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Fixed‐target serial crystallography has become an important method for the study of protein structure and dynamics at synchrotrons and X‐ray free‐electron lasers. However, sample homogeneity, consumption and the physical stress on samples remain major challenges for these high‐throughput experiments, which depend on high‐quality protein microcrystals. The batch crystallization procedures that are typically applied require time‐ and sample‐intensive screening and optimization. Here, a simple protein crystallization method inside the features of the HARE serial crystallography chips is reported that circumvents batch crystallization and allows the direct transfer of canonical vapor‐diffusion conditions to in‐chip crystallization. Based on conventional hanging‐drop vapor‐diffusion experiments, the crystallization solution is distributed into the wells of the HARE chip and equilibrated against a reservoir with mother liquor. Using this simple method, high‐quality microcrystals were generated with sufficient density for the structure determination of four different proteins. A new protein variant was crystallized using the protein concentrations encountered during canonical crystallization experiments, enabling structure determination from ∼55 µg of protein. Additionally, structure determination from intracellular crystals grown in insect cells cultured directly in the features of the HARE chips is demonstrated. In cellulo crystallization represents a comparatively unexplored space in crystallization, especially for proteins that are resistant to crystallization using conventional techniques, and eliminates any need for laborious protein purification. This in‐chip technique avoids harvesting the sensitive crystals or any further physical handling of the crystal‐containing cells. These proof‐of‐principle experiments indicate the potential of this method to become a simple alternative to batch crystallization approaches and also as a convenient extension to canonical crystallization screens. [ABSTRACT FROM AUTHOR]

The biomineralization process is a mechanism inherent to all organisms of the Earth. Throughout the decades, diverse works have reported that the origin of life is tied to crystals, specifically to biominerals of silica that catalyzed RNA, and had some influence in the homochirality. Although the mechanism by which crystals surfaces (minerals) gave origin to life has not yet been proven, the truth is that, up to the present, biominerals are being synthetized by the organisms of different kingdoms in two basic ways: biologically induced and biologically controlled biomineralization. Paradoxically, this fact makes a fundamental difference between inorganic materials and those formed by living organisms, as the latter are associated with macromolecules that are bound to the mineral phase. Conserving growth and formation of these biogenic organic crystals inside cells is a fascinating subject that has been studied mainly in some of the kingdoms, like Monera (bacteria), Fungi (yeasts), and Animalia (Homo sapiens). Notwithstanding in the Plantae kingdom, the formation, conservation, and functions of crystals has not yet been completely elucidated and described, which is of particular relevance because life on Earth, as we know it, would not be possible without plants. The aim of the present work is to revise the different crystals of calcium oxalate synthetized inside the cells of plants, as well as to identify the mechanism of their formation and their possible functions in plants. The last part is related to the existence of certain proteins called phototropins, which not only work as the blue-light sensors, but they also play an important role on the accumulation of calcium in vacuoles. This new trend is shortly reviewed to explain the characteristics and their plausible role in the calcium uptake along with the biomineralization processes.

Protein crystallization in living cells has been observed surprisingly often as a native assembly process during the past decades, and emerging evidence indicates that this phenomenon is also accessible for recombinant proteins. But only recently the advent of high-brilliance synchrotron sources, X-ray free-electron lasers, and improved serial data collection strategies has allowed the use of these micrometer-sized crystals for structural biology. Thus, in cellulo crystallization could offer exciting new possibilities for proteins that do not crystallize applying conventional approaches. In this review, we comprehensively summarize the current knowledge of intracellular protein crystallization. This includes an overview of the cellular functions, the physical properties, and, if known, the mode of regulation of native in cellulo crystal formation, complemented with a discussion of the reported crystallization events of recombinant proteins and the current method developments to successfully collect X-ray diffraction data from in cellulo crystals. Although the intracellular protein self-assembly mechanisms are still poorly understood, regulatory differences between native in cellulo crystallization linked to a specific function and accidently crystallizing proteins, either disease associated or recombinantly introduced, become evident. These insights are important to systematically exploit living cells as protein crystallization chambers in the future. [ABSTRACT FROM AUTHOR]

Polyhedra represent an ancient system used by a number of insect viruses to protect virions during long periods of environmental exposure. We present high resolution crystal structures of polyhedra for seven previously uncharacterised types of cypoviruses, four using ab initio selenomethionine phasing (two of these required over 100 selenomethionine crystals each). Approximately 80% of residues are structurally equivalent between all polyhedrins (pairwise rmsd ⩽1.5 Å), whilst pairwise sequence identities, based on structural alignment, are as little as 12%. These structures illustrate the effect of 400 million years of evolution on a system where the crystal lattice is the functionally conserved feature in the face of massive sequence variability. The conservation of crystal contacts is maintained across most of the molecular surface, except for a dispensable virus recognition domain. By spreading the contacts over so much of the protein surface the lattice remains robust in the face of many individual changes. Overall these unusual structural constraints seem to have skewed the molecule’s evolution so that surface residues are almost as conserved as the internal residues. [ABSTRACT FROM AUTHOR]

The biomineralization process is a mechanism inherent to all organisms of the Earth. Throughout the decades, diverse works have reported that the origin of life is tied to crystals, specifically to biominerals of silica that catalyzed RNA, and had some influence in the homochirality. Although the mechanism by which crystals surfaces (minerals) gave origin to life has not yet been proven, the truth is that, up to the present, biominerals are being synthetized by the organisms of different kingdoms in two basic ways: biologically induced and biologically controlled biomineralization. Paradoxically, this fact makes a fundamental difference between inorganic materials and those formed by living organisms, as the latter are associated with macromolecules that are bound to the mineral phase. Conserving growth and formation of these biogenic organic crystals inside cells is a fascinating subject that has been studied mainly in some of the kingdoms, like Monera (bacteria), Fungi (yeasts), and Animalia (Homo sapiens). Notwithstanding in the Plantae kingdom, the formation, conservation, and functions of crystals has not yet been completely elucidated and described, which is of particular relevance because life on Earth, as we know it, would not be possible without plants. The aim of the present work is to revise the different crystals of calcium oxalate synthetized inside the cells of plants, as well as to identify the mechanism of their formation and their possible functions in plants. The last part is related to the existence of certain proteins called phototropins, which not only work as the blue-light sensors, but they also play an important role on the accumulation of calcium in vacuoles. This new trend is shortly reviewed to explain the characteristics and their plausible role in the calcium uptake along with the biomineralization processes. [ABSTRACT FROM AUTHOR]

Lahey-Rudolph and co-workers [ J. Appl. Cryst. (2020), 53 , 1169-1180] have reported a rapid and sensitive method to screen for crystals in cellulo - a welcome addition to the structural biology toolbox., (© Bergfors and Majumdar 2020.)