Facing the phase problem.

The marvel of X-ray crystallography is the beauty and precision of the atomic structures deduced from diffraction patterns. Since these patterns record only amplitudes, phases for the diffracted waves must also be evaluated for systematic structure determination. Thus, we have the phase problem as a central complication, both intellectually for the field and practically so for many analyses. Here, I discuss how we - myself, my laboratory and the diffraction community - have faced the phase problem, considering the evolution of methods for phase evaluation as structural biology developed to the present day. During the explosive growth of macromolecular crystallography, practice in diffraction analysis evolved from a universal reliance on isomorphous replacement to the eventual domination of anomalous diffraction for de novo structure determination. As the Protein Data Bank (PDB) grew and familial relationships among proteins became clear, molecular replacement overtook all other phasing methods; however, experimental phasing remained essential for molecules without obvious precedents, with multi- and single-wavelength anomalous diffraction (MAD and SAD) predominating. While the mathematics-based direct methods had proved to be inadequate for typical macromolecules, they returned to crack substantial selenium substructures in SAD analyses of selenomethionyl proteins. Native SAD, exploiting the intrinsic S and P atoms of biomolecules, has become routine. Selenomethionyl SAD and MAD were the mainstays of structural genomics efforts to populate the PDB with novel proteins. A recent dividend has been paid in the success of PDB-trained artificial intelligence approaches for protein structure prediction. Currently, molecular replacement with AlphaFold models often obviates the need for experimental phase evaluation. For multiple reasons, we are now unfazed by the phase problem. Cryo-EM analysis is an attractive alternative to crystallography for many applications faced by today's structural biologists. It simply finesses the phase problem; however, the principles and procedures of diffraction analysis remain pertinent and are adopted in single-particle cryo-EM studies of biomolecules.

A Novel Fluorescence Probe Based on Azamonardine for Detecting and Imaging Cysteine in Cells and Zebrafish with High Selectivity and Sensitivity.

A novel fluorescent probe based on azamonardine (Aza) fluorophore was designed and synthesized for the highly selective detection of cysteine (Cys) in vivo and in vitro. After reacting with acryloyl chloride, the fluorescence of Aza is effectively quenched, resulting in the formation of the Aza-acryl probe. Upon the addition of Cys, the ester bond of Aza-acryl is cleaved, releasing a new compound (Compound 1) with strong fluorescence, thereby achieving fluorescence turn-on detection of Cys. The structure of Aza-acryl was characterized using X-ray crystallography and NMR spectroscopy. Additionally, density functional theory was employed to elucidate the quenching mechanism of the acyl group on the Aza. Aza-acryl exhibits high selectivity towards Cys and distinguishes it from other biothiols such as homocysteine (Hcy) and glutathione (GSH). The mechanism of Aza-acryl for detecting Cys was investigated through HPLC, NMR spectroscopy, high-resolution mass spectrometry, and reaction kinetics experiments. Aza-acryl demonstrates excellent imaging capabilities for Cys in cells and zebrafish, providing a reliable and selectable tool for the detection and imaging of Cys in biological systems.

C-H Groups as Donors in Hydrogen Bonds: A Historical Overview and Occurrence in Proteins and Nucleic Acids.

Hydrogen bonds constitute a unique type of non-covalent interaction, with a critical role in biology. Until fairly recently, the canonical view held that these bonds occur between electronegative atoms, typically O and N, and that they are mostly electrostatic in nature. However, it is now understood that polarized C-H groups may also act as hydrogen bond donors in many systems, including biological macromolecules. First recognized from physical chemistry studies, C-H…X bonds were visualized with X-ray crystallography sixty years ago, although their true significance has only been recognized in the last few decades. This review traces the origins of the field and describes the occurrence and significance of the most important C-H…O bonds in proteins and nucleic acids.

Structure Determination of SH2-Phosphopeptide Complexes by X-Ray Crystallography: The Example of p120RasGAP.

The p120RasGAP protein contains two Src homology 2 (SH2) domains, each with phosphotyrosine-binding activity. We describe the crystallization of the isolated and purified p120RasGAP SH2 domains with phosphopeptides derived from a binding partner protein, p190RhoGAP. Purified recombinant SH2 domain protein is mixed with synthetic phosphopeptide at a stoichiometric ratio to form the complex in vitro. Crystallization is then achieved by the hanging drop vapor diffusion method over specific reservoir solutions that yield single macromolecular co-crystals containing SH2 domain protein and phosphopeptide. This protocol yields suitable crystals for X-ray diffraction studies, and our recent X-ray crystallography studies of the two SH2 domains of p120RasGAP demonstrate that the N-terminal SH2 domain binds phosphopeptide in a canonical interaction. In contrast, the C-terminal SH2 domain binds phosphopeptide via a unique atypical binding mode. The crystallographic studies for p120RasGAP illustrate that although the three-dimensional structure of SH2 domains and the molecular details of their binding to phosphotyrosine peptides are well defined, careful structural analysis can continue to yield new molecular-level insights.

Structures of the Insecticidal Toxin Complex Subunit XptA2 Highlight Roles for Flexible Domains.

The Toxin Complex (Tc) superfamily consists of toxin translocases that contribute to the targeting, delivery, and cytotoxicity of certain pathogenic Gram-negative bacteria. Membrane receptor targeting is driven by the A-subunit (TcA), which comprises IgG-like receptor binding domains (RBDs) at the surface. To better understand XptA2, an insect specific TcA secreted by the symbiont X. nematophilus from the intestine of entomopathogenic nematodes, we determined structures by X-ray crystallography and cryo-EM. Contrary to a previous report, XptA2 is pentameric. RBD-B exhibits an indentation from crystal packing that indicates loose association with the shell and a hotspot for possible receptor binding or a trigger for conformational dynamics. A two-fragment XptA2 lacking an intact linker achieved the folded pre-pore state like wild type (wt), revealing no requirement of the linker for protein folding. The linker is disordered in all structures, and we propose it plays a role in dynamics downstream of the initial pre-pore state.

Structural Motifs in Aryl Organogermanium Ge-O Derivatives for Material Design.

The aim of this work was to understand the main structural features and ways of formation of Ge-O bonds in organogermanium compounds under the conditions of ArnGeHal4-n (Hal = halide) hydrolysis. The structural types of these compounds were considered, providing 11 blocks (A-K). The molecular structures of the novel compounds [(p-FC6H4)3Ge]2O (1), [(p-F3CC6H4)3Ge]2O (2), and cyclo-[(p-F3CC6H4)2GeO]4 (3) were studied through XRD (X-ray diffraction) analysis. The molecular structure of [(p-F3CC6H4)3GeO]4Ge (4), representing a novel structural type, was also investigated. The data presented in this study will be important in the design of materials with useful properties based on group 14 element derivatives with element-oxygen bonding.

Structural insights into functional properties of the oxidized form of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes. The turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized to the metastable OH state, and a reductive phase, in which OH is reduced back to the R state. During each phase, two protons are translocated across the membrane. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX), we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide insights into the proton translocation mechanism of CcO.

ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography.

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an aqueous environment is typically achieved by employing detergents. These detergents disintegrate the lipid bilayer and solubilize the proteins. The intrinsic habitat of membrane proteins within the lipid bilayer poses a challenge in maintaining their stability and uniformity in solution for structural characterization. Bicelles, which comprise a blend of long and short-chain phospholipids and detergents, replicate the natural lipid structure. The utilization of lipid bicelles and detergents serves as a suitable model system for obtaining high-quality diffraction crystals, specifically to determine the high-resolution structure of membrane proteins. Through these synthetic microenvironments, membrane proteins preserve their native conformation and functionality, facilitating the formation of three-dimensional crystals. In this approach, the detergent-solubilized heterodimeric ABCG5/G8 was reintegrated into DMPC/CHAPSO bicelles, supplemented with cholesterol. This setup was employed in the vapor diffusion experimental procedure for protein crystallization.

Cryo-EM technique and its application: Structure of steroid hormone receptors.

In recent years, cryo-electron microscopy (cryo-EM) has become one of the most powerful tools to solve the 3-D structure of macromolecules. Unlike X-ray crystallography, the cryo-EM method has advantage of providing an in-depth insight into the dynamic behavior of macromolecules, which is particularly useful to determine 3-D structural analyses of large protein complexes. Due to recent technical advancements, cryo-EM has become the method of choice for the determination of protein structures. Among other proteins, solving 3-D structure of steroid hormone receptors (SHRs) complexed with DNA and coactivators has been a challenge for decades. The limitations with X-ray crystallography and NMR to solve SHR protein structures prompted investigators to move towards cryo-EM technique. The cryo-EM structural analyses have been successful in revealing structural dynamics of several SHRs in recent years. Though, limited by low-resolution, the structural analyses of these SHRs may be useful in understanding many receptor functions as well as provide a platform to refine high-resolution structural analyses in future. This review article discusses the cryo-EM technique in general as well as structural information gained for SHRs using cryo-EM.

A biophysical framework for double-drugging kinases.

Selective orthosteric inhibition of kinases has been challenging due to the conserved active site architecture of kinases and emergence of resistance mutants. Simultaneous inhibition of distant orthosteric and allosteric sites, which we refer to as "double-drugging", has recently been shown to be effective in overcoming drug resistance. However, detailed biophysical characterization of the cooperative nature between orthosteric and allosteric modulators has not been undertaken. Here, we provide a quantitative framework for double-drugging of kinases employing isothermal titration calorimetry, Förster resonance energy transfer, coupled-enzyme assays, and X-ray crystallography. We discern positive and negative cooperativity for Aurora A kinase (AurA) and Abelson kinase (Abl) with different combinations of orthosteric and allosteric modulators. We find that a conformational equilibrium shift is the main principle governing cooperativity. Notably, for both kinases, we find a synergistic decrease of the required orthosteric and allosteric drug dosages when used in combination to inhibit kinase activities to clinically relevant inhibition levels. X-ray crystal structures of the double-drugged kinase complexes reveal the molecular principles underlying the cooperative nature of double-drugging AurA and Abl with orthosteric and allosteric inhibitors. Finally, we observe a fully closed conformation of Abl when bound to a pair of positively cooperative orthosteric and allosteric modulators, shedding light on the puzzling abnormality of previously solved closed Abl structures. Collectively, our data provide mechanistic and structural insights into rational design and evaluation of double-drugging strategies.

Abietane diterpenoids with anti-inflammatory activities from Callicarpa bodinieri.

Nine undescribed abietane diterpenoids (1-9) and eleven known abietane analogs (10-20) were isolated from Callicarpa bodinieri. Their structures were characterized by interpreting spectroscopic data, X-ray crystallography, and ECD analysis. The anti-inflammatory activities of these compounds were tested by evaluation of their inhibitory effect on NO production by lipopolysaccharide in RAW 264.7 macrophages, and compounds 3 and 8 exhibited potent anti-inflammatory activities with IC50 values of 36.35 ± 1.12 and 37.21 ± 0.92 µM. The western blotting studies demonstrated that compound 3 inhibited the expression of nitric oxide synthase and p65 that involved in the NF-κB pathway.

Architecture of the rings of 5-arylidenerhodanine derivatives versus P-gp inhibition.

5-Arylidene derivatives of rhodanine show various biological activities. The new crystal structures of five derivatives investigated towards ABCB1 efflux pump modulation are reported, namely, 2-[5-([1,1'-biphenyl]-4-ylmethylidene]-4-oxo-2-thioxothiazolidin-3-yl)acetic acid dimethyl sulfoxide monosolvate, C18H13NO3S2·C2H6OS (1), 4-[5-([1,1'-biphenyl]-4-ylmethylidene]-4-oxo-2-thioxothiazolidin-3-yl)butanoic acid, C20H17NO3S2 (2), 5-[4-(benzyloxy)benzylidene]-2-thioxothiazolidin-4-one, C17H13NO2S2 (3), 4-{5-[4-(benzyloxy)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}butanoic acid, C21H19NO4S2 (4), and 5-[4-(diphenylamino)benzylidene]-2-thioxothiazolidin-4-one, C22H16N2OS2 (5). Compounds 1 and 3-5 crystallize in the triclinic space group P-1, while 2 crystallizes in the monoclinic space group P21/n, where the biphenyl moiety is observed in two positions (A and B). Two molecules are present in the asymmetric unit of 5 and, for the other four compounds, there is only one molecule; moreover, 1 crystallizes with one dimethyl sulfoxide molecule. The packing of the molecules containing a carboxyl group (1, 2 and 4) is determined by O-H...O hydrogen bonds, while in the other two compounds (3 and 5), the packing is determined by N-H...O hydrogen bonds. Additionally, induced-fit docking studies have been performed for the active compounds to investigate their putative binding mode inside the human glycoprotein P (P-gp) binding pocket.

CryoEM Status Update and Innovative Developments.

ARTICLES DISCUSSED: Truong, C. D. et al. Sample preparation using a lipid monolayer method for electron crystallographic studies. Journal of Visualized Experiments. (177), e63015 (2021). Johnson, M. C., Grant, A. J., Schmidt-Krey, I. The peel-blot technique: A cryo-EM sample preparation method to separate single layers from multi-layered or concentrated biological samples. Journal of Visualized Experiments. (184), e64099 (2022). Chang, Y.-C., Chen, C.-Y., Tsai, M.-D. Preparation of high-temperature sample grids for cryo-EM. Journal of Visualized Experiments. (173), e62772 (2021). Kang, M.-H., Lee, M., Kang, S., Park, J. Fabrication of micro-patterned chip with controlled thickness for high-throughput cryogenic electron microscopy. Journal of Visualized Experiments. (182), e63739 (2022). Bieber, A., Capitanio, C., Wilfling, F., Plitzko, J., Erdmann, P. S. Sample preparation by 3D-correlative focused ion beam milling for high-resolution cryo-electron tomography. Journal of Visualized Experiments. (176), e62886 (2021). DiCecco, L.-A. et al. Advancing high-resolution imaging of virus assemblies in liquid and ice. Journal of Visualized Experiments. (185), e63856 (2022). Kumar, A., P, S., Gulati, S., Dutta, S. User-friendly, high-throughput, and fully automated data acquisition software for single-particle cryo-electron microscopy. Journal of Visualized Experiments. (173), e62832 (2021).

Structural insights into histone binding and nucleosome assembly by chromatin assembly factor-1.

Chromatin inheritance entails de novo nucleosome assembly after DNA replication by chromatin assembly factor-1 (CAF-1). Yet direct knowledge about CAF-1's histone binding mode and nucleosome assembly process is lacking. In this work, we report the crystal structure of human CAF-1 in the absence of histones and the cryo-electron microscopy structure of CAF-1 in complex with histones H3 and H4. One histone H3-H4 heterodimer is bound by one CAF-1 complex mainly through the p60 subunit and the acidic domain of the p150 subunit. We also observed a dimeric CAF-1-H3-H4 supercomplex in which two H3-H4 heterodimers are poised for tetramer assembly and discovered that CAF-1 facilitates right-handed DNA wrapping of H3-H4 tetramers. These findings signify the involvement of DNA in H3-H4 tetramer formation and suggest a right-handed nucleosome precursor in chromatin replication.

Experimental and Theoretical Biological Probing of Schiff Bases as Esterase Inhibitors: Structural, Spectral and Molecular Insights.

The present study was designed to evaluate the in vitro and in silico potential of the Schiff bases (Z)-4-ethoxy-N-((5-nitrothiophen-2-yl)methylene)benzenamine (1) and (Z)-2,4-diiodo-6-((2-methyl-3-nitrophenylimino)methyl)phenol (2). These Schiff bases were synthesized according to a reported method using ethanol as a solvent, and each reaction was monitored on a TLC until completion of the reaction. The structures of both compounds were elucidated using spectroscopic techniques such as UV-Vis, FTIR, 1H NMR and 13C NMR. Molecular structure was determined using single-crystal XRD, which revealed that compounds 1 and 2 were monoclinic and triclinic, respectively. Hirshfeld surface analysis (HS) and 2D fingerprint plots were used to determine the intermolecular interactions along the contact contribution in the crystalline molecules. The structures of both compounds were optimized through a hybrid functional method B3LYP using the 6-31G(d,p) basis set, and various structural parameters were studied. The experimental and theoretical parameters (bond angle and bond length) of the compounds were compared with each other and are in close agreement. The in vitro esterase potential of the synthesized compounds was checked using a spectrophotometric model, while in silico molecular docking studies were performed with AutoDock against two enzymes of the esterase family. The docking studies and the in vitro assessment predicted that such molecules could be used as enzyme inhibitors against the tested enzymes: acetylcholine esterase (AChE) and butyrylcholine esterase (BChE).

A Conformationally Restricted Gold(III) Complex Elicits Antiproliferative Activity in Cancer Cells.

Diamine ligands are effective structural scaffolds for tuning the reactivity of transition-metal complexes for catalytic, materials, and phosphorescent applications and have been leveraged for biological use. In this work, we report the synthesis and characterization of a novel class of cyclometalated [C^N] Au(III) complexes bearing secondary diamines including a norbornane backbone, (2R,3S)-N2,N3-dibenzylbicyclo[2.2.1]heptane-2,3-diamine, or a cyclohexane backbone, (1R,2R)-N1,N2-dibenzylcyclohexane-1,2-diamine. X-ray crystallography confirms the square-planar geometry and chirality at nitrogen. The electronic character of the conformationally restricted norbornane backbone influences the electrochemical behavior with redox potentials of -0.8 to -1.1 V, atypical for Au(III) complexes. These compounds demonstrate promising anticancer activity, particularly, complex 1, which bears a benzylpyridine organogold framework, and supported by the bicyclic conformationally restricted diaminonorbornane, shows good potency in A2780 cells. We further show that a cellular response to 1 evokes reactive oxygen species (ROS) production and does not induce mitochondrial dysfunction. This class of complexes provides significant stability and reactivity for different applications in protein modification, catalysis, and therapeutics.

Biomimetic catalysis of nitrite reductase enzyme using copper complexes in chemical and electrochemical reduction of nitrite.

Copper nitrite reductase mimetics were synthesized using three new tridentate ligands sharing the same N,N,N motif of coordination. The ligands were based on L-proline modifications, attaching a pyridine and a triazole to the pyrrolidine ring, and differ by a pendant group (R = phenyl, n-butyl and n-propan-1-ol). All complexes coordinate nitrite, as evidenced by cyclic voltammetry, UV-Vis, FTIR and electron paramagnetic resonance (EPR) spectroscopies. The coordination mode of nitrite was assigned by FTIR and EPR as κ2O chelate mode. Upon acidification, EPR experiments indicated a shift from chelate to monodentate κO mode, and 15N NMR experiments of a Zn2+ analogue, suggested that the related Cu(II) nitrous acid complex may be reasonably stable in solution, but in equilibrium with free HONO under non catalytic conditions. Reduction of nitrite to NO was performed both chemically and electrocatalytically, observing the highest catalytic activities for the complex with n-propan-1-ol as pendant group. These results support the hypothesis that a hydrogen bond moiety in the secondary coordination sphere may aid the protonation step.

Crystal structure of a GCN5-related N-acetyltransferase from Lactobacillus curiae.

Members of the GCN5-related N-acetyltransferase (GNAT) family are found in all domains of life and are involved in processes ranging from protein synthesis and gene expression to detoxification and virulence. Due to the variety of their macromolecular targets, GNATs are a highly diverse family of proteins. Currently, 3D structures of only a small number of GNAT representatives are available and thus the family remains poorly characterized. Here, the crystal structure of the guanidine riboswitch-associated GNAT from Lactobacillus curiae (LcGNAT) that acetylates canavanine, a structural analogue of arginine with antimetabolite properties, is reported. LcGNAT shares the conserved fold of the members of the GNAT superfamily, but does not contain an N-terminal ß0 strand and instead contains a C-terminal ß7 strand. Its P-loop, which coordinates the pyrophosphate moiety of the acetyl-coenzyme A cosubstrate, is degenerated. These features are shared with its closest homologues in the polyamine acetyltransferase subclass. Site-directed mutagenesis revealed a central role of the conserved residue Tyr142 in catalysis, as well as the semi-conserved Tyr97 and Glu92, suggesting that despite its individual substrate specificity LcGNAT performs the classical reaction mechanism of this family.

Multicomponent Assembly of Bicyclic Aza-peroxides Catalyzed by Samarium Complexes and Their Cytotoxic Activity.

An original strategy toward bridged tetraoxazaspirobicycloalkanes was developed. The synthesis is based on a three-component condensation-cyclization reaction of primary arylamines with 1,1'-peroxybis (1-hydroperoxycycloalkanes) and pentane-1,5-dial catalyzed by Sm(NO3)3·6H2O. The structures and conformations of the products were determined by X-ray diffraction analysis and 1H and 13C NMR spectroscopy. High cytotoxic activity and biological potential toward ferroptosis induction were found for the synthesized bicyclic aza-peroxides.

Structure of the imine reductase from Ajellomyces dermatitidis in three crystal forms.

The NADPH-dependent imine reductase from Ajellomyces dermatitidis (AdRedAm) catalyzes the reductive amination of certain ketones with amine donors supplied in an equimolar ratio. The structure of AdRedAm has been determined in three forms. The first form, which belongs to space group P3121 and was refined to 2.01 Šresolution, features two molecules (one dimer) in the asymmetric unit in complex with the redox-inactive cofactor NADPH4. The second form, which belongs to space group C21 and was refined to 1.73 Šresolution, has nine molecules (four and a half dimers) in the asymmetric unit, each complexed with NADP+. The third form, which belongs to space group P3121 and was refined to 1.52 Šresolution, has one molecule (one half-dimer) in the asymmetric unit. This structure was again complexed with NADP+ and also with the substrate 2,2-difluoroacetophenone. The different data sets permit the analysis of AdRedAm in different conformational states and also reveal the molecular basis of stereoselectivity in the transformation of fluorinated acetophenone substrates by the enzyme.