Under severe or prolonged stress, bacteria produce a non‐specific DNA‐binding protein (Dps), which effectively protects DNA against damaging agents both in vitro and in vivo by forming intracellular biocrystals. The phenomenon of protective crystallization of DNA in living cells has been intensively investigated during the last two decades; however, the results of studies are somewhat contradictory, and up to now, there has been no direct determination of a Dps‐DNA crystal structure. Here, we report the in vitro analysis of the vital process of Dps‐DNA co‐crystallization using two complementary structural methods: synchrotron small‐angle X‐ray scattering in solution and cryo‐electron tomography. Importantly, for the first time, the DNA in the co‐crystals was visualized, and the lattice parameters of the crystalline Dps‐DNA complex were determined.

The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane. We showed that with excitation at 445 nm, the fluorescence lifetime of both SypHer3s and SypHerExtra strongly depend on pH. Using FLIM microscopy in live eukaryotic cells, we demonstrated that SypHerExtra can be successfully used to determine extracellular pH, while SypHer3s can be applied to measure intracellular pH. Thus, these two sensors are suitable for quantitative measurements using the FLIM method, to determine intracellular and extracellular pH in a range from pH 7.5 to 9.5 in different biological systems.

Rapid increase of intracellular synthesis of specific histone-like Dps protein that binds DNA to protect the genome against deleterious factors leads to in cellulo crystallization—one of the most curious processes in the area of life science at the moment. However, the actual structure of the Dps–DNA co-crystals remained uncertain in the details for more than two decades. Cryo-electron tomography and small-angle X-ray scattering revealed polymorphous modifications of the co-crystals depending on the buffer parameters. Two different types of the Dps–DNA co-crystals are formed in vitro: triclinic and cubic. Three-dimensional reconstruction revealed DNA and Dps molecules in cubic co-crystals, and the unit cell parameters of cubic lattice were determined consistently by both methods.

Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg²⁺ ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe²⁺ in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cations using SAXS and cryo-EM. Additionally, the single particle analysis of Dps with accumulated iron performed by cryo-EM shows localization of iron nanoparticles inside the Dps cavity next to the acidic (hydrophobic) pore, near three glutamate residues.

Insulin receptor–related receptor (IRR) is a receptor tyrosine kinase of the insulin receptor family and functions as an extracellular alkali sensor that controls metabolic alkalosis in the regulation of the acid–base balance. In the present work, we sought to analyze structural features of IRR by comparing them with those of the insulin receptor, which is its closest homolog but does not respond to pH changes. Using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM), we investigated the overall conformation of the recombinant soluble IRR ectodomain (ectoIRR) at neutral and alkaline pH. In contrast to the well-known inverted U-shaped (or λ-shaped) conformation of the insulin receptor, the structural models reconstructed at different pH values revealed that the ectoIRR organization has a “droplike” shape with a shorter distance between the fibronectin domains of the disulfide-linked dimer subunits within ectoIRR. We detected no large-scale pH-dependent conformational changes of ectoIRR in both SAXS and AFM experiments, an observation that agreed well with previous biochemical and functional analyses of IRR. Our findings indicate that ectoIRR's sensing of alkaline conditions involves additional molecular mechanisms, for example engagement of receptor juxtamembrane regions or the surrounding lipid environment.