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In the tutorial of RNAfold it states,

RNAfold reads RNA sequences from stdin, calculates their minimum free energy (MFE) structure, prints the MFE structure in dot-bracket notation and its free energy to stdout.

In the example using the -p --MEA options, the

  • MFE = -5.00 and the
  • ensemble Free Energy = -5.72.

How can the free energy of the ensemble be lower than the minimum free energy?

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  • $\begingroup$ @MichaelG. First, thank you. I read and learn from your comments and answers. Second: I tried with CUACGGCGCGGCGC and I get: structure=.....(((...))) with -0.40 kcal/mol for the MFE and for the probab. representation of the ensemble, structu=.....,,,...)), with -1.10 kcal/mol. I don't see pseudoknots there (should I?). $\endgroup$
    – aerijman
    Apr 15, 2019 at 18:49
  • $\begingroup$ @MichaelG. 1st: Thank you! I feel honored!! 2nd: I think (and I could be wrong) that the only tool in Vienna that predicts pseudoknots is RNAPKplex (I am too far from being expert in the topic, I just googled it -couldn't find that info as fast in Vienna documentation-). 3rd: I am perplexed... I don't know how is this possible $\endgroup$
    – aerijman
    Apr 22, 2020 at 16:26

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