starting structures: a random collapsed conformer gener- ated from a standard MD simulation at 300 K and the crystal conformer from the ribosome. Convergence of the data is conﬁrmed by calculating the PMFs with increasing simulation time. For example, PMFs were calculated for 20–40 ns, 20–60 ns and 20–80 ns for the hairpin (see Figure S1, Supplementary Data). Data analysis Standard parameters such as radius of gyration (Rg), root mean square deviation (RMSD) and number of hydrogen bonds are calculated using Carnal and Ptraj from Amber8 package (47). A hydrogen bond is formed when the hydrogen bond donor (the heavy-atom to whom the hydrogen is attached to) and acceptor (heavy-atom) are separated by <3.3 A˚ from each other. Additionally, the hydrogen bond donor (heavy-atom), the hydrogen atom, and the hydrogen bond acceptor (heavy-atom) are required to be aligned in a <208 angle. Native hydrogen bonds (NH) are deﬁned as the ones present in the crystal conformer dissected from the ribosome. The number of non-native hydrogen bonds (NN) is calculated by subtracting the number of native hydrogen bond from the number of total hydrogen bond calculated based on the criteria mentioned above. Structures are visualized using Pymol (53) and UCSF Chimera (54). The free-energy landscape for folding was characterized by determining potential of mean force (PMF) as a function of one or more order parameters at a given temperature. PMF has the units of energy and it gives information about the probability of being in a particular state or conformer such that lower energy corresponds to higher probability. Weighted Histogram Analysis Methods (WHAM) (55–57) is used to calculate PMFs. Data from all temperatures were taken into account during the PMF calculation. Data from the ﬁrst 10 ns of each simulation were excluded from PMF calculations. For visual convenience, the energy of the lowest basin in each PMF has been shifted to zero. For the T-loop in isolation, the histogram grid size is 0.1 A˚ (RMSD) and 0.1 A˚ (Rg). For the hairpin, the histogram grid size is 0.2 A˚ (RMSD), 0.1 A˚ (Rg), 1 (NH) or 1 (NN). RESULTS T-loop in isolation and as part of a Hairpin In order to compare the folding of the T-loop sub-motif in the presence and absence of a local context (the stem), we determined potentials of mean force (PMF) of both the T-loop in isolation and as part of a hairpin as a function of the all-atom RMSD of the T-loop (with respect to the crystal conformer) and Radius of gyration (Rg). The T-loop in isolation shows a PMF at 300 K with one major basin centered around Rg values of 7.4 A˚ and all-atom RMSD ranking between 5.5 and 6 A˚ (Figure 2A). Visual analysis of the structures within the cluster reveals that the T-loop in isolation tends to be in a coil conformation such that base stacking interactions are optimized. The presence of U-turn sub-motif does not seem to have any Nucleic Acids Research, 2007, Vol. 35, No. 20 6997 Figure 2. (A) The PMF of T-loop in isolation plotted as a function of T-loop Rg versus RMSD at 300 K. T-loop in isolation shows a single minimum in the PMF. A representative structure is shown at the top left of the plot. The native state is not populated and it is not shown as a minimum on the plot. The PMF is plotted such that the energy of the global minimum is at 0 kcal/mol. (B) The PMF of the T-loop in the context of a hairpin at 300 K is plotted as a function of the T-loop RMSD versus Rg. For clarity, stems are not shown in the images. The PMF shows multiple distinct minima. All sample structures shown in the plot exhibit characteristic features (discussed in text) shared by the ensemble of structures found in the clusters. The energy of the global minimum (native) is 0 kcal/mol. The energy of the second minimum (expanded) is 0.4 kcal/mol. The energy of the third minimum (coil) is 0.6 kcal/mol. Each contour line represents an increase of 0.2 kcal/mol or a reduction in probability by 28%. A local minimum is considered to be signiﬁcant when it contains more than 2% of the population and is separated by a barrier of at least 1.5 kcal/mol. All RMSD and Rg values are in units of Angstroms (A˚ ). The PMF is plotted in units of kcal/mol, where 1 kcal roughly equals to 0.6 KT at 300 K. eﬀects on the conformation of the T-loop in isolation. Kinks found in the backbone of T-loop are often necessary to fulﬁll optimal base stacking interactions. Overall, the coil structures deviate signiﬁcantly from the T-loop crystal conformation and contain none of the signature hydrogen bonds belonging to this particular motif. These results indicate that the T-loop in isolation has no tendency to fold into its well-recognized crystal conformers (18). Neither of its structural components (U-turn or U:A trans W/H bp) is capable of directing the folding of the T-loop motif in this context. A suitable local environment appears critical for the formation of a U-shaped loop.