Possibilities on the four levels of modeling detail. The figure illustrates the four nested levels of modeling detail (rows) of particle-based reaction-diffusion simulation and the effects than can be captured on each level (columns). The free diffusion level (level 1) contains basic 3D diffusion of point particles and reactions between them in a simulation box. The confined diffusion level (level 2) adds the definition of cellular geometry and diffusion in compartments. The excluded volume level (level 3) replaces point particles with volumetric entities. The particle-particle potential level (level 4) allows the definition of potentials between particles. The levels are shown using an illustration model of synaptic vesicle release (the tutorial example of ReaDDy ) that demands all four levels of modeling detail: Syntaxin (syx, blue, cyan), snap25 (grey) and syx-snap25 (red) are bound to a 2D disk membrane (gray disk) while synaptic vesicles (large, yellow, red, orange) diffuse in 3D cytoplasm. Self-clustering of syx and snap25 are modeled by an attractive particle-particle potential. The same simulation parameters are used on each level. The columns show: A snapshot of the simulated trajectory, the mean squared displacement (MSD) evolution of syx, the radial distribution function (RDF) of syx, the particle number evolution in time due to reactions and the software packages available on each level. On level 1, pure 3D diffusion prevents the modeling of membrane bound proteins. Level 2 allows 2D confinement. Level 3 prevents particle-particle overlaps, visible in the RDF. Level 4 allows to model syx-syx attraction (the resulting clustering appears as solvation shells in the RDF). Note, the non-trivial influence of confinement, excluded volume and clustering on the apparent diffusion constant (i.e. slope of MSD) and the reactions, underlining the importance to choose the correct level of modeling for the biological system at hand.