &#8220;HETA&#8221; residue name was updated to &#8220;CARA.&#8221;<\/p>\n\n\n\n<p><strong>step3_input_modified.pdb<\/strong><\/p>\n\n\n\n<pre class=\"prettyprint\">ATOM  13624  H62 BGLC    1      71.007  48.075  80.029  1.00  0.00      CARB H\nATOM  13625  O6  BGLC    1      72.397  49.312  79.163  1.00  0.00      CARB O\nATOM  13626  HO6 BGLC    1      72.514  49.786  78.333  1.00  0.00      CARB H\nTER\nATOM  13627  C1  AGLC    1      64.666  41.110  70.306  1.00  0.00      CARA C\nATOM  13628  H1  AGLC    1      65.508  40.620  70.843  1.00  0.00      CARA H\nATOM  13629  O1  AGLC    1      64.261  42.275  71.033  1.00  0.00      CARA O\nATOM  13630  HO1 AGLC    1      63.972  41.976  71.903  1.00  0.00      CARA H\nATOM  13631  C5  AGLC    1      62.444  40.803  69.577  1.00  0.00      CARA C\nATOM  13632  H5  AGLC    1      62.104  41.710  70.140  1.00  0.00      CARA H\nATOM  13633  O5  AGLC    1      63.579  40.198  70.187  1.00  0.00      CARA O\nATOM  13634  C2  AGLC    1      65.120  41.488  68.900  1.00  0.00      CARA C\nATOM  13635  H2  AGLC    1      65.461  40.556  68.391  1.00  0.00      CARA H\nATOM  13636  O2  AGLC    1      66.213  42.403  68.974  1.00  0.00      CARA O\nATOM  13637  HO2 AGLC    1      65.972  43.129  69.565  1.00  0.00      CARA H\nATOM  13638  C3  AGLC    1      63.984  42.118  68.111  1.00  0.00      CARA C\nATOM  13639  H3  AGLC    1      63.726  43.108  68.553  1.00  0.00      CARA H\nATOM  13640  O3  AGLC    1      64.416  42.299  66.762  1.00  0.00      CARA O\nATOM  13641  HO3 AGLC    1      65.340  41.994  66.681  1.00  0.00      CARA H\nATOM  13642  C4  AGLC    1      62.760  41.227  68.152  1.00  0.00      CARA C\nATOM  13643  H4  AGLC    1      62.925  40.308  67.542  1.00  0.00      CARA H\nATOM  13644  O4  AGLC    1      61.627  41.949  67.690  1.00  0.00      CARA O\nATOM  13645  C6  AGLC    1      61.293  39.812  69.593  1.00  0.00      CARA C\nATOM  13646  H61 AGLC    1      61.544  38.851  69.092  1.00  0.00      CARA H\nATOM  13647  H62 AGLC    1      60.407  40.284  69.159  1.00  0.00      CARA H\nATOM  13648  O6  AGLC    1      60.893  39.625  70.945  1.00  0.00      CARA O\nATOM  13649  HO6 AGLC    1      61.722  39.574  71.442  1.00  0.00      CARA H\nATOM  13650  C1  AGLC    2      61.574  41.975  66.260  1.00  0.00      CARA C\nATOM  13651  H1  AGLC    2      62.495  42.191  65.705  1.00  0.00      CARA H\nATOM  13652  C5  AGLC    2      59.800  40.395  66.308  1.00  0.00      CARA C\nATOM  13653  H5  AGLC    2      59.842  40.325  67.393  1.00  0.00      CARA H\nATOM  13654  O5  AGLC    2      61.055  40.757  65.739  1.00  0.00      CARA O\nATOM  13655  C2  AGLC    2      60.621  43.093  65.877  1.00  0.00      CARA C\nATOM  13656  H2  AGLC    2      60.526  43.143  64.768  1.00  0.00      CARA H\nATOM  13657  O2  AGLC    2      61.111  44.342  66.353  1.00  0.00      CARA O\nATOM  13658  HO2 AGLC    2      61.356  44.301  67.297  1.00  0.00      CARA H\nATOM  13659  C3  AGLC    2      59.270  42.804  66.503  1.00  0.00      CARA C\nATOM  13660  H3  AGLC    2      59.357  42.759  67.616  1.00  0.00      CARA H\nATOM  13661  O3  AGLC    2      58.345  43.836  66.175  1.00  0.00      CARA O\nATOM  13662  HO3 AGLC    2      58.631  44.627  66.649  1.00  0.00      CARA H\nATOM  13663  C4  AGLC    2      58.774  41.468  65.981  1.00  0.00      CARA C\nATOM  13664  H4  AGLC    2      58.641  41.524  64.876  1.00  0.00      CARA H\nATOM  13665  O4  AGLC    2      57.518  41.134  66.572  1.00  0.00      CARA O\nATOM  13666  HO4 AGLC    2      57.343  41.641  67.380  1.00  0.00      CARA H\nATOM  13667  C6  AGLC    2      59.367  39.040  65.772  1.00  0.00      CARA C\nATOM  13668  H61 AGLC    2      59.501  38.989  64.669  1.00  0.00      CARA H\nATOM  13669  H62 AGLC    2      58.297  38.845  66.005  1.00  0.00      CARA H\nATOM  13670  O6  AGLC    2      60.140  38.032  66.418  1.00  0.00      CARA O\nATOM  13671  HO6 AGLC    2      61.044  38.025  66.070  1.00  0.00      CARA H\nTER\nATOM  13672  SOD SOD     1      47.130  96.053  99.155  1.00  0.00      IONSNa\nATOM  13673  SOD SOD     2      77.410  92.297   2.916  1.00  0.00      IONSNa\nATOM  13674  SOD SOD     3      41.610  89.017  71.986  1.00  0.00      IONSNa\n<\/pre>\n\n\n\n<p><strong>topol.top<\/strong> (include downloaded charmm36 force field and replace &#8220;HETA&#8221; with &#8220;CARA&#8221;)<\/p>\n\n\n\n<pre class=\"prettyprint\">; Include forcefield parameters\n#include &#34;charmm36-feb2026_cgenff-5.0.ff&#47;forcefield.itp&#34; ; modified\n#include &#34;toppar&#47;PROA.itp&#34;\n#include &#34;toppar&#47;CARA.itp&#34;  ; modified\n#include &#34;toppar&#47;SOD.itp&#34;\n#include &#34;toppar&#47;TIP3.itp&#34;\n\n&#91; system &#93;\n; Name\nTitle\n\n&#91; molecules &#93;\n; Compound\t#mols\nPROA  \t           1\nCARA  \t           1  ; modified\nSOD   \t          23\nTIP3  \t       42746\n<\/pre>\n\n\n\n<pre class=\"prettyprint\">cp -r charmm-gui-&#91;numbers&#93;&#47;gromacs .\ncd gromacs\nsource &#47;usr&#47;local&#47;gromacs&#47;2026.1_cuda_torchcu128&#47;bin&#47;GMXRC\ngmx editconf -f step3_input_modified.pdb -bt cubic -box 11.7 -o box.gro\ngmx make_ndx -f box.gro &lt;&lt;EOF\n1 | 13 | 14\n15 | 16 | 17\nname 18 SOLU\nname 19 SOLV\na 3242-3253\nname 20 TYR214\na 5010-5015\nname 21 ASP327\na 5647-5653\nname 22 ASPP366\na 6223-6240\nname 23 TRP406\na 6828-6833\nname 24 ASP443\na 6843-6850\nname 25 MET444\na 8100-8111\nname 26 ARG526\na 8277-8294\nname 27 TRP539\na 8327-8333\nname 28 ASPP542\na 14190-14192\nname 29 HOH\n14|20|21|22|23|24|25|26|27|28|29\nname 30 QMatoms\na 6833\na 8333\na 13644\na 13650\nname 31 ASP443_OD2\nname 32 ASP542_HD2\nname 33 AGL1_O4\nname 34 AGL2_C1\nq\nEOF\n\n<\/pre>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-large is-resized\"><a href=\"https:\/\/manual.gromacs.org\/2020.4\/reference-manual\/special\/qmmm.html\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"793\" src=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-1024x793.jpg\" alt=\"\" class=\"wp-image-6954\" style=\"aspect-ratio:1.2913143735588009;width:471px;height:auto\" srcset=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-1024x793.jpg 1024w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-300x232.jpg 300w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-768x594.jpg 768w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-1536x1189.jpg 1536w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-2048x1585.jpg 2048w\" sizes=\"auto, (max-width: 1024px) 100vw, 1024px\" \/><\/a><figcaption class=\"wp-element-caption\">Atoms of the QM region.  As per the <a href=\"https:\/\/manual.gromacs.org\/2020.4\/reference-manual\/special\/qmmm.html\" target=\"_blank\" rel=\"noreferrer noopener\">GROMACS Manual<\/a>, chemical bonds connecting the two subsystems are capped with hydrogen link atoms. The force on these atoms is distributed over the two atoms forming the bond.<\/figcaption><\/figure>\n<\/div>\n\n\n<h3 class=\"wp-block-heading\">2. MD Simulation Protocol<\/h3>\n\n\n\n<h3 class=\"wp-block-heading\">2.1 Minimization and NVT Equilibration (303.15 K, 125 ps)<\/h3>\n\n\n\n<p>To maintain a complex geometry similar to the acarbose-bound state, flat-bottom distance restraints and angle restraints were applied using the GROMACS pull code (pull rate = 0):<\/p>\n\n\n\n<p><strong>Angle:<\/strong> ASP443_OD2 \u2013 AGL2_C1 \u2013 AGL1_O4 (nucleophile\u2013anomeric carbon\u2013leaving atom)<\/p>\n\n\n\n<p><strong>Distance 1:<\/strong> ASP443_OD2 \u2013 AGL2_C1<\/p>\n\n\n\n<p><strong>Distance 2:<\/strong> AGL1_O4 \u2013 ASP542_HD2<\/p>\n\n\n\n<pre class=\"prettyprint\">; PULL CODE settings\npull                    = yes\npull-ngroups            = 4\npull-ncoords            = 3\n\n; Define the groups by their names in the index file\npull-group1-name        = ASP443_OD2\npull-group2-name        = AGL2_C1\npull-group3-name        = AGL1_O4\npull-group4-name        = ASP542_HD2\n\n; --- Pull Coordinate 1 (Atom 1 and Atom 2) ---\npull-coord1-groups      = 1 2         ; References pull-group1 and pull-group2\npull-coord1-type        = flat-bottom    ; Or &#39;constant-force&#39;, &#39;constraint&#39;, etc.\npull-coord1-geometry    = distance    ; Pulling along the vector connecting them\npull-coord1-dim         = Y Y Y       ; Pulling in all dimensions (3D distance)\npull-coord1-init       = 0.33         ; Use initial distance as reference\npull-coord1-k           = 1000        ; Force constant &#91;kJ&#47;(mol*nm^2)&#93;\npull-coord1-rate        = 0        ; Pulling rate &#91;nm&#47;ps&#93;\n\n; --- Pull Coordinate 2 (Atom 1, Atom 2, and Atom 3) ---\npull-coord2-groups       = 2 1 2 3        ; vector 2-1 and vector 2-3\npull-coord2-type         = umbrella    ; Applies a harmonic restraint\npull-coord2-geometry     = angle        ; Specifies an angle (deg)\npull-coord2-dim          = Y Y Y        ; Dimensions are ignored for angle\npull-coord2-start        = no           ; Do not use the initial MD angle as reference\npull-coord2-init         = 180.0        ; The target angle in degrees\npull-coord2-k            = 500.0        ; Force constant (kJ&#47;mol&#47;rad^2)\n\n; --- Pull Coordinate 3 (Atom 3 and Atom 4) ---\npull-coord3-groups      = 3 4         ; References pull-group3 and pull-group4\npull-coord3-type        = flat-bottom\npull-coord3-geometry    = distance\npull-coord3-dim         = Y Y Y\npull-coord3-init       = 0.2\npull-coord3-k           = 1000\npull-coord3-rate        = 0\n<\/pre>\n\n\n\n<pre class=\"prettyprint\">wget -O charmm36-feb2026_cgenff-5.0.ff.tgz https:&#47;&#47;mackerell.umaryland.edu&#47;download.php?filename=CHARMM_ff_params_files&#47;charmm36-feb2026_cgenff-5.0.ff.tgz\ntar xvzf charmm36-feb2026_cgenff-5.0.ff.tgz\n\ngmx grompp -f step4.0_minimization_pull.mdp -c box.gro -r box.gro -n index.ndx -o step4.0\ngmx mdrun -v -deffnm step4.0 -ntmpi 1 -ntomp 9\ngmx grompp -f step4.1_equilibration_pull.mdp -c step4.0.gro -r step4.0.gro -n index.ndx -o step4.1\ngmx mdrun -v -deffnm step4.1 -ntmpi 1 -ntomp 9 # 125 ps\ngmx grompp -f step5_production_pull.mdp -c step4.1.gro -t step4.1.cpt -n index.ndx -o step5_1\ngmx mdrun -v -deffnm step5_1 -ntmpi 1 -ntomp 9 # 10 ns\n\nIn PyMOL,\nselect near_contacts, byres ((polymer.protein or resn TIP3) within 5 of resn AGLC)\nIn VMD,\n(protein or resname TIP3) and same residue as within 5 of resname AGLC\n<\/pre>\n\n\n\n<p><strong>run_equilibration.sh<\/strong><\/p>\n\n\n\n<pre class=\"prettyprint\">#!&#47;bin&#47;bash \n#SBATCH --job-name=step5_1_2_3 \n#SBATCH --output=job_%j.out \n#SBATCH --error=job_%j.err \n#SBATCH --partition=main \n#SBATCH --nodes=1 \n#SBATCH --ntasks=1 \n#SBATCH --cpus-per-task=9 # number of CPU core\n#SBATCH --mem=2G # memory\n#SBATCH --gres=gpu:1 # \n#SBATCH --time=48:00:00 # max job time\n\ncd $SLURM_SUBMIT_DIR\n\nsource &#47;usr&#47;local&#47;gromacs&#47;2026.1_cuda_torchcu128&#47;bin&#47;GMXRC\n\nOMP=9\n\ngmx grompp -f step5_production_pull.mdp -c step4.1.gro -t step4.1.cpt -n index.ndx -o step5_1\ngmx mdrun -deffnm step5_1 -ntmpi 1 -ntomp 9 # with three restraints, 10 ns\ngmx grompp -f step5_production_pull_2.mdp -c step5_1.gro -t step5_1.cpt -n index.ndx -o step5_2\ngmx mdrun -deffnm step5_2 -ntmpi 1 -ntomp 9 # with two restraints between ASP443 and maltose, 10 ns\ngmx grompp -f step5_production_10ns.mdp -c step5_2.gro -t step5_2.cpt -n index.ndx -o step5_3\ngmx mdrun -deffnm step5_3 -ntmpi 1 -ntomp 9 # without restraint, 10 ns\n\n<\/pre>\n\n\n\n<pre class=\"prettyprint\">sudo systemctl start slurmctld\nsudo systemctl enable slurmctld\nsudo systemctl start slurmd\nsudo systemctl enable slurmd\nsudo scontrol update nodename=mas-i9wx state=resume\n\nsbatch run_equilibration.sh\n<\/pre>\n\n\n\n<h3 class=\"wp-block-heading\">2.2 NPT Production with Triple Restraints (303.15 K, 1 bar, 10 ns)<\/h3>\n\n\n\n<p>The system was stabilized with all three restraints active.<\/p>\n\n\n\n<pre class=\"prettyprint\">gmx grompp -f step5_production_pull.mdp -c step4.1.gro -t step4.1.cpt -n index.ndx -o step5_1\ngmx mdrun -v -deffnm step5_1 -ntmpi 1 -ntomp 9 # with three restraints, 10 ns<\/pre>\n\n\n\n<h3 class=\"wp-block-heading\">2.3 NPT Production with Dual Restraints (10 ns)<\/h3>\n\n\n\n<p>The restraint on the <strong>ASP542_HD2 \u2013 AGL1_O4<\/strong> distance (Coordinate 3) was removed.<\/p>\n\n\n\n<p><strong>Observation:<\/strong> Without this restraint, the ASP542 side chain reoriented away from the maltose.<\/p>\n\n\n\n<pre class=\"prettyprint\">gmx grompp -f step5_production_pull_2.mdp -c step5_1.gro -t step5_1.cpt -n index.ndx -o step5_2\ngmx mdrun -v -deffnm step10_2 -ntmpi 1 -ntomp 9 # 10 ns<\/pre>\n\n\n\n<h3 class=\"wp-block-heading\">2.4 Unrestrained NPT Run (10 ns)<\/h3>\n\n\n\n<p>A fully unrestrained simulation was performed.<\/p>\n\n\n\n<p><strong>Correction:<\/strong> Consequently, the coordinates from the <strong>NPT simulation with three restraints (Section 2.2)<\/strong> were used as the starting point for the subsequent pulling simulations.<\/p>\n\n\n\n<p><strong>Result:<\/strong> Similar to section 2.3, the ASP542 side chain maintained an incorrect orientation relative to the substrate.<\/p>\n\n\n\n<pre class=\"prettyprint\">gmx grompp -f step5_production_10ns.mdp -c step5_2.gro -t step5_2.cpt -n index.ndx -o step5_3\ngmx mdrun -v -deffnm step5_3 -ntmpi 1 -ntomp 9 # 10 ns<\/pre>\n\n\n\n<h3 class=\"wp-block-heading\">2.5 Steered MD: Pulling along the Reaction Coordinate (0.2 ps)<\/h3>\n\n\n\n<p>Due to memory limitations encountered when using GROMACS 2026.1 with CP2K 2026.1 on a local workstation, subsequent steered MD (SMD) simulations were performed using GROMACS 2025.4 and CP2K 2024.3 at the <strong>Research Center for Computational Science<\/strong> (Okazaki, Japan). This version compatibility necessitated restricting the DFT functionals to <strong>PBE\/TZVP-MOLOPT<\/strong> and <strong>BLYP\/TZVP-MOLOPT<\/strong>.<\/p>\n\n\n\n<p>We performed pulling along the <strong>ASP443_OD2 \u2013 AGL2_C1<\/strong> coordinate to model the first glycosylation step. pull-coord1-k, 50000 [kJ\/(mol*nm^2)]; pull-coord1-rate, -1.0 [nm\/ps]. Per Br\u00e1s <em>et al<\/em>. (2018), the target distances for this step are:<\/p>\n\n\n\n<p><strong>Reactant (R):<\/strong> 3.26 \u00c5<\/p>\n\n\n\n<p><strong>Transition State (TS1):<\/strong> 2.05 \u00c5<\/p>\n\n\n\n<p><strong>Intermediate (INT1):<\/strong> 1.49 \u00c5<\/p>\n\n\n\n<p><strong>step6_qmmm_pulling.mdp<\/strong><\/p>\n\n\n\n<pre class=\"prettyprint\">integrator              = md\ndt                      = 0.001\nnsteps                  = 400\nnstxout-compressed      = 1\nnstxout                 = 0\nnstvout                 = 0\nnstfout                 = 0\nnstcalcenergy           = 1\nnstenergy               = 1\nnstlog                  = 1\n;\ncutoff-scheme           = Verlet\nnstlist                 = 20\nvdwtype                 = Cut-off\nvdw-modifier            = Force-switch\nrvdw_switch             = 1.0\nrvdw                    = 1.2\nrlist                   = 1.2\nrcoulomb                = 1.2\ncoulombtype             = PME\n;\ntcoupl                  = v-rescale\ntc_grps                 = SOLU SOLV\ntau_t                   = 1.0 1.0\nref_t                   = 303.15 303.15\n;\n;\nconstraints             = none\nconstraint_algorithm    = LINCS\ncontinuation            = yes\n;\nnstcomm                 = 100\ncomm_mode               = linear\ncomm_grps               = SOLU SOLV\n;\n; CP2K QMMM parameters\nqmmm-cp2k-active              = true   ; Activate QMMM MdModule\nqmmm-cp2k-qmgroup             = QMatoms ; Index group of QM atoms\nqmmm-cp2k-qmmethod            = PBE    ; Method to use\nqmmm-cp2k-qmcharge            = -1      ; Charge of QM system\nqmmm-cp2k-qmmultiplicity      = 1      ; Multiplicity of QM system\n\n; PULL CODE settings\npull                    = yes\npull-ngroups            = 2\npull-ncoords            = 1\n\n; Define the groups by their names in the index file\npull-group1-name        = ASP443_OD2\npull-group2-name        = AGL2_C1\n\n; --- Pull Coordinate 1 (Atom 1 and Atom 2) ---\npull-coord1-groups      = 1 2         ; References pull-group1 and pull-group2\npull-coord1-type        = umbrella    ; Or &#39;constant-force&#39;, &#39;constraint&#39;, etc.\npull-coord1-geometry    = distance    ; Pulling along the vector connecting them\npull-coord1-dim         = Y Y Y       ; Pulling in all dimensions (3D distance)\npull-coord1-start       = yes\npull-coord1-k           = 50000        ; Force constant &#91;kJ&#47;(mol*nm^2)&#93;\npull-coord1-rate        = -1.0        ; Pulling rate &#91;nm&#47;ps&#93;\npull-nstxout  \t      \t= 1\npull-nstfout  \t      \t= 1\n<\/pre>\n\n\n\n<h4 class=\"wp-block-heading\">Execution and Performance<\/h4>\n\n\n\n<p>The simulation was executed using the following commands:<\/p>\n\n\n\n<pre class=\"prettyprint\">source &#47;usr&#47;local&#47;gromacs&#47;2026.1_mpi_d_cp2k&#47;bin&#47;GMXRC\ngmx_mpi_d_cp2k grompp -f step6_qmmm_pulling_test.mdp -c step5_1.gro -t step5_1.cpt -n index.ndx -o step6_qmmm_test\nmpirun -np 32 gmx_mpi_d_cp2k mdrun -v -deffnm step6_qmmm_test -ntomp 1 # 5 fs (5 steps)<\/pre>\n\n\n\n<p>The calculation required <strong>6.5 hours<\/strong> to complete <strong>300 steps (0.3 ps)<\/strong> using 32 CPU cores.<\/p>\n\n\n\n<p><strong>Conclusion and Visualization<\/strong><\/p>\n\n\n\n<p>While QM\/MM MD simulations provide a reliable framework for estimating free energy changes, their computational cost remains high. Consequently, static QM\/MM methods employing the <strong>ONIOM scheme<\/strong> remain a practical alternative. Snapshots extracted from this pulling simulation can serve as initial coordinates for subsequent ONIOM calculations.<\/p>\n\n\n\n<figure class=\"wp-block-video\"><video height=\"1024\" style=\"aspect-ratio: 1024 \/ 1024;\" width=\"1024\" controls src=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/output.mp4\"><\/video><figcaption class=\"wp-element-caption\"><strong>Animation<\/strong>: Pull simulation trajectory. Atoms in the QM region are shown as sticks (excluding MET444). MM region atoms within 5 \u00c5 of the substrate are shown as lines.<\/figcaption><\/figure>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-full is-resized\"><img loading=\"lazy\" decoding=\"async\" width=\"792\" height=\"612\" src=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/pullx.png\" alt=\"\" class=\"wp-image-6964\" style=\"aspect-ratio:1.2941251563816478;width:496px;height:auto\" srcset=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/pullx.png 792w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/pullx-300x232.png 300w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/pullx-768x593.png 768w\" sizes=\"auto, (max-width: 792px) 100vw, 792px\" \/><figcaption class=\"wp-element-caption\"><strong>Plot 1<\/strong>: ASP443_OD2 \u2013 AGL2_C1 distance (nm) versus simulation time.<\/figcaption><\/figure>\n<\/div>\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-full is-resized\"><img loading=\"lazy\" decoding=\"async\" width=\"792\" height=\"612\" src=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/step6_qmmm_pulling_veryfast_50000_pullf.png\" alt=\"\" class=\"wp-image-6965\" style=\"aspect-ratio:1.2941251563816478;width:474px;height:auto\" srcset=\"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/step6_qmmm_pulling_veryfast_50000_pullf.png 792w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/step6_qmmm_pulling_veryfast_50000_pullf-300x232.png 300w, https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/step6_qmmm_pulling_veryfast_50000_pullf-768x593.png 768w\" sizes=\"auto, (max-width: 792px) 100vw, 792px\" \/><figcaption class=\"wp-element-caption\"><strong>Plot 2<\/strong>: Force exerted on the harmonic string (kJ\/mol\/nm) over time.<\/figcaption><\/figure>\n<\/div>\n\n\n<hr \/>\n\n\n\n<p>This blog post was copyedited with the assistance of Gemini (Google LLC). The author has reviewed and verified the final content for accuracy.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>This note details the reproduc &#8230;<\/p>\n","protected":false},"author":1,"featured_media":6954,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"om_disable_all_campaigns":false,"_uag_custom_page_level_css":"","_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"_uf_show_specific_survey":0,"_uf_disable_surveys":false,"_locale":"en_US","_original_post":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/?p=6974","footnotes":""},"categories":[5],"tags":[],"class_list":["post-7006","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-5","en-US"],"aioseo_notices":[],"uagb_featured_image_src":{"full":["https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-scaled.jpg",2560,1981,false],"thumbnail":["https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-150x150.jpg",150,150,true],"medium":["https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-300x232.jpg",300,232,true],"medium_large":["https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-768x594.jpg",768,594,true],"large":["https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-1024x793.jpg",800,620,true],"1536x1536":["https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-1536x1189.jpg",1536,1189,true],"2048x2048":["https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-2048x1585.jpg",2048,1585,true],"onepress-blog-small":["https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-300x150.jpg",300,150,true],"onepress-small":["https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-480x300.jpg",480,300,true],"onepress-medium":["https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp\/wp-content\/uploads\/2026\/04\/QMregion-640x400.jpg",640,400,true]},"uagb_author_info":{"display_name":"RCY","author_link":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/author\/charlesy\/"},"uagb_comment_info":0,"uagb_excerpt":"This note details the reproduc ...","_links":{"self":[{"href":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp-json\/wp\/v2\/posts\/7006","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp-json\/wp\/v2\/comments?post=7006"}],"version-history":[{"count":1,"href":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp-json\/wp\/v2\/posts\/7006\/revisions"}],"predecessor-version":[{"id":7007,"href":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp-json\/wp\/v2\/posts\/7006\/revisions\/7007"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp-json\/wp\/v2\/media\/6954"}],"wp:attachment":[{"href":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp-json\/wp\/v2\/media?parent=7006"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp-json\/wp\/v2\/categories?post=7006"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.ag.kagawa-u.ac.jp\/charlesy\/wp-json\/wp\/v2\/tags?post=7006"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}