Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.

The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These `ultralong\' CDR3Hs form β-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6 Å resolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing.

The manuscript `Modeling a unit cell: crystallographic refinement procedure using the biomolecular MD simulation platform Amber\' presents a novel protein structure refinement method claimed to offer improvements over traditional techniques like Refmac5 and Phenix. Our re-evaluation suggests that while the new method provides improvements, traditional methods achieve comparable results with less computational effort.

In the folded state, biomolecules exchange between multiple conformational states crucial for their function. However, most structural models derived from experiments and computational predictions only encode a single state. To represent biomolecules accurately, we must move towards modeling and predicting structural ensembles. Information about structural ensembles exists within experimental data from X-ray crystallography and cryo-electron microscopy. Although new tools are available to detect conformational and compositional heterogeneity within these ensembles, the legacy PDB data structure does not robustly encapsulate this complexity. We propose modifications to the macromolecular crystallographic information file (mmCIF) to improve the representation and interrelation of conformational and compositional heterogeneity. These modifications will enable the capture of macromolecular ensembles in a human and machine-interpretable way, potentially catalyzing breakthroughs for ensemble-function predictions, analogous to the achievements of AlphaFold with single-structure prediction.

A series of events underscoring the significant advancements in micro-crystallization and in vivo crystallography were held during the 26th IUCr Congress in Melbourne, positioning microcrystallography as a pivotal field within structural biology. Through collaborative discussions and the sharing of innovative methodologies, these sessions outlined frontier approaches in macromolecular crystallography. This review provides an overview of this rapidly moving field in light of the rich dialogues and forward-thinking proposals explored during the congress workshop and microsymposium. These advances in microcrystallography shed light on the potential to reshape current research paradigms and enhance our comprehension of biological mechanisms at the molecular scale.

Fibulin-2 is a multidomain, disulfide-rich, homodimeric protein which belongs to a broader extracellular matrix family. It plays an important role in the development of elastic fiber structures. Malfunction of fibulin due to mutation or poor expression can result in a variety of diseases including synpolydactyly, limb abnormalities, eye disorders leading to blindness, cardiovascular diseases and cancer. Traditionally, fibulins have either been produced in mammalian cell systems or were isolated from the extracellular matrix, a procedure that results in poor availability for structural and functional studies. Here, we produced seven fibulin-2 constructs covering 62% of the mature protein (749 out of 1195 residues) using a prokaryotic expression system. Biophysical studies confirm that the purified constructs are folded and that the presence of disulfide bonds within the constructs makes them extremely thermostable. In addition, we solved the first crystal structure for any fibulin isoform, a structure corresponding to the previously suggested three motifs related to anaphylatoxin. The structure reveals that the three anaphylatoxins moieties form a single-domain structure.

Determining short-lived intermediate structures in chemical reactions is challenging. Although ultrafast spectroscopic methods can detect the formation of transient intermediates, real-space structures cannot be determined directly from such studies. Time-resolved serial femtosecond crystallography (TR-SFX) has recently proven to be a powerful method for capturing molecular changes in proteins on femtosecond timescales. However, the methodology has been mostly applied to natural proteins/enzymes and limited to reactions promoted by synthetic molecules due to structure determination challenges. This work demonstrates the applicability of TR-SFX for investigations of chemical reaction mechanisms of synthetic metal complexes. We fix a light-induced CO-releasing Mn(CO)3 reaction center in porous hen egg white lysozyme (HEWL) microcrystals. By controlling light exposure and time, we capture the real-time formation of Mn-carbonyl intermediates during the CO release reaction. The asymmetric protein environment is found to influence the order of CO release. The experimentally-observed reaction path agrees with quantum mechanical calculations. Therefore, our demonstration offers a new approach to visualize atomic-level reactions of small molecules using TR-SFX with real-space structure determination. This advance holds the potential to facilitate design of artificial metalloenzymes with precise mechanisms, empowering design, control and development of innovative reactions.

An aqueous solution of 2,3-cis gallate type catechin (-)-epigallocatechin-3-O-gallate (EGCg) and caffeine afforded a precipitate of Creaming-down Phenomenon, which crystallized slowly for about three months to give a colorless block crystal. By X-ray crystallographic analysis, the crystal was determined to be a 2 : 2 complex of EGCg and caffeine, in which caffeine molecules were captured in a hydrophobic space formed with three aromatic A, B, and B\' rings of EGCg. It was considered that the solubility of the 2 : 2 complex in water rapidly decreased and the 2 : 2 complex precipitated from aqueous solution. The hydrophobic spaces of EGCg captured a variety of heterocyclic compounds, and the molecular capture abilities of heterocyclic compounds using EGCg from the aqueous solutions were evaluated. Since the C ring of EGCg has two chiral carbon atoms, C2 and C3, the hydrophobic space of EGCg was a chiral space. EGCg captured diketopiperazine cyclo(Pro-Xxx) (Xxx=Phe, Tyr) and pharmaceuticals with a xanthine skeleton, proxyphylline and diprophylline, in the hydrophobic space, and recognized their chirality.

Structural biology research of terpene synthases (TSs) has provided a useful basis to understand their catalytic mechanisms in producing diverse terpene products with polycyclic ring systems and multiple chiral centers. However, compared to the large numbers of>95,000 terpenoids discovered to date, few structures of TSs have been solved and the understanding of their catalytic mechanisms is lagging. We here (i) introduce the basic catalytic logic, the structural architectures, and the metal-binding conserved motifs of TSs; (ii) provide detailed experimental procedures, in gene cloning and plasmid construction, protein purification, crystallization, X-ray diffraction data collection and structural elucidation, for structural biology research of TSs; and (iii) discuss the prospects of structure-based engineering and de novo design of TSs in generating valuable terpene molecules, which cannot be easily achieved by chemical synthesis.

Magnesium ions (Mg2+) are crucial in class II terpene cyclases that utilize substrates with diphosphate groups. Interestingly, these enzymes catalyze reactions without cleaving the diphosphate group, instead initiating the reaction through protonation. In our recent research, we discovered a novel class II sesquiterpene cyclase in Streptomyces showdoensis. Notably, we determined its crystal structure and identified Mg2+ within its active site. This finding has shed light on the previously elusive question of Mg2+ binding in class II terpene cyclases. In this chapter, we outline our methods for discovering this novel enzyme, including steps for its purification, crystallization, and kinetic analysis.