Clustering cryo-EM images of helical protein polymers for helical reconstructions.

Helical protein polymers are often dynamic and complex assemblies, with many conformations and flexible domains possible within the helical assembly. During cryo-electron microscopy reconstruction, classification of the image data into homogeneous subsets is a critical step for achieving high resolution, resolving different conformations and elucidating functional mechanisms. Hence, methods aimed at improving the homogeneity of these datasets are becoming increasingly important. In this paper, we introduce a new algorithm that uses results from 2D image classification to sort 2D classes into groups of similar helical polymers. We show that our approach is able to distinguish helical polymers that differ in conformation, composition, and helical symmetry. Our results on test and experimental cases - actin filaments and amyloid fibrils - illustrate how our approach can be useful to improve the homogeneity of a data set. This method is exclusively applicable to helical polymers and other limitations are discussed.

Systematic Excited State Studies of Reversibly Switchable Fluorescent Proteins.

The reversibly switchable fluorescent proteins Dronpa, rsFastLime, rsKame, Padron, and bsDronpa feature the same chromophore but display a 40 nm variation in absorption maxima and an only 18 nm variation in emission maxima. In the present contribution, we employ QM/MM models to investigate the mechanism of such remarkably different spectral variations, which are caused by just a few amino acid replacements. We show that the models, which are based on CASPT2//CASSCF level of QM theory, reproduce the observed trends in absorption maxima, with only a 3.5 kcal/mol blue-shift, and in emission maxima, with an even smaller 1.5 kcal/mol blue-shift with respect to the observed quantities. In order to explain the variations across the series, we look at the chromophore's electronic structure change during absorption and emission. Such analysis indicates that a change in charge-transfer character, which is more pronounced during absorption, triggers a cascade of hydrogen-bond-network rearrangements, suggesting preparation to an isomerization event. We also show how the contribution of Arg 89 and Arg 64 residues to the chromophore conformational changes correlate with the spectral variations in absorption and emission. Furthermore, we describe how the conical intersection stability is related to the protein's photophysical properties. While for the Dronpa, rsFastLime, and rsKame triad, the stability correlates with the photoswitching speed, this does not happen for bsDronpa and Padron, suggesting a less obvious photoisomerization mechanism.

Molecular Dynamics of Double Stranded Xylo-Nucleic Acid.

Xylo-nucleic acid (XyloNA) is a synthetic analogue of ribo-nucleic acid (RNA), where the ribose sugar has been replaced by xylose. We present a molecular dynamics study of the conformational evolution of XyloNA double strand oligomers derived from A-RNA through the substitution of ß-d-ribofuranose by ß-d-xylofuranose and having lengths of 8, 16, and 29 base pairs, using a set of independent all-atom simulations performed at various time scales ranging from 55 to 100 ns, with one long 500 ns simulation of the 29-mer. In order to validate the robustness of XyloNA conformation, a set of simulations using various cutoff distances and solvation box dimensions has also been performed. These independent simulations reveal the uncoiling or elongation of the initial conformation to form an open ladder type transient state conformation and the subsequent formation of a highly flexible duplex with a tendency to coil in a left-handed fashion. The observed open ladder conformation is in line with recently obtained NMR data on the XyloNA 8-mer derived using 5'-d(GUGUACAC)-3'. The observed negative interbase pair twist leads to the observed highly flexible left-handed duplex, which is significantly less rigid than the stable left-handed dXyloNA duplex having a strong negative twist. A comparison between the xylo-analogues of DNA and RNA shows a clear distinction between the helical parameters, with implications for the pairing mechanism.

Thermal Isomerization Mechanism in Dronpa and Its Mutants.

The photoswitching speed of the reversibly switchable fluorescent proteins (RSFPs) from the family of green fluorescent proteins (GFPs) changes upon mutation which is of direct importance for various high-resolution techniques. Dronpa is one of the most used RSFPs. Its point mutants rsFastLime (Dronpa V157G) and rsKame (Dronpa V157L) exhibit a striking difference in their photoswitching speed. Here the QM/MM on-the-fly string method is used in order to explore the details of the thermal isomerization mechanism. The four principal ways in which isomerization may occur have been scrutinized for each of the three proteins. It has been shown that thermal isomerization occurs via a one-bond-flip mechanism in all three proteins, although, in rsKame, where the chromophore is constrained more, the activation free energy difference between hula-twist and one-bond-flip is significantly smaller. Functional mode analysis has been applied to examine the motions of the amino acids during the isomerization. It clearly identifies the importance of Val/Leu 157 as well as the amino acids in the α-helix during the isomerization.

Molecular Dynamic Indicators of the Photoswitching Properties of Green Fluorescent Proteins.

Reversibly photoswitchable fluorescent proteins (RSFPs) are highly useful probes for a range of applications including diffraction-unlimited fluorescence microscopy. It was previously shown that reversible photoswitching not only involves cis-trans isomerization and protonation-deprotonation of the chromophore but also results in a marked difference in ß-barrel flexibility. In this work, we performed flexibility profiling and functional mode analysis (FMA) using molecular dynamics calculations to study how the flexibility of the RSFP ß-barrel influences the photoswitching properties of several fluorescent proteins. We also used Partial Least-Squared (PLS) FMA to detect promising mutation sites for the modulation of photoswitching properties of RSFPs. Our results show that the flexibility of RSFP does depend on its state with a systematically higher flexibility in the dark state compared to the bright state. In particular our method highlights the importance of Val157 in Dronpa, which upon mutation yields a striking difference in the collective motions of the two mutants. Overall, we show that PLS-FMA yields information, complementary to static structures, that can guide the rational design of fluorescent proteins.