Single particle imaging with FEL using photon correlations

Scattering experiments with femtosecond high-intensity free-electron laser pulses provide a new route to macromolecular structure determination without the need for crystallization at low material usage. In these experiments, the X-ray pulses are scattered with high repetition on a stream of identical single biomolecules and the scattered photons are recorded on a pixelized detector. The main challenges are the unknown random orientation of the molecule in each shot and the extremely low signal to noise ratio due to the very low expected photon count per scattering image, typically well below the number of over 100 photons required by available analysis methods. The latter currently limits the scattering experiments to nano-crystals or larger virus particles, but the ultimate goal remains to retrieve the atomic structure of single biomolecules. Here, we use photon correlations to overcome the issue with low photon counts and present an approach that can determine the molecular structure de novo from as few as three coherently scattered photons per image. We further validate the method with a small protein (46 residues), show that near-atomic resolution of 3.3 Å is within experimental reach and demonstrate structure determination in the presence of isotropic noise from various sources, indicating that the number of disordered solvent molecules attached to the macromolecular surface should be kept at a minimum. Our correlation method allows to infer structure from images containing multiple particles, potentially opening the method to other types of experiments such as fluctuation X-ray scattering (FXS)

Active role of elongation factor G in maintaining the mRNA reading frame during translation.

During translation, the ribosome moves along the mRNA one codon at a time with the help of elongation factor G (EF-G). Spontaneous changes in the translational reading frame are extremely rare, yet how the precise triplet-wise step is maintained is not clear. Here, we show that the ribosome is prone to spontaneous frameshifting on mRNA slippery sequences, whereas EF-G restricts frameshifting. EF-G helps to maintain the mRNA reading frame by guiding the A-site transfer RNA during translocation due to specific interactions with the tip of EF-G domain 4. Furthermore, EF-G accelerates ribosome rearrangements that restore the ribosome's control over the codon-anticodon interaction at the end of the movement. Our data explain how the mRNA reading frame is maintained during translation

ATP-magnesium coordination: Protein structure-based force field evaluation and corrections

In the numerous molecular recognition and catalytic processes across biochemistry involving adenosine triphosphate (ATP), the common bioactive form is its magnesium chelate, ATP·Mg2+. In aqueous solution, two chelation geometries predominate, distinguished by bidentate and tridentate Mg2+–phosphate coordination. These are approximately isoenergetic but separated by a high energy barrier. Force field-based atomistic simulation studies of this complex require an accurate representation of its structure and energetics. Here we focused on the energetics of ATP·Mg2+ coordination. Applying an enhanced sampling scheme to circumvent prohibitively slow sampling of transitions between coordination modes, we observed striking contradictions between Amber and CHARMM force field descriptions, most prominently in opposing predictions of the favored coordination mode. Through further configurational free energy calculations, conducted against a diverse set of ATP·Mg2+–protein complex structures to supplement otherwise limited experimental data, we quantified systematic biases for each force field. The force field calculations were strongly predictive of experimentally observed coordination modes, enabling additive corrections to the coordination free energy that deliver close agreement with experiment. We reassessed the applicability of the thus corrected force field descriptions of ATP·Mg2+ for biomolecular simulation and observed that, while the CHARMM parameters display an erroneous preference for overextended triphosphate configurations that will affect many common biomolecular simulation applications involving ATP, the force field energy landscapes broadly agree with experimental measurements of solution geometry and the distribution of ATP·Mg2+ structures found in the Protein Data Bank. Our force field evaluation and correction approach, based on maximizing consistency with the large and heterogeneous collection of structural information encoded in the PDB, should be broadly applicable to many other systems

Nanomechanics combined with HDX reveals allosteric drug binding sites of CFTR NBD1.

Cystic fibrosis (CF) is a frequent genetic disease in Caucasians that is caused by the deletion of F508 (DF508) in the nucleotide binding domain 1 (NBD1) of the CF transmembrane conductance regulator (CFTR). The DF508 compromises the folding energetics of the NBD1, as well as the folding of three other CFTR domains. Combination of FDA approved corrector molecules can efficiently but incompletely rescue the DF508-CFTR folding and stability defect. Thus, new pharmacophores that would reinstate the wild-type-like conformational stability of the DF508-NBD1 would be highly beneficial. The most prominent molecule, 5-bromoindole-3-acetic acid (BIA) that can thermally stabilize the NBD1 has low potency and efficacy. To gain insights into the NBD1 (un)folding dynamics and BIA binding site localization, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen- deuterium exchange (HDX) experiments. We found that the NBD1 a-subdomain with three adjacent strands from the b-subdomain plays an important role in early folding steps, when crucial non-native interactions are formed via residue F508. Our AFM and HDX experiments showed that BIA associates with this a-core region and increases the resistance of the DF508-NBD1 against mechanical unfolding, a phenomenon that could be exploited in future developments of folding correctors

Scaling of the GROMACS 4.6 molecular dynamics code on SuperMUC.

Here we report on the performance of GROMACS 4.6 on the SuperMUC cluster at the Leibniz Rechenzentrum in Garching. We carried out benchmarks with three biomolecular systems consisting of eighty thousand to twelve million atoms in a strong scaling test each. The twelve million atom simulation system reached a performance of 49 nanoseconds per day on 32,768 cores