It is diffuculty to say. Typically from my experience this could be related
to initial packing of chains and water
Arun
On Fri, 5 Dec 2025, 18:56 cai.hanfeng--- via AMBER, <amber.ambermd.org>
wrote:
> Dear Amber community,
>
> We are trying to prepare an rbLDH (rabbit lactate dehydrogenase) with
> ligands in 1 M trehalose (TRE), and have encountered an unexpected increase
> in box size after the parametrization step. This change alters the
> trehalose concentration from the intended 1 M to ~0.28 M. We would
> appreciate help understanding why the box expands and how to keep the
> concentration constant.
>
> Summary of our workflow:
> We first dissolved the complex [rbLDH + pyruvate (PYR) + NADH (NAI)] into
> TIP3P water, and parameterized the system in water. From the output
> topology and coordinate files, we extracted the box size (890,346 ų,
> truncated octahedron) and calculated the number of trehalose molecules that
> we need to add to achieve 1 M (need 537 TRE molecules). To prepare the
> system with trehalose, we first removed all water and ions from the
> original system, loaded the solute-only structure (rbLDH + PYR + NAI) into
> tleap, saved a PDB with a temporary box (90°), and then manually fixed
> CRYST1 to our original truncated octahedron. Then we used AddToBox tool to
> add 537 molecules of trehalose into the solute and kept the box size
> consistent. After that, we performed parameterization for the whole system
> (rbLDH + PYR + NAI + TRE), in which we also dissolved the system with TIP3P
> water. But after that we found the box was changed into a larger size
> (3.13922e+06 ų), and therefore the concentration of trehalose is no longer
> 1 M. We wonder why the box size will increase after the last step
> parameterization, or how to maintain the original box size, or how to keep
> the trehalose concentration constant after parameterization?
>
> Here are the step-by-step detail that we did:
> 1) We prepare trehalose structure and parameters for trehalose, create a
> PDB with residue name TRE for trehalose in tleap.
> 2) To perform parametrization in water for rbLDH, with ligand pyruvate
> (PYR) and NADH (NAI), then we obtained two files “rbLDH_PYR_NAI.prmtop” and
> “rbLDH_PYR_NAI.inpcrd” files.
> 3) We measure the box volume (ų) based on these output files and the box
> volume is V = 890,346 ų.
> 4) To calculate how many moles of trehalose we need to add, we use the
> molarity formula with the right unit conversion N_molecules=C(mol/L ) ×
> V(L) × NA.
> For 1.0 M and V = 890,346 ų, need to add 537 of trehalose
> 5) We extracted the current solvated LDH box by converting the
> “rbLDH_PYR_NAI.prmtop” and “rbLDH_PYR_NAI.inpcrd” files to a PDB file
> “ldh_box.pdb” and looking for the CRYST1 line (first line in the obtained
> file):
> “CRYST1 104.969 104.969 104.969 109.47 109.47 109.47 P 1 1”
> 6) We strip all waters and common ions and obtain “solute_only.pdb”,
> containing the protein complex without waters and ions with the same box.
> 7) We produce a solute-only PDB file with a temporary box (90°):
> source leaprc.protein.ff14SB
> source leaprc.gaff
> loadamberparams PYR.frcmod
> loadoff PYR.lib
> loadamberparams NAI.frcmod
> loadoff NAI.lib
> LDH = loadpdb solute_only.pdb
> set LDH box { 109.47122 109.47122 109.47122 }
> savepdb LDH solute_only_oct.pdb
> quit
>
> 8) Manually fix CRYST1 to the original truncated octahedron by changing
> the first line in the “solute_only_oct.pdb” from
> CRYST1 109.471 109.471 109.471 90.00 90.00 90.00 P 1
> To box that we obtained in step 5):
> CRYST1 104.969 104.969 104.969 109.47 109.47 109.47 1
>
> 9) Then we use AddToBox tool to add 537 molecules trehalose into the
> solute.
> AddToBox -c solute_only_oct.pdb -a tre_single.pdb -na 537 -o
> solute_TRE_oct.pdb -RP 2.0 -RW 1.8 -V
> In the output file “solute_TRE_oct.pdb”, the box is still with the first
> line “CRYST1 104.970 104.970 104.970 109.47 109.47 109.47”.
>
> 10) Then we performed parameterization for the whole system
> “solute_TRE_oct.pdb”:
> tleap
> source leaprc.protein.ff14SB
> source leaprc.GLYCAM_06j-1
> source leaprc.gaff2
> source leaprc.water.tip3p
> loadamberparams frcmod.ionsjc_tip3p
> loadamberparams PYR.frcmod
> loadoff PYR.lib
> loadamberparams NAI.frcmod
> loadoff NAI.lib
> loadoff trehalose.lib
> LDH = loadpdb solute_TRE_oct.pdb # Load the packed solute+TRE PDB (has
> CRYST1)
> solvateoct LDH TIP3PBOX 0.0
> charge LDH
> addIons LDH Cl- 16
> saveamberparm LDH rbLDH_PYR_NAI_TRE1M.prmtop rbLDH_PYR_NAI_TRE1M.inpcrd
> quit
>
> 11) After this step, we convert the output files
> “rbLDH_PYR_NAI_TRE1M.prmtop” and “rbLDH_PYR_NAI_TRE1M.inpcrd” into the PDB
> file “rbLDH_PYR_NAI_TRE1M_tleap.pdb”, and check the box size.
> The first line in this PDB file was changed into “CRYST1 159.765
> 159.765 159.765 109.47 109.47 109.47 P 1 1”, therefore the box
> volume was also changed into 3.13922e+06 ų.
> In this case, the concentration of trehalose was changed into C_eff =
> 0.284054471834518 M instead of 1 M.
>
> We wonder why the box size will increase after the last step
> parameterization, or how to maintain the original box size, or how to keep
> the trehalose concentration constant after parameterization?
>
> Thank you for your guidance!
> Best regards,
> Hanfeng
>
>
>
> Hanfeng Cai
>
> Ph.D. student
>
> Prof. Ayelet Fishman’s Laboratory of Molecular and Applied Biocatalysis
>
> Dept. of Biotechnology and Food and Engineering
>
> Technion-Israel Institute of Technology
>
> Haifa, 3200003
>
> Israel
>
> Tel: (+972) 058-7562823
>
>
>
>
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Received on Fri Dec 05 2025 - 12:30:05 PST
