### abstract ###
A longstanding question in molecular biology is the extent to which the behavior of macromolecules observed in vitro accurately reflects their behavior in vivo.
A number of sophisticated experimental techniques now allow the behavior of individual types of macromolecule to be studied directly in vivo; none, however, allow a wide range of molecule types to be observed simultaneously.
In order to tackle this issue we have adopted a computational perspective, and, having selected the model prokaryote Escherichia coli as a test system, have assembled an atomically detailed model of its cytoplasmic environment that includes 50 of the most abundant types of macromolecules at experimentally measured concentrations.
Brownian dynamics simulations of the cytoplasm model have been calibrated to reproduce the translational diffusion coefficients of Green Fluorescent Protein observed in vivo, and snapshots of the simulation trajectories have been used to compute the cytoplasm's effects on the thermodynamics of protein folding, association and aggregation events.
The simulation model successfully describes the relative thermodynamic stabilities of proteins measured in E. coli, and shows that effects additional to the commonly cited crowding effect must be included in attempts to understand macromolecular behavior in vivo.
### introduction ###
While reductionist biophysical studies continue to contribute important insights into the properties and functions of biological macromolecules, research attention is increasingly being directed at uncovering the extent to which behavior observed in vitro is likely to reflect that occurring in vivo CITATION, CITATION.
In a physiological setting, all biomolecules must inevitably experience non-specific, unintended interactions with the intracellular milieu and there are good theoretical reasons to expect that, even if such interactions are only steric in nature, significant alterations in macromolecular folding and association equilibria may result CITATION, CITATION.
In order to allow macromolecules to be directly interrogated in vivo therefore, a number of important developments have been made in the experimental fields of hydrogen exchange CITATION, nuclear magnetic resonance CITATION, CITATION, and fluorescence spectroscopies CITATION CITATION .
An alternative to the use of experimental techniques is to assemble a molecular model of an intracellular environment in silico and to use molecular simulation techniques to explore its behavior; if such a model could be shown to be realistic and that is a big if it would have the important advantage of allowing the simultaneous, direct observation of all molecules in the system.
In fact, at least two simulation studies that attempt to model the bacterial cytoplasm have already been reported CITATION, CITATION, producing a number of intriguing results.
Both of these previous studies, however, modeled all cytoplasmic molecules as spheres and it is perhaps to be anticipated therefore that simulations that include structurally detailed macromolecular models might lead to additional insights.
In pursuit of this strategy, we have chosen the gram-negative prokaryote Escherichia coli as a test system, combining quantitative proteomic CITATION and high-resolution structural data CITATION to build a first structurally detailed molecular model of the bacterial cytoplasm.
