PyRETIS 3: Conquering rare and slow events without boundaries.

We present and discuss the advancements made in PyRETIS 3, the third instalment of our Python library for an efficient and user-friendly rare event simulation, focused to execute molecular simulations with replica exchange transition interface sampling (RETIS) and its variations. Apart from a general rewiring of the internal code towards a more modular structure, several recently developed sampling strategies have been implemented. These include recently developed Monte Carlo moves to increase path decorrelation and convergence rate, and new ensemble definitions to handle the challenges of long-lived metastable states and transitions with unbounded reactant and product states. Additionally, the post-analysis software PyVisa is now embedded in the main code, allowing fast use of machine-learning algorithms for clustering and visualising collective variables in the simulation data.

Highly parallelizable path sampling with minimal rejections using asynchronous replica exchange and infinite swaps.

Capturing rare yet pivotal events poses a significant challenge for molecular simulations. Path sampling provides a unique approach to tackle this issue without altering the potential energy landscape or dynamics, enabling recovery of both thermodynamic and kinetic information. However, despite its exponential acceleration compared to standard molecular dynamics, generating numerous trajectories can still require a long time. By harnessing our recent algorithmic innovations-particularly subtrajectory moves with high acceptance, coupled with asynchronous replica exchange featuring infinite swaps-we establish a highly parallelizable and rapidly converging path sampling protocol, compatible with diverse high-performance computing architectures. We demonstrate our approach on the liquid-vapor phase transition in superheated water, the unfolding of the chignolin protein, and water dissociation. The latter, performed at the ab initio level, achieves comparable statistical accuracy within days, in contrast to a previous study requiring over a year.

Extensive Angular Sampling Enables the Sensitive Localization of Macromolecules in Electron Tomograms.

Cryo-electron tomography provides 3D images of macromolecules in their cellular context. To detect macromolecules in tomograms, template matching (TM) is often used, which uses 3D models that are often reliable for substantial parts of the macromolecules. However, the extent of rotational searches in particle detection has not been investigated due to computational limitations. Here, we provide a GPU implementation of TM as part of the PyTOM software package, which drastically speeds up the orientational search and allows for sampling beyond the Crowther criterion within a feasible timeframe. We quantify the improvements in sensitivity and false-discovery rate for the examples of ribosome identification and detection. Sampling at the Crowther criterion, which was effectively impossible with CPU implementations due to the extensive computation times, allows for automated extraction with high sensitivity. Consequently, we also show that an extensive angular sample renders 3D TM sensitive to the local alignment of tilt series and damage induced by focused ion beam milling. With this new release of PyTOM, we focused on integration with other software packages that support more refined subtomogram-averaging workflows. The automated classification of ribosomes by TM with appropriate angular sampling on locally corrected tomograms has a sufficiently low false-discovery rate, allowing for it to be directly used for high-resolution averaging and adequate sensitivity to reveal polysome organization.

Exchanging Replicas with Unequal Cost, Infinitely and Permanently.

We developed a replica exchange method that is effectively parallelizable even if the computational cost of the Monte Carlo moves in the parallel replicas are considerably different, for instance, because the replicas run on different types of processor units or because of the algorithmic complexity. To prove detailed-balance, we make a paradigm shift from the common conceptual viewpoint in which the set of parallel replicas represents a high-dimensional superstate, to an ensemble-based criterion in which the other ensembles represent an environment that might or might not participate in the Monte Carlo move. In addition, based on a recent algorithm for computing permanents, we effectively increase the exchange rate to infinite without the steep factorial scaling as a function of the number of replicas. We illustrate the effectiveness of this replica exchange methodology by combining it with a quantitative path sampling method, replica exchange transition interface sampling (RETIS), in which the costs for a Monte Carlo move can vary enormously as paths in a RETIS algorithm do not have the same length and the average path lengths tend to vary considerably for the different path ensembles that run in parallel. This combination, coined ∞RETIS, was tested on three model systems.

Path Sampling Simulations Reveal How the Q61L Mutation Alters the Dynamics of KRas.

Flexibility is essential for many proteins to function, but can be difficult to characterize. Experiments lack resolution in space and time, while the time scales involved are prohibitively long for straightforward molecular dynamics simulations. In this work, we present a multiple state transition path sampling simulation study of a protein that has been notoriously difficult to characterize in its active state. The GTPase enzyme KRas is a signal transduction protein in pathways for cell differentiation, growth, and division. When active, KRas tightly binds guanosine triphosphate (GTP) in a rigid state. The protein-GTP complex can also visit more flexible states, in which it is not active. KRas mutations can affect the conversion between these rigid and flexible states, thus prolonging the activation of signal transduction pathways, which may result in tumor formation. In this work, we apply path sampling simulations to investigate the dynamic behavior of KRas-4B (wild type, WT) and the oncogenic mutant Q61L (Q61L). Our results show that KRas visits several conformational states, which are the same for WT and Q61L. The multiple state transition path sampling (MSTPS) method samples transitions between the different states in a single calculation. Tracking which transitions occur shows large differences between WT and Q61L. The MSTPS results further reveal that for Q61L, a route to a more flexible state is inaccessible, thus shifting the equilibrium to more rigid states. The methodology presented here enables a detailed characterization of protein flexibility on time scales not accessible with brute-force molecular dynamics simulations.

A Comprehensive Guide for Assessing Covalent Inhibition in Enzymatic Assays Illustrated with Kinetic Simulations.

Covalent inhibition has become more accepted in the past two decades, as illustrated by the clinical approval of several irreversible inhibitors designed to covalently modify their target. Elucidation of the structure-activity relationship and potency of such inhibitors requires a detailed kinetic evaluation. Here, we elucidate the relationship between the experimental read-out and the underlying inhibitor binding kinetics. Interactive kinetic simulation scripts are employed to highlight the effects of in vitro enzyme activity assay conditions and inhibitor binding mode, thereby showcasing which assumptions and corrections are crucial. Four stepwise protocols to assess the biochemical potency of (ir)reversible covalent enzyme inhibitors targeting a nucleophilic active site residue are included, with accompanying data analysis tailored to the covalent binding mode. Together, this will serve as a guide to make an educated decision regarding the most suitable method to assess covalent inhibition potency. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol I: Progress curve analysis of substrate association competition Basic Data Analysis Protocol 1A: Two-step irreversible covalent inhibition Basic Data Analysis Protocol 1B: One-step irreversible covalent inhibition Basic Data Analysis Protocol 1C: Two-step reversible covalent inhibition Basic Data Analysis Protocol 1D: Two-step irreversible covalent inhibition with substrate depletion Basic Protocol II: Incubation time-dependent potency IC50 (t) Basic Data Analysis Protocol 2: Two-step irreversible covalent inhibition Basic Protocol III: Preincubation time-dependent inhibition without dilution Basic Data Analysis Protocol 3: Preincubation time-dependent inhibition without dilution Basic Data Analysis Protocol 3Ai: Two-step irreversible covalent inhibition Alternative Data Analysis Protocol 3Aii: Two-step irreversible covalent inhibition Basic Data Analysis Protocol 3Bi: One-step irreversible covalent inhibition Alternative Data Analysis Protocol 3Bii: One-step irreversible covalent inhibition Basic Data Analysis Protocol 3C: Two-step reversible covalent inhibition Basic Protocol IV: Preincubation time-dependent inhibition with dilution/competition Basic Data Analysis Protocol 4: Preincubation time-dependent inhibition with dilution Basic Data Analysis Protocol 4Ai: Two-step irreversible covalent inhibition Alternative Data Analysis Protocol 4Aii: Two-step irreversible covalent inhibition Basic Data Analysis Protocol 4Bi: One-step irreversible covalent inhibition Alternative Data Analysis Protocol 4Bii: One-step irreversible covalent inhibition.

Chemistrees: Data-Driven Identification of Reaction Pathways via Machine Learning.

We propose to analyze molecular dynamics (MD) output via a supervised machine learning (ML) algorithm, the decision tree. The approach aims to identify the predominant geometric features which correlate with trajectories that transition between two arbitrarily defined states. The data-driven algorithm aims to identify these features without the bias of human "chemical intuition". We demonstrate the method by analyzing the proton exchange reactions in formic acid solvated in small water clusters. The simulations were performed with ab initio MD combined with a method to efficiently sample the rare event, path sampling. Our ML analysis identified relevant geometric variables involved in the proton transfer reaction and how they may change as the number of solvating water molecules changes.

PyRETIS 2: An improbability drive for rare events.

The algorithmic development in the field of path sampling has made tremendous progress in recent years. Although the original transition path sampling method was mostly used as a qualitative tool to sample reaction paths, the more recent family of interface-based path sampling methods has paved the way for more quantitative rate calculation studies. Of the exact methods, the replica exchange transition interface sampling (RETIS) method is the most efficient, but rather difficult to implement. This has been the main motivation to develop the open-source Python-based computer library PyRETIS that was released in 2017. PyRETIS is designed to be easily interfaced with any molecular dynamics (MD) package using either classical or ab initio MD. In this study, we report on the principles and the software enhancements that are now included in PyRETIS 2, as well as the recent developments on the user interface, improvements of the efficiency via the implementation of new shooting moves, easier initialization procedures, analysis methods, and supported interfaced software. © 2019 The Authors. Journal of Computational Chemistry published by Wiley Periodicals, Inc.