Living cells are brimming with the activity of macromolecular complexes carrying out their assigned tasks. Structures of these complexes can be resolved with cryo-electron microscopy (cryo-EM), wherein the complexes are first freeze-shocked into states characterizing their action and subsequently imaged by detection cameras. Recent advances in direct detection camera technology enable today's cryo-EM laboratories to image the macromolecular complexes at high-resolution, giving us a better view of the cell than ever before. Computational techniques like molecular dynamics flexible fitting (MDFF) are a key tool for producing atomic models of the imaged molecules, providing greater insight into their structure and function. The increased resolution of EM maps, which contain sharp valleys capable of trapping structures, presents a challenge to MDFF which was originally developed for maps in a lower resolution range. However, a recent study unveils two new techniques called cascade (cMDFF) and resolution exchange (ReMDFF) molecular dynamics flexible fitting to overcome the hurdles posed by high-resolution maps. The refinement is achieved by interpreting a range of cryo-EM images, starting with an image of fuzzy resolution and progressively improving the image's contrast until near-atomic resolution is reached. These techniques were employed to solve the structure of the proteasome, the recycling machine of the human cell. New analysis schemes that look at the flexibility of the obtained structure provide a measure of model uncertainty within the near-atomic EM images, improving their contrast. All the tools are available on cloud computing platforms allowing community-wide usage at low monetary cost; the complex computations can now be performed at the cost of a cup of coffee.

While waste recycling in daily life has become popular only recently, living cells have been recycling their protein content since the very beginning. Recycling of unneeded protein molecules in cells is performed by a molecular machine called the proteasome, which cuts these proteins into smaller pieces for reuse as building blocks for new proteins. Proteins that need to be recycled are labeled by tags made of poly-ubiquitin protein chains. The proteasome machine recognizes and binds to these tags, pulls the tagged protein close, then unwinds it, and finally cuts it into pieces. Despite its substantial role in the cell's life cycle, the proteasome's atomic structure and function still remain elusive. In our recent study, we obtained an atomic structure of the human 26S proteasome by combining computational modeling techniques, through molecular dynamics flexible fitting (MDFF) of the cryo-electron microscopy (cryo-EM) data. The features observed in the resulting structure are important for coordinating the proteasomal subunits during protein recycling. One of the key advances is that for the first time the nucleotides bound to the ATPase motor of the proteasome are resolved. The atomic resolution of the structure permits to perform molecular dynamics simulations to investigate the detailed proteasomal function, in particular the protein unwinding process of the ATPase motor. Furthermore, our obtained structure will serve as a starting point for structure-guided drug discovery, developing the proteasome as a crucial drug target. The atomic models are deposited in the protein data bank (PDB) with the PDB IDs 5L4G and 5L4K and the 3.9 Å resolution cryo-EM density is deposited in the electron microscopy data bank EMD-4002. More information about our proteasome projects is available on our proteasome website. Easy access to our modeling techniques is provided through QwikMD, which was employed here for the first time.

While waste recycling became popular in our daily life, living cells mastered waste recycling of their protein content since their very beginning. Recycling of unneeded protein molecules in cells is performed by a molecular machine called 26S proteasome, which cuts these proteins into smaller pieces and releases the pieces into the cell interior for reuse as building blocks for new protein. Proteins that need to be recycled are usually those that are misfolded. Proteins are recognized as such by the cells' so-called quality control system. This system labels misfolded proteins by a tag made of tetra-ubiquitin protein chains. The 26S proteasome machine recognizes and binds to these tags via its subunit Rpn10. After Rpn10 binds to the tetra-ubiquitin tag and pulls the protein close, the 26S proteasome unwinds the tagged protein and cuts it into pieces. A recent study, based on molecular dynamics simulations with NAMD, sheds light onto how 26S proteasome and Rpn10 recognize the tetra-ubiquitin tag in three stages: In stage 1 of the recognition process conserved complementary electrostatic patterns of Rpn10 and ubiquitins guide protein association; stage 2 induces refolding of Rpn10 and tetra-ubiquitin tag; stage 3 facilitates formation of hydrophobic contacts between the tag and Rpn10. More information is available on our 26S proteasome website.

RNA molecules are continuously synthesized in living cells as carriers of biological information written in the sequence of basic RNA units, called nucleotides. To keep cells healthy, RNA molecules not longer needed or with errors have to be removed. A large barrel-like protein complex, the RNA exosome, is a molecular machine that degrades unneeded RNA molecules, pulling them inside its long internal channel and cutting them sequentially into single nucleotides. A new molecular dynamics study, employing NAMD, shows that a special active protein subunit of the exosome, called Rrp44, grips tightly the RNA molecule throughout its extended channel. Rrp44 grips RNA molecules with five or more nucleotides in length while their ending nucleotides get sequentially cut, whereas shorter RNAs are only weakly bound and unlikely to be cut. The simulations reveal how the exosome can act both as a molecular motor that pulls RNA, without energy input other than the one released in nucleotide cleavage, as well as an enzyme that cuts RNA. More information is available on our RNA exosome website.

Many processes in living cells require molecular motors. Examples are transport of cargo within a cell, degrading misfolded proteins, and controlling gene expression. In the latter case acts a motor, called Rho, that moves along messenger RNA. The energy of the cell's motors stems from molecules of ATP that are converted to ADP, release thereby energy and drive motor action. How exactly this happens remained largely a mystery, despite decades of study and despite the availability of detailed molecular structures of the motors. A molecular dynamics study employing NAMD has achieved a great breakthrough in resolving the mechanism by which ATP-to-ADP conversion drives Rho to translocate along RNA. While molecular dynamics simulation, in principle, is well suited to explain Rho's motor action, the problem was that the action takes about a millisecond which is a time period beyond such simulations' reach. Employing new sampling methods, a recent publication, reported in new, complete and fascinating detail how Rho works. The simulations permitted literally to look under the hood of Rho's engine and see how it pulls itself along RNA and coordinates a cyclic and repeated motor action. It turned out that Rho, a ring of six identical protein subunits, engages in an ATP-to-ADP conversion-induced periodic motion of the subunits that pushes RNA electrostatically through the ring center. A completely surprising finding was the existence of coordination switches that make each ATP-to-ADP conversion lead to exactly one forward step along the RNA and keep the six subunits strictly synchronized, turning a randomly moving protein system into a well-behaved engine. More on our molecular motor website.