A Suite of Tutorials for the WESTPA 2.0 Rare-Events Sampling Software [Article v2.0].

The weighted ensemble (WE) strategy has been demonstrated to be highly efficient in generating pathways and rate constants for rare events such as protein folding and protein binding using atomistic molecular dynamics simulations. Here we present two sets of tutorials instructing users in the best practices for preparing, carrying out, and analyzing WE simulations for various applications using the WESTPA software. The first set of more basic tutorials describes a range of simulation types, from a molecular association process in explicit solvent to more complex processes such as host-guest association, peptide conformational sampling, and protein folding. The second set ecompasses six advanced tutorials instructing users in the best practices of using key new features and plugins/extensions of the WESTPA 2.0 software package, which consists of major upgrades for larger systems and/or slower processes. The advanced tutorials demonstrate the use of the following key features: (i) a generalized resampler module for the creation of "binless" schemes, (ii) a minimal adaptive binning scheme for more efficient surmounting of free energy barriers, (iii) streamlined handling of large simulation datasets using an HDF5 framework, (iv) two different schemes for more efficient rate-constant estimation, (v) a Python API for simplified analysis of WE simulations, and (vi) plugins/extensions for Markovian Weighted Ensemble Milestoning and WE rule-based modeling for systems biology models. Applications of the advanced tutorials include atomistic and non-spatial models, and consist of complex processes such as protein folding and the membrane permeability of a drug-like molecule. Users are expected to already have significant experience with running conventional molecular dynamics or systems biology simulations.

WESTPA 2.0: High-Performance Upgrades for Weighted Ensemble Simulations and Analysis of Longer-Timescale Applications.

The weighted ensemble (WE) family of methods is one of several statistical mechanics-based path sampling strategies that can provide estimates of key observables (rate constants and pathways) using a fraction of the time required by direct simulation methods such as molecular dynamics or discrete-state stochastic algorithms. WE methods oversee numerous parallel trajectories using intermittent overhead operations at fixed time intervals, enabling facile interoperability with any dynamics engine. Here, we report on the major upgrades to the WESTPA software package, an open-source, high-performance framework that implements both basic and recently developed WE methods. These upgrades offer substantial improvements over traditional WE methods. The key features of the new WESTPA 2.0 software enhance the efficiency and ease of use: an adaptive binning scheme for more efficient surmounting of large free energy barriers, streamlined handling of large simulation data sets, exponentially improved analysis of kinetics, and developer-friendly tools for creating new WE methods, including a Python API and resampler module for implementing both binned and "binless" WE strategies.

A Suite of Tutorials for the WESTPA Rare-Events Sampling Software [Article v1.0].

The weighted ensemble (WE) strategy has been demonstrated to be highly efficient in generating pathways and rate constants for rare events such as protein folding and protein binding using atomistic molecular dynamics simulations. Here we present five tutorials instructing users in the best practices for preparing, carrying out, and analyzing WE simulations for various applications using the WESTPA software. Users are expected to already have significant experience with running standard molecular dynamics simulations using the underlying dynamics engine of interest (e.g. Amber, Gromacs, OpenMM). The tutorials range from a molecular association process in explicit solvent to more complex processes such as host-guest association, peptide conformational sampling, and protein folding.

Real-time digital median frequency estimator for surface myoelectric signals.

A prototype instrument is described that uses a digital signal processor to estimate the median frequency of the myoelectric signal in real time. A set of tenth-order digital low-pass filters is used to track the median frequency. An estimate of frequency is obtained once every 256 ms with a resolution of 1 Hz.

Photoaffinity labelling of isopenicillin N synthetase.

The alpha-aminoadipoyl group of the natural substrate of isopenicillin N synthetase (IPNS), L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV), has been replaced by a diazirinyl-containing group, which can be photoactivated. This has allowed investigation of the substrate binding site of IPNS by photoaffinity labelling. Laser flash photolysis of this analogue, [3H]DCV, in the presence of IPNS leads to the incorporation of radioactivity into the enzyme. Tryptic digestion of this labelled enzyme, followed by separation and sequencing of the resultant fragments, identified two labelled regions of the protein. These are the fragments Asp-40 to Arg-78 and Thr-237 to Gly-256.

Photoaffinity labelling of isopenicillin N synthetase by laser-flash photolysis.

Isopenicillin N synthetase (IPNS) from Acremonium chrysogenum was photolabelled by laser-flash photolysis in the presence of a diazirinyl-containing substrate, 2-[3-(3-trifluoromethyl-3H-diazirin-3-yl)-phenoxy]acetyl-S- methyloxycarbonylsulphenyl-L-cysteinyl-D-valine (DCV). Labelling of IPNS by DCV is partially inhibited in the presence of an excess of L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV), the natural substrate. In the absence of light, DCV is converted into the corresponding penicillin with comparable Km but significantly depressed Vmax relative to ACV. Selective incorporation of [14C]DCV into IPNS has been demonstrated by fluorography of IPNS analysed by SDS/polyacrylamide-gel electrophoresis. Scintillation counting of labelled IPNS purified on an ion-exchange f.p.l.c. column confirms this result. This methodology may be applicable for studies aimed at investigating the binding of substrates to IPNS.

Purification and characterization of cloned isopenicillin N synthetase.

Isopenicillin N synthetase (IPS) cloned from Cephalosporium acremonium has been isolated from transformed Escherichia coli and purified to homogeneity. The resulting, abundant, recombinant protein, whilst undergoing slightly different N-terminal processing to that observed for the fungally-derived protein, has identical kinetics for the conversion of LLD-aminoadipoyl-cysteinyl-valine to isopenicillin N. Recombinant IPS converts analogue substrates into unusual beta-lactam antibiotics in exactly the same way as the fungal protein.

Biosynthetic thiolase from zoogloea ramigera. I. Preliminary characterization and analysis of proton transfer reaction.

The biosynthetic thiolase, from Zoogloea ramigera, involved in generation of acetoacetyl-CoA for poly-beta-hydroxybutyrate synthesis, has been prepared pure in quantity for initial structural characterization of this homotetrameric enzyme. Edman degradation provided the sequence of the NH2 terminal 25 residues and an active site cysteine-containing nonapeptide labeled on stoichiometric inactivation by iodoacetamide. Both sequences were used to align the encoding DNA sequence of the cloned gene as described in an accompanying paper. Synthetic analogs of acetoacetyl-S-CoA, modified in the CoA moiety, were prepared and tested, and acetoacetyl-S-pantetheine 11-pivalate 1 was shown to have a kcat/Km of 6.4 X 10(6) M-1 s-1, comparable to the kcat/Km of 2 X 10(7) M-1 s-1 for acetoacetyl-S-CoA. The pantetheine pivalate group facilitates nonaqueous synthetic manipulations and may be generally useful as a CoA replacement. We have also prepared the carba analog of 1, with CH2 replacing S, to yield a beta-diketone analog 10 of acetoacetyl-S-CoA and the corresponding methyl ketone analog 9 of acetyl-S-CoA. These analogs have been used to prove the ability of Z. ramigera thiolase to catalyze proton abstraction from the C-2 methyl group of the acetyl portion of substrate in a transition state separate from C-C bond formation. NMR studies in D2O show exchange only when condensation is possible. Further studies with [2-3H]acetyl-CoA show there is neither pre-equilibrium washout nor detectable kH/kT expressed in turnover and provide no evidence for a discrete acetyl-CoA C-2 carbanion or a nonconcerted reaction.