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Welcome to ExiFRET

ExiFRET is a simple and flexible program that calculates FRET efficiencies between fluorophores in complex geometries, to help design and understand FRET experiments. This page allows you to run the program ExiFRET over the web and retrieve the output files for your own use. A short background to the program, as well as a more detailed description of the input parameters, examples of how to use the program and publications describing the method can be found by following the tabs above.

Example input files for the Figures found in the newest ExiFRET publication can be found in examples 2 .

To run the program simply enter the required input parameters in the form below and click on the "Run ExiFRET" button. The output of the program will be displayed below. You can also download the calculated FRET efficiencies as a text file.

For cases where fluorophores have constrained orientations, please use thetaFRET.

ExiFRET is developed in the Corry Research Group at the Australian National University.

Input Parameters

external coordinate generation:
user_coordinates: true false Choose true if you want to upload a file with fluorophore coordinates. In this case you can skip to the FRET calculation parameters but have to provide a file of coordinates. see also: user provided coordinates . Choose false to use the built in fluorophore coordinate generation
internal coordinate generation:
system_dimension: 2D 3D Use 2D for membrane systems, 3D for solution
nmer_size: How many fluorophores are attached to each host molecule? (Use 1 if donors and acceptors not on same host)
da_stoichiometry: true false Do you want to enforce a specific stoichiometry amongst fluorophores on each host?
d_per_nmer: if you are enforcing a specific stoichiometry, how many donors and how many acceptors should be on each host?
a_per_nmer: d_per_nmer + a_per_nmer = nmer_size
da_separate: true false Do you want hosts to contain either only donors or only acceptors such that only intermoleculr FRET can occur?
oligomers: true false For hexagonally packed fluorophores in each nmer (rather than the normal case of rings). Only works in 2D.
n_densities: The number of host molecule concentrations at which to calculate FRET (One of n_densities and n_radius needs to be 1)
density_min: The minimum host molecule concentration (host molecules per square angstrom in 2D or cubic angstrom in 3D; in 3D, 1 mM = 6.023e-7 )
density_max: The maximum host molecule concentration
n_radius: Number of host molecule (nmer) radii for which to calculate E (One of n_densities and n_radius needs to be 1)
radius_min: The minimum nmer radius
radius_max: The maximum nmer radius
second_radius: true false uses different radii for nmers and avoiding overlap. see userguide for details.
radius_diff: the difference between the radius used for avoiding overlaps and nmer radius
avoid_overlap: true false Prevent host molecules from overlapping (assumes them to be circular in 2D or spherical in 3D)
planar_nmers: true false If system dimension is 3, do you want each nmer to be distributed in a plane (true) or on a sphere (false)?
n_particles: The number of host molecules (nmers) in each calculation system
n_configurations: The number of different configurations of host molecules to average before outputting FRET efficiency
P_donor: The probability that any given fluorophore is a donor (should lie between 0 and 1).
labeling_efficiency: The fraction of potential sites that contain a fluorophore. 100% labelling = 1.0
clusters: true false distributes fluorophores in local regions of high density rather than randonly throughout space. only works in 2D.
n clusters: number of clusters
cluster radius: size of each cluster
FRET calculation parameters:
R0: The Forster distance of the donor / acceptor pair
n_photons: The number of photons to simulate interacting with each test configuration
buffer_size: To avoid edge effects, fluorophores within buffer_size x R0 are not included in efficiency calculation
irradiance: Irradiance of illuminating laser in units of Watts per square um
wavelength: Wavelength of illuminating laser in nm
extinction_coefficient: Extinction coefficient of donor fluorophores in cm-1 M-1
donor_lifetime: The lifetime of donor excitation in the absence of acceptors (ns)
acceptor_lifetime: The lifetime of acceptor excitation (ns)

This may take time - especially if n_densities, n_radius, n_particles, n_configurations or n_photons are large
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