Dear Harvey,
Yes, your issue is in the implementation of the 5’-phosphate parameters. If I were to guess you have one of two issues (or both).
a) your starting structure isn’t quite right.
b) your force field parameters and/or lib files aren’t quit right (this is most likely your issue). To see if this is the case, I would honestly systematically write out the chemical structure of the area where you have a 5’-phosphate. Make sure that you have those simulation parameters in the lib files that you have loaded. Make sure that you have the force field parameters you intend for this section.
c) you have a typo in your restraints (not a weird typo that doesn’t include the terminal phosphate, the terminal phosphate might be a separate residue number).
d) you need to ease off your restraints more slowly. Sometimes I do this when I have a troublesome system but I would guess the issue is (b) or (c) because your structure adopts a “weirdly distorted geometry.”
Maria
====
Maria Nagan, PhD
Professor of Practice, Chemistry Department
Associate Dean for Curriculum, College of Arts and Sciences
Stony Brook University
> On Mar 21, 2023, at 7:28 PM, Harvey Dale via AMBER <amber.ambermd.org> wrote:
>
> Hi,
>
> I have recently been attempting to simulate some simple RNA-based systems in explicit solvent using Amber (OL3/TIP3P), including RNA strands that terminate with a 5'-monophosphate. These 5'-phosphorylated strands have seemingly been causing issues, and I was hoping someone may be able to offer some advice on this count. I note a similar recent post on what appears to be a related issue.
>
> Specifically, I have been attempting to run simulations starting from an RNA duplex comprising two strands of different lengths (i.e., a nicked loop). The shorter strand is required by experimental considerations to be monophosphorylated at the 5'-terminus.
>
> I have prepared the topology and coordinate files via several different approaches - including as detailed in section 3.2.3 in the Amber22 manual, and via loadoff terminal_monophosphate.lib - starting from non-experimental pdb files, and have not been threatened with any obvious warnings from leap when generating these files (and parmed checkvalidity does not appear to highlight any issues with the prmtop file). I can minimise my system, and run constrained NVT/NPT simulations, but in the absence of constraints my production runs usually crash after several hundred ps, depending on the options I use.
>
> The crashes are typically preceded by various 'vlimit exceeded for step...' warnings and, sometimes, obvious temperature spikes; visual inspection would appear to implicate the terminal monophosphate in the instability (i.e., in the last frames prior to crashing, often the terminal phosphate in particular appears to have a weirdly distorted geometry, etc.), but I don't yet have enough experience with these simulations to be confident here.
>
> I have had no such problems in simulating an analogous system devoid of a 5'-monophosphate, but seem to get the same issue when attempting to simulate the single 5'-phosphorylated strand independently.
>
> To "fix" this issue I have tried running longer minimisations and constrained equilibration steps, using different barostat settings, different engines (sander.MPI vs pmemd.MPI, Amber16/22), shorter timesteps (1 fs vs 2 fs), but without luck. Turning SHAKE off and using a reduced timestep led to successful constrained equilibrations, as usual, but then resulted in a massive temperature spike and a complete meltdown during the production run prior to crashing (presumably SHAKE failure is causing the crashes when turned on)...
>
> Any help would be much appreciated. As a trivial error on my part is highly possible, I have put a typical production input file below.
>
> Cheers,
>
> Harvey
>
> Production.in:
> Overhang test
> &cntrl
> imin = 0,
> ig = -1,
> irest = 1,
> ntx = 5,
> ntb = 2,
> ntr = 0,
> cut = 8.0,
> ntc = 2,
> ntf = 2,
> ntt = 3,
> gamma_ln = 1.0,
> tempi = 300.0,
> temp0 = 300.0,
> ntp = 1,
> pres0 = 1.0,
> taup = 2.0,
> nstlim = 50000000, dt = 0.002,
> ntpr = 1000, ntwx = 1000, ntwr = 10000
> /
>
> ________________________________________________
>
> Dr Harvey J. A. Dale
>
> Research Fellow
> Royal Commission for the Exhibition of 1851
>
> John Henry Coates Research Fellow
> Emmanuel College | Cambridge | CB2 3AP | hd487.cam.ac.uk<mailto:hd487.cam.ac.uk>
>
> Postdoctoral Researcher
> MRC Lab of Molecular Biology | Cambridge | CB2 0QH
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Received on Tue Mar 21 2023 - 17:30:02 PDT
