As for the density question, the initial densities, as you can see from the
tleap output, are not that far off from one another, so tleap is getting
the initial densities just fine, it is just adding a lot more water in
amber26.
------
Joseph Baker, PhD
Barbara Meyers Pelson Chair in Faculty-Student Engagement (AY 2024-2027)
Professor, Department of Chemistry
The College of New Jersey
Ewing, NJ 08628
Office: C212 Science Complex
Phone: (609) 771-3173
Web: www.bakercompchemlab.com
Social Media: Instagram <https://www.instagram.com/bakerlabtcnj/>, LinkedIn
<
https://www.linkedin.com/in/joseph-baker-6b444758/>
Pronouns: he/him/his
Chair-elect (2026), Trenton Local Section of the ACS
Chemistry Division Representative (2024-2027), The Council on Undergraduate
Research
On Tue, Jun 16, 2026 at 4:07 PM Baker, Joseph <bakerj.tcnj.edu> wrote:
> Hi Patricio,
>
> Very standard script, pasted at the bottom below. So we haven't been using
> the iso/aniso options. As you can see we are using the ff19SB and OPC water
> combo. As I mentioned, this same script run through multiple versions of
> amber only starts adding a large number of extra waters when we hit the
> amber26 version. As you can see the AmberTools25 and AmberTools23 versions
> of tleap both give a smaller number of OPC water residues added, and the
> bottom of the rst7 file for each of these agree identically. It is only in
> AmberTools26 where things start to change. Thanks for your help looking
> into this! - Joe
>
> For example when I use AmberTools26 it gives me:
>
> Loading PDB file: ../../00-structure/fold_full_sequence_model_0.pdb
>
> total atoms in file: 5271
>
> Leap added 4881 missing atoms according to residue templates:
>
> 4881 H / lone pairs
>
> Scaling up box by a factor of 1.260286 to meet diagonal cut criterion
>
> Solute vdw bounding box: 137.890 98.975 70.390
>
> Total bounding box for atom centers: 209.069 209.069 209.069
>
> (box expansion for 'iso' is 81.5%)
>
> Solvent unit box: 18.865 18.478 19.006
>
> Volume: 4635036.174 A^3 (oct)
>
> Total mass 2620962.702 amu, Density 0.939 g/cc
>
> Added 141329 residues.
>
>
> and then the bottom of the rst7 is: 181.9246991 181.9246991 181.9246991
> 109.4712190 109.4712190 109.4712190
>
> For example when I use AmberTools25 it gives me:
>
> Loading PDB file: ../../00-structure/fold_full_sequence_model_0.pdb
>
> total atoms in file: 5271
>
> Leap added 4881 missing atoms according to residue templates:
>
> 4881 H / lone pairs
>
> Scaling up box by a factor of 1.464352 to meet diagonal cut criterion
>
> Solute vdw bounding box: 137.890 98.975 70.390
>
> Total bounding box for atom centers: 178.892 178.892 178.892
>
> (box expansion for 'iso' is 105.2%)
>
> Solvent unit box: 18.865 18.478 19.006
>
> Volume: 2910739.196 A^3 (oct)
>
> Total mass 1628046.894 amu, Density 0.929 g/cc
>
> Added 86216 residues.
>
> and then the bottom of the rst7 is: 155.7907688 155.7907688 155.7907688
> 109.4712190 109.4712190 109.4712190
>
>
> For example when I use AmberTools23 it gives me:
>
> Loading PDB file: ../../00-structure/fold_full_sequence_model_0.pdb
>
> total atoms in file: 5271
>
> Leap added 4881 missing atoms according to residue templates:
>
> 4881 H / lone pairs
>
> Scaling up box by a factor of 1.464352 to meet diagonal cut criterion
>
> Solute vdw bounding box: 137.890 98.975 70.390
>
> Total bounding box for atom centers: 178.892 178.892 178.892
>
> (box expansion for 'iso' is 105.2%)
>
> Solvent unit box: 18.865 18.478 19.006
>
> Volume: 2910739.196 A^3 (oct)
>
> Total mass 1628046.894 amu, Density 0.929 g/cc
>
> Added 86216 residues.
>
>
> and then the bottom of the rst7 is: 155.7907688 155.7907688
> 155.7907688 109.4712190 109.4712190 109.4712190
>
>
> ----SCRIPT----
>
> #Load in the necessary forcefields
>
> source leaprc.protein.ff19SB
>
> source leaprc.water.opc
>
>
> #Load the PDBs
>
> prot = loadpdb ../00-structure/fold_full_sequence_model_0.pdb
>
>
> #solvate system
>
> solvateoct prot OPCBOX 14
>
>
> #Adding Ions
>
> addIonsRand prot Na+ 0
>
> addIonsRand prot Cl- 0
>
>
> #save files
>
> saveAmberParm prot sys.parm7 sys.rst7
>
>
> # Quit
>
> quit
>
> ------
> Joseph Baker, PhD
> Barbara Meyers Pelson Chair in Faculty-Student Engagement (AY 2024-2027)
> Professor, Department of Chemistry
> The College of New Jersey
> Ewing, NJ 08628
> Chair-elect (2026), Trenton Local Section of the ACS
> Chemistry Division Representative (2024-2027), The Council on
> Undergraduate Research
>
>
>
> On Tue, Jun 16, 2026 at 3:46 PM Patricio Barletta via AMBER <
> amber.ambermd.org> wrote:
>
>> *
>> Can I see the tleap script you are running? I'm curious about the water
>> model.
>> *
>> If you do tail -n1 <OUTPUT RST7 FILE> , do you get the same box size in
>> all cases?
>> *
>> If you run NVT on the output systems, do you get the same starting
>> density?
>>
>> The coordinates for the pre-equilibrated water molecules are at
>> `amber/dat/leap/lib/`, and I don't see any change on files like
>> 'tip3pbox.off'.
>> The other factor that may affect the final result is the usage of the
>> keyword "aniso", that prevents tleap from centering and reorienting the
>> solute before adding the box. This increases the box size for a given
>> buffer length.
>>
>> Best,
>>
>> ________________________________
>> From: amber-request.ambermd.org <amber-request.ambermd.org>
>> Sent: Tuesday, June 16, 2026 3:00 PM
>> To: amber.ambermd.org <amber.ambermd.org>
>> Subject: AMBER Digest, Vol 4976, Issue 1
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>> Today's Topics:
>>
>> 1. solvateoct behavior in Amber26 (Baker, Joseph)
>>
>>
>> ----------------------------------------------------------------------
>>
>> Message: 1
>> Date: Tue, 16 Jun 2026 14:55:03 -0400
>> From: "Baker, Joseph" <bakerj.tcnj.edu>
>> To: AMBER Mailing List <amber.ambermd.org>
>> Subject: [AMBER] solvateoct behavior in Amber26
>> Message-ID:
>> <
>> CAA5VDpDSCstP8vGa_cQ3Nj9eWtZCx3vpwkNSvTuWcxJsukYpTw.mail.gmail.com>
>> Content-Type: text/plain; charset="UTF-8"
>>
>> Hello,
>>
>> One of my students noticed that their simulations were running much more
>> slowly suddenly, and when looking at our tleap output we observed that the
>> solvateoct command seems to be adding a significantly larger number of
>> water molecules to the system than it has in the past (with the same
>> padding value, using the same initial structure). We went back and checked
>> the behavior in 25,24,23,22 versions and they were all consistent with the
>> number of water molecules being placed by solvateoct. Has there been some
>> change in the way solvateoct handles building the water shell?
>>
>> Thanks!
>> Joe
>>
>> ------
>> Joseph Baker, PhD
>> Barbara Meyers Pelson Chair in Faculty-Student Engagement (AY 2024-2027)
>> Professor, Department of Chemistry
>> The College of New Jersey
>> Ewing, NJ 08628
>> Chair-elect (2026), Trenton Local Section of the ACS
>> Chemistry Division Representative (2024-2027), The Council on
>> Undergraduate
>> Research
>>
>>
>> ------------------------------
>>
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