Fig. 1: Experimental design of MalE as a protein model system for smFRET studies.
a, Crystal structure of MalE in its ligand-free apo state (PDB ID 1OMP) with domains D1 and D2 linked by flexible beta sheets (highlighted in blue). b, The crystal structure of MalE (rotated by 90° as compared to a in the apo (gray, PDB ID 1OMP) and holo (green, PDB ID 1ANF) states with mutations at K29C-S352C (MalE-1), D87C-A186C (MalE-2) and A134C-A186C (MalE-3) indicated in black. Note, each mutant only contains one cysteine pair and was measured using the Alexa546–Alexa647 FRET pair. The estimated mean position of the fluorophores from AV calculations are shown as red spheres. c, FRET efficiency E histograms for three MalE mutants, MalE-1 (left), MalE-2 (middle) and MalE-3 (right), in the absence and presence of 1 mM maltose (bottom, green) for one exemplary dataset measured in laboratory 1. The distribution is fitted to a Gaussian distribution. The reported mean FRET efficiencies for 16 laboratories are shown below (due to experimental difficulties, the results of three laboratories were excluded; Supplementary Table 1). The mean FRET efficiency and the standard deviation of all 16 laboratories are given by the black line and gray area. d, Individual FRET efficiency differences for each laboratory, between the apo and holo states, \(\left\langle {E_{{{{\mathrm{holo}}}}}}\right\rangle - \left\langle{E_{{{{\mathrm{apo}}}}}} \right\rangle\), for MalE-1 (left), MalE-2 (middle) and MalE-3 (right). The mean FRET efficiency difference and the standard deviation of all 16 laboratories are given by the black line and gray area.
