The main requirement for using thetaFRET is to get familiar with the input parameters and to be able to define the geometry of the fluorophores.

Coordinate File

The main input is a coordinate file defining the geometry of the fluorophores. The coordinate file should contain the location, type, average transition moment direction and fluctuation angle. The file needs to have to following format.

x     y     z     fluorophore_type     vector-x     vector-y     vector-z     θ

In this, x, y and z refer to the position of each fluorphore, The fluorophore type defines if the fluorophore is a donor or acceptor where 1 = donor and 0 = acceptor. The vector components (vector-x, vector-y, vector-z) define a vector pointing in the direction of the transition dipole. The model assumes that the fluorophore is freely oscilating in a cone defined by θ.

The position and type of each fluorophore needs to be on a separate line and the file should not contain any emtpy lines ie if there are 20 fluorophores the file needs to exactly 20 lines plus a carriage return at the end of the last line. The field deliminator is a space, no comma is required.

The following is an example of an input file for a simulation with 1 donor and 2 acceptors, arranged in 3 dimensions.

60.00	10.00	0.00	0	1.00	0.00	1.00	30
0.00	0.00	0.00	1	1.00	0.00	0.00	10
0.00	-50.00	-20.00	0	0.00	1.00	0.00	10

n_configurations

When calculating FRET, the program generates many fluorophore orientations within the cone defined by the mean ransition moment vector and the cone angle θ. The number of orientations for each fluorophore is given by n_configurations. Obviously you will get a better average FRET value if you use a larger number of configurations.

n_excitons

For calculating FRET according to the static average regime, the FRET efficiency is calculated for each set of fluorophore orientations using the monte-carlo scheme developed in the program exiFRET . Again, a better static average FRET value will come with a greater number of excitons, although this will increase the compute time.

R0

This is the characteristic Forster distance of the fluorophore pair assuming an orientation factor of 2/3. Note that this value is not used directly in the calculation, but rather is modified according to the orientation factors found for the specific fluorophore configurations.

Output

The program will output the calcualted FRET efficiency according to 2 averaging regimes.

The static averaging regime assumes that the relative orientations of the fluorophores do not change during the exctited lifetime of the donor, ie that the orientational correlation time is much larger than the fluorescence lifetime.

The dynamic averaging regime assumes that the fluorophores can change their relative orientation during the time the conor is excited, ie that the orientational correlation time is much smaller than the fluorescence lifetime