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 - 11:00:03 PST
