RNAstructure logo

RNAstructure Command Line Help
efn2 and efn2-smp

efn2 (Energy Function 2) is used to calculate the folding free energy change of a structure or structures in a CT file or Dot-Bracket file. The output from efn2 can be either a simple list of energies or a detailed breakdown of the calculation.

efn2 now supports structures that contain pseudoknots (as of version 6.0).

efn2 now by default reports error estimates for free energy calculations for RNA (as of version 6.1; this can be disabled using --ne). This is described in Zuber and Mathews (2018).

efn2-smp is a parallel processing version for use on multi-core computers, built using OpenMP.

Important Note: efn2 uses a different energy function for estimating multibranch loop initiation free energy changes than the dynamic programming algorithms, e.g. Allsub, Fold, partition, and stochastic. The efn2 energy function has an additional term that penalizes asymmetry in the distribution of unpaired nucleotides around the loop. Therefore, folding free energies from efn2 can differ from other programs in RNAstructure. See the -s, -S, --simple flag below.

USAGE: efn2 <ct file> <energy file> [options]

OR: efn2-smp <ct file> <energy file> [options]

Required parameters:

<ct file> The name of a CT file or Dot-Bracket file containing the input structure.
<energy file> The energy file to which output is written.
The energy file can be written in one of two forms:
  1. Simple List
    Lists free energy for each structure, lowest first.
    This file type is written by default.
  2. Thermodynamic Details
    Writes details of every substructure in each structure, and corresponding free energy of each.
    This file type is only written if "-w," "-W," or "--writedetails" (see below) is specified, and replaces a simple list file.

Options that do not require added values:

-d, -D, --DNA Specify that the sequence is DNA, and DNA parameters are to be used.
Default is to use RNA parameters.
-h, -H, --help Display the usage details message.
--ne Do not calculate experimental uncertainties.
-p, -P, --print Print the output file to standard output.
This won't override default behavior of writing to a file.
Thermodynamic files (if written) are not printed, even if this option is specified, because they can be very large.
-s, -S, --simple Use the simple energy function for multibranch loops that is the same used by the dynamic programming algorithms (Fold, partition, stochastic, AllSub, etc.). If this is not specified, an more sophisticated energy function is used, and the energies might not match those estimated for structures during structure prediction.
-w, -W, --writedetails Write a thermodynamic details file.
The thermodynamic details file replaces a standard output (list) file.

Options that require added values:

-a, -A, --alphabet Specify the name of a folding alphabet and associated nearest neighbor parameters. The alphabet is the prefix for the thermodynamic parameter files, e.g. "rna" for RNA parameters or "dna" for DNA parameters or a custom extended/modified alphabet. The thermodynamic parameters need to reside in the at the location indicated by environment variable DATAPATH.
The default is "rna" (i.e. use RNA parameters). This option overrides the --DNA flag.
-c, -C, --count Specify a file where parameter usage counts will be exported
-sh, -SH, --SHAPE Specify a SHAPE constraints file to be applied. These constraints specificially use SHAPE pseudoenergy constraints.
Default is no SHAPE constraint file specified.
-si, -SI, --SHAPEintercept Specify an intercept used with SHAPE constraints.
Default is -0.6 kcal/mol.
-sm, -SM, --SHAPEslope Specify a slope used with SHAPE constraints.
Default is 1.8 kcal/mol.
-t, -T, --temperature Specify the temperature at which the free energy free should be calculated in Kelvin.
Default is 310.15 K, which is 37 degrees C.

Notes for smp:

efn2-smp, by default, will use all available compute cores for processing. The number of cores used can be controlled by setting the OMP_NUM_THREADS environment variable.

Notes for uncertainty calculations:

  • The calculation of uncertainty in folding free energy changes is only enabled in efn2, not efn2-smp.
  • The calculation of uncertainty can only be done for RNA sequences, since the required data tables are not available for DNA.
  • The error estimates calculated by efn2 currently do not take into account uncertainty in the pseudoknot or SHAPE energy models.


  1. Zuber, J and Mathews, D.H.
    "Estimating Uncertainty in Predicted Folding Free Energy Changes of RNA Secondary Structures"
    RNA, Submitted. (2018).
  2. Reuter, J.S. and Mathews, D.H.
    "RNAstructure: software for RNA secondary structure prediction and analysis."
    BMC Bioinformatics, 11:129. (2010).
  3. Mathews, D.H., Sabina, J., Zuker, M. and Turner, D.H.
    "Expanded sequence dependence of thermodynamic parameters provides improved prediction of RNA secondary structure."
    J. Mol. Biol., 288:911-940. (1999).