The receptor ability of diethyl N,N'-(1,3-phenyl­ene)dicarbamate (1) to form host–guest com­plexes with theophylline (TEO) and caffeine (CAF) by mechanochemistry was evaluated. The formation of the 1–TEO com­plex (C12H16N2O4·C7H8N4O2) was preferred and involves the conformational change of one of the ethyl carbamate groups of 1 from the endo conformation to the exo conformation to allow the formation of inter­molecular inter­actions. The formation of an N—H⋯O=C hydrogen bond between 1 and TEO triggers the conformational change of 1. CAF mol­ecules are unable to form an N—H⋯O=C hydrogen bond with 1, making the conformational change and, therefore, the formation of the com­plex impossible. Conformational change and selective binding were monitored by IR spectroscopy, solid-state 13C nuclear magnetic resonance and single-crystal X-ray diffraction. The 1–TEO com­plex was characterized by IR spectroscopy, solid-state 13C nuclear magnetic resonance, powder X-ray diffraction and single-crystal X-ray diffraction.

A section in the Acta Crystallographica Section C article by Raymond & Girolami [Acta Cryst. (2023), C79, 445–455] stated that the product of the reaction of [(Cp*Rh)2(μ-OH)3]+ (Cp* is 1,2,3,4,5-penta­methyl­cyclo­penta­diene) with 1-methyl­thymine (1-MT) at pH 10 and 60 °C, to synthesize the anionic com­ponent [RhI(η1-N3-1-MT)2]−, was not an RhI com­plex, but rather an AgI com­plex, due to the use of silver triflate (AgOTf) to remove Cl− from [Cp*RhCl2]2 to synthesize [Cp*Rh(H2O)3](OTf)2, a water-soluble crystalline com­plex. We will clearly show that this premise, as stated, is invalid, while the authors have simply avoided several important facts, including that Cp*OH, a reductive elimination product, at pH 10 and 60 °C, was unequivocally identified, thus leading to the RhI anionic com­ponent [RhI(η1-N3-1-MT)2]−. More importantly, AgOH, from the reaction of NaOH at pH 10 with any potentially remaining AgOTf, after the AgCl was filtered off, would be insoluble in water. Furthermore, a control experiment with the inorganic com­plex Rh(OH)3, reacting with 1-methyl­thymine at pH 10, provided no product, and this bodes well for a similar fate with AgOTf and 1-methyl­thymine, i.e. at pH 10, AgOTf would again be converted to the water-insoluble AgOH; therefore, no reaction would occur! Finally, a 1H NMR spectroscopy experiment was carried out with synthesized and crystallized [Cp*Rh(H2O)3](OTf)2 in D2O at various pD values; at pD 8.65 no reaction took place, while at pD 13.6, and at 60 °C for 2 h, a reductive elimination reaction caused the precipitation of Cp*OH. The subsequent 1H NMR spectrum clearly demonstrated, in the absence of any AgI com­plexes, that the solution structure and the X-ray crystals in D2O were similar. A postulated mechanism for this novel anionic com­ponent structure, as published previously [Smith et al. (2014). Organometallics, 33, 2389–2404], will be presented, along with the experimental data, to insure the credibility of our results. We will also answer the comments in the response of Drs Raymond and Girolami to this rebuttal.

We stand fully behind our earlier suggestion [Raymond & Girolami (2023). Acta Cryst. C79, 445–455] that the claim by Fish and co-workers [Chen et al. (1995). J. Am. Chem. Soc. 117, 9097–9098; Smith et al. (2014). Organometallics, 33, 2389–2404] of a linear two-coordinate rhodium(I) species is incorrect, and that the putative rhodium atom is in fact silver.

Using a 1:1 cocrystal of (E)-N-(3,4-di­fluoro­phen­yl)-1-(pyridin-4-yl)methanimine with acetic acid, C12H8F2N2·C2H4O2, we investigate the influence of F atoms introduced to the aromatic ring on promoting π–π inter­actions. The cocrystal crystallizes in the triclinic space group P1. Through crystallographic analysis and com­putational studies, we reveal the mol­ecular arrangement within this co­crystal, demonstrating the presence of hydrogen bonding between the acetic acid mol­ecule and the pyridyl group, along with π–π inter­actions between the aromatic rings. Our findings highlight the importance of F atoms in promoting π–π inter­actions without necessitating full halogenation of the aromatic ring.

Geminal and vicinal bis­(tri­fluoro­methane­sulfonate) esters are highly reactive alkyl­ene synthons used as potent electrophiles in the macrocyclization of imid­azoles and the transformation of bypyridines to diquat derivatives via nucleophilic substitution reactions. Herein we report the crystal structures of methyl­ene (C3H2F6O6S2) and ethyl­ene bis­(tri­fluoro­methane­sulfonate) (C4H4F6O6S2), the first examples of a geminal and vicinal bis­(tri­fluoro­methane­sulfonate) ester characterized by single-crystal X-ray diffraction (SC-XRD). With melting points slightly below ambient temperature, both reported bis­(tri­fluoro­methane­sulfonate)s are air- and moisture-sensitive oils and were crys­tallized at 277 K to afford two-com­ponent non-merohedrally twinned crystals. The dominant inter­actions present in both com­pounds are non-classical C—H⋯O hydrogen bonds and inter­molecular C—F⋯F—C inter­actions between tri­fluoro­methyl groups. Mol­ecular electrostatic potential (MEP) cal­culations by DFT-D3 helped to qu­antify the polarity between O⋯H and F⋯F contacts to rationalize the self-sorting of both bis­(tri­fluoro­methane­sulfonate) esters in polar (non-fluorous) and non-polar (fluorous) domains within the crystal structure.

The salt ammonium 2-am­ino­mal­on­ate (systematic name: ammonium 2-aza­niumyl­propane­dioate), NH4+·C3H4NO4−, was synthesized in diethyl ether from the starting materials malonic acid, ammonia and bromine. The salt was recrystallized from water as colourless blocks. In the solid state, intra­molecular medium–strong N—H⋯O, weak C—H⋯O and weak C—H⋯N hydrogen bonds build a three-dimensional network.

The synthesis, crystal structure and magnetic properties of an ox­am­ate-con­taining erbium(III) com­plex, namely, tetra­butyl­ammonium aqua­[N-(2,4,6-tri­methyl­phen­yl)oxamato]erbium(III)–di­methyl sulfoxide–water (1/3/1.5), (C16H36N)[Er(C11H12NO3)4(H2O)]·3C2H6OS·1.5H2O or n-Bu4N[Er(Htmpa)4(H2O)]·3DMSO·1.5H2O (1), are reported. The crystal structure of 1 reveals the occurrence of an erbium(III) ion, which is surrounded by four N-phenyl-substituted ox­am­ate ligands and one water mol­ecule in a nine-coordinated environment, together with one tetra­butyl­ammonium cation acting as a counter-ion, and one water and three dimethyl sulfoxide (DMSO) mol­ecules of crystallization. Variable-temperature static (dc) and dynamic (ac) magnetic mea­sure­ments were carried out for this mononuclear com­plex, revealing that it behaves as a field-induced single-ion magnet (SIM) below 5.0 K.

The highly cytotoxic macrocyclic trichothecene Isororidin A (C29H40O9) was isolated from the fungus Myrothesium verrucaria endophytic on the wild medicinal plant `Datura' (Datura stramonium L.) and was characterized by one- (1D) and two-dimensional (2D) NMR spectroscopy. The three-dimensional structure of Isororidin A has been confirmed by X-ray crystallography at 0.81 Å resolution from crystals grown in the ortho­rhom­bic space group P212121, with one mol­ecule per asymmetric unit. Isororidin A is the epimer of previously described (by X-ray crystallography) Roridin A at position C-13' of the macrocyclic ring.

Chalcopyrite, the world's primary copper ore mineral, is abundant in Latin America. Copper extraction offers significant economic and social benefits due to its strategic importance across various industries. However, the hydro­metallurgical route, considered more environmentally friendly for processing low-grade chal­co­py­rite ores, remains challenging, as does its concentration by froth flotation. This limited understanding stems from the poorly understood structure and reactivity of chal­co­py­rite surfaces. This study reviews recent contributions using density functional theory (DFT) calculations with periodic boundary conditions and slab models to elucidate chal­co­py­rite surface properties. Our analysis reveals that reconstructed surfaces preferentially expose S atoms at the topmost layer. Furthermore, some studies report the formation of di­sulfide groups (S22−) on pristine sulfur-terminated surfaces, accom­panied by the reduction of Fe3+ to Fe2+, likely due to surface oxidation. Additionally, Fe sites are consistently identified as favourable adsorption locations for both oxygen (O2) and water (H2O) mol­ecules. Finally, the potential of com­puter modelling for investigating collector–chal­co­py­rite surface inter­actions in the context of selective froth flotation is discussed, highlighting the need for further research in this area.

The reaction of titanium(IV) chloride with sodium hexa­fluoro­iso­pro­pox­ide, carried out in hexa­fluoro­iso­propanol, produces titanium(IV) hexa­fluoro­iso­pro­pox­ide, which is a liquid at room temperature. Recrystallization from coordinating solvents, such as aceto­nitrile or tetra­hydro­furan, results in the formation of bis-solvate com­plexes. These com­pounds are of inter­est as possible Ziegler–Natta polymerization catalysts. The aceto­nitrile com­plex had been structurally characterized previously and adopts a distorted octahedral structure in which the nitrile ligands adopt a cis configuration, with nitro­gen lone pairs coordinated to the metal. The low-melting tetra­hydro­furan com­plex has not provided crystals suitable for single-crystal X-ray analysis. However, the structure of chlorido­tris­(hexa­fluoro­isopropoxido-κO)bis­(tetra­hydro­furan-κO)titanium(IV), [Ti(C3HF6O)3Cl(C4H8O)2], has been obtained and adopts a distorted octa­hedral coordination geometry, with a facial arrangement of the alkoxide ligands and adjacent tetra­hydro­furan ligands, coordinated by way of metal–oxygen polar coordinate inter­actions.

A series of organotin heterocycles of general formula [{Me2C(C6H3CH2)2O}SnR2] [R = methyl (Me, 4), n-butyl (n-Bu, 5), benzyl (Bn, 6) and phenyl (Ph, 7)] was easily synthesized by a Barbier-type reaction assisted by the sonochemical activation of metallic magnesium. The 119Sn{1H} NMR data for all four com­pounds confirm the presence of a central Sn atom in a four-coordinated environment in solution. Single-crystal X-ray diffraction studies for 17,17-dimethyl-7,7-di­phenyl-15-oxa-7-stanna­tetra­cyclo­[11.3.1.05,16.09,14]hepta­deca-1,3,5(16),9(14),10,12-hexa­­ene, [Sn(C6H5)2(C17H16O)], 7, at 100 and 295 K con­firmed the formation of a mono­nuclear eight-membered heterocycle, with a conformation depicted as boat–chair, resulting in a weak Sn⋯O inter­action. The Sn and O atoms are surrounded by hydro­phobic C—H bonds. A Hirshfeld surface analysis of 7 showed that the eight-membered heterocycles are linked by weak C—H⋯π, π–π and H⋯H noncovalent inter­actions. The pairwise inter­action energies showed that the cohesion between the heterocycles are mainly due to dispersion forces.

The crystal structures of 16 boron sub­phthalo­cy­an­ines (BsubPcs) with structurally diverse axial groups were analyzed and com­pared to elucidate the impact of the axial group on the inter­molecular π–π inter­actions, axial-group inter­actions, axial bond length and BsubPc bowl depth. π–π inter­actions between the iso­indole units of adjacent BsubPc mol­ecules most often involve concave–concave packing, whereas axial-group inter­actions with adjacent BsubPc mol­ecules tend to favour the convex side of the BsubPc bowl. Furthermore, axial groups that contain O and/or F atoms tend to have significant hy­dro­gen-bonding inter­actions, while axial groups containing arene site(s) can participate in π–π inter­actions with the BsubPc bowl, both of which can strongly influence the crystal packing. Bulky axial groups did tend to disrupt the π–π inter­actions and/or axial-group inter­actions, preventing some of the close packing that is seen in BsubPcs with less bulky axial groups. The atomic radius of the heteroatom bonded to boron directly influences the axial bond length, whereas the axial group has minimal impact on the BsubPc bowl depth. Finally, the crystal growth method did not generally appear to have a significant impact on the solid-state arrangement, with the exception of water occasionally being incorporated into crystal structures when hygroscopic solvents were used. These insights can help with the design and fine-tuning of the solid-state structures of BsubPcs as they continue to be developed as functional materials in organic electronics.

The incommensurately modulated structure of (2S,3S)-2-amino-3-hy­droxy-3-methyl-4-phen­oxy­butanoic acid dihydrate (C11H15NO4·2H2O or I·2H2O) is described in the (3+1)-dimensional superspace group P212121(0β0)000 (β = 0.357). The loss of the three-dimensional periodicity is ascribed to the occupational modulation of one positionally disordered solvent water mol­ecule, where the two positions are related by a small translation [ca 0.666 (9) Å] and ∼168 (5)° rotation about one of its O—H bonds, with an average 0.624 (3):0.376 (3) occupancy ratio. The occupational modulation of this mol­ecule arises due to the com­petition between the different hy­dro­gen-bonding motifs associated with each position. The structure can be very well refined in the average approximation (all satellite reflections disregarded) in the space group P212121, with the water mol­ecule refined as disordered over two positions in a 0.625 (16):0.375 (16) ratio. The refinement in the commensurate threefold supercell approximation in the space group P1121 is also of high quality, with the six corresponding water mol­ecules exhibiting three different occupancy ratios averaging 0.635:0.365.

Herein we report the crystal structures of two ben­zo­di­az­e­pines obtained by reacting N,N'-(4,5-di­amino-1,2-phenyl­ene)bis­(4-methyl­ben­zene­sul­fon­am­ide) (1) or 4,5-(4-methyl­ben­zene­sul­fon­am­ido)­ben­zene-1,2-diaminium dichloride (1·2HCl) with acetone, giving 2,2,4-trimethyl-8,9-bis­(4-methyl­ben­zene­sul­fon­am­ido)-2,3-di­hydro-5H-1,5-ben­zo­di­az­e­pine, C26H30N4O4S2 (2), and 2,2,4-tri­methyl-8,9-bis­(4-methyl­ben­zene­sul­fon­am­ido)-2,3-di­hydro-5H-1,5-ben­zo­di­az­e­pin-1-ium chloride 0.3-hydrate, C26H31N4O4S2+·Cl−·0.3H2O (3). Compounds 2 and 3 were first obtained in attempts to recrystallize 1 and 1·2HCl using acetone as solvent. This solvent reacted with the vicinal di­amines present in the mol­ecular structures, forming a 5H-1,5-ben­zo­di­az­e­pine ring. In the crystal structure of 2, the seven-membered ring of ben­zo­di­az­e­pine adopts a boat-like conformation, while upon protonation, observed in the crystal structure of 3, it adopts an envelope-like conformation. In both crystalline com­pounds, the tosyl­amide N atoms are not in resonance with the arene ring, mainly due to hy­dro­gen bonds and steric hindrance caused by the large vicinal groups in the aromatic ring. At a supra­molecular level, the crystal structure is maintained by a combination of hy­dro­gen bonds and hydro­phobic inter­actions. In 2, amine-to-tosyl N—H⋯O and amide-to-imine N—H⋯N hy­dro­gen bonds can be observed. In contrast, in 3, the chloride counter-ion and water mol­ecule result in most of the hy­dro­gen bonds being of the amide-to-chloride and ammonium-to-chloride N—H⋯Cl types, while the amine inter­acts with the tosyl group, as seen in 2. In conclusion, we report the synthesis of 1, 1·2HCl and 2, as well as their chemical characterization. For 2, two synthetic methods are described, i.e. solvent-mediated crystallization and synthesis via a more efficient and cleaner route as a polycrystalline material. Salt 3 was only obtained as presented, with only a few crystals being formed.

The crystal structures of two coordination com­pounds, (acetato-κO)(2,2'-bi­py­ri­dine-κ2N,N')(1,10-phenanthroline-κ2N,N')copper(II) acetate hexa­hydrate, [Cu(C2H3O2)(C10H8N2)(C12H8N2)](C2H3O2)·6H2O or [Cu(bipy)(phen)Ac]Ac·6H2O, and (acetato-κO)bis­(2,2'-bi­py­ri­dine-κ2N,N')copper(II) acetate–acetic acid–water (1/1/3), [Cu(C2H3O2)(C10H8N2)2](C2H3O2)·C2H4O2·3H2O or [Cu(bipy)2Ac]Ac·HAc·3H2O, are reported and com­pared with the previously published structure of [Cu(phen)2Ac]Ac·7H2O (phen is 1,10-phenanthroline, bipy for 2,2'-bi­py­ri­dine, ac is acetate and Hac is acetic acid). The geometry around the metal centre is penta­coordinated, but highly distorted in all three cases. The coordination number and the geometric distortion are both discussed in detail, and all com­plexes belong to the space group Poverline{1}. The analysis of the geometric parameters and the Hirshfeld surface properties dnorm and curvedness provide information about the metal–ligand inter­actions in these com­plexes and allow com­parison with similar systems.

Historically, knowledge of the mol­ecular packing within the crystal structures of organic semi­con­duc­tors has been instrumental in understanding their solid-state electronic properties. Nowadays, crystal structures are thus becoming increasingly important for enabling engineering properties, understanding poly­mor­phism in bulk and in thin films, exploring dynamics and elucidating phase-transition mech­a­nisms. This review article introduces the most salient and recent results of the field.

Starting from (p-tolyl­sulfin­yl)fer­ro­cene (1), a mixture of the complete series [CpFe{C5H5–n(SOTol-p)n}] (n = 2–4) (2–4) in all regioisomers was obtained. After chromatographic separation, crystals of 1,2-bis­[(4-methyl­benzene)­sulfin­yl]fer­ro­cene, 2a, and 1,3-bis­[(4-methyl­benzene)­sulfin­yl]fer­ro­cene, 2b, both [Fe(C5H5)(C19H17O2S2)], as well as of 1,2,3-tris­[(4-methyl­benzene)­sulfin­yl]fer­ro­cene, [Fe(C5H5)(C26H23O3S3)], 3a, and 1,2,3,4-tetra­kis­[(4-methyl­benzene)­sul­fin­yl]fer­ro­cene ethyl acetate 0.75-solvate, [Fe(C5H5)(C33H29O4S4)]·0.75C4H8O2, 4, could be isolated. Their mol­ecular and crystal structures are compared with each other and also with the so far un­reported structures of related 1,2-bis­(phenyl­sulfan­yl)fer­ro­cene, [Fe(C5H5)(C17H13S2)], 5, and 1,2,3,4-tetra­kis­(phenyl­sulfan­yl)fer­ro­cene, [Fe(C5H5)(C29H21S4)], 6. In all the sulfinyl structures, the O atoms of the S=O groups are in equatorial positions, except for that in tetrasubstituted 4. All the arene rings of these com­pounds (except for one ring in 4) are in axial positions directed away from the Fe atom, mostly in a near perpendicular orientation with respect to the plane of the cyclo­penta­di­en­yl ring. The main inter­molecular inter­actions in the crystals are C—H⋯H—C, C—H⋯π and C—H⋯O, while C—H⋯S inter­actions are much less important, except for tetra­sul­fan­yl com­pound 6. π–π inter­actions (intra­molecular) are only important in com­pound 3a. Hirshfeld analysis shows that dispersion terms are dominant for the inter­action energies of all six com­pounds. In general, the calculated total inter­action energies increase with increasing number of substituents and are higher for the sulfinyl than for the sul­fan­yl groups.

Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent structures have been solved using molecular replacement as a phasing method, requiring an a priori structure that is closely related to the target protein to serve as a search model; when no such search model exists, molecular replacement is not possible. New advances in computational machine-learning methods, however, have resulted in major advances in protein structure predictions from sequence information. Methods that generate predicted structural models of sufficient accuracy provide a powerful approach to molecular replacement. Taking advantage of these advances, AlphaFold predictions were applied to enable structure determination of a bacterial protein of unknown function (UniProtKB Q63NT7, NCBI locus BPSS0212) based on diffraction data that had evaded phasing attempts using MIR and anomalous scattering methods. Using both X-ray and micro-electron (microED) diffraction data, it was possible to solve the structure of the main fragment of the protein using a predicted model of that domain as a starting point. The use of predicted structural models importantly expands the promise of electron diffraction, where structure determination relies critically on molecular replacement.

Low-molecular-weight (LMW) thiols are involved in many processes in all organisms, playing a protective role against reactive species, heavy metals, toxins and antibiotics. Actinobacteria, such as Mycobacterium tuberculosis, use the LMW thiol mycothiol (MSH) to buffer the intracellular redox environment. The NADPH-dependent FAD-containing oxidoreductase mycothiol disulfide reductase (Mtr) is known to reduce oxidized mycothiol disulfide (MSSM) to MSH, which is crucial to maintain the cellular redox balance. In this work, the first crystal structures of Mtr are presented, expanding the structural knowledge and understanding of LMW thiol reductases. The structural analyses and docking calculations provide insight into the nature of Mtrs, with regard to the binding and reduction of the MSSM substrate, in the context of related oxidoreductases. The putative binding site for MSSM suggests a similar binding to that described for the homologous glutathione reductase and its respective substrate glutathione disulfide, but with distinct structural differences shaped to fit the bulkier MSSM substrate, assigning Mtrs as uniquely functioning reductases. As MSH has been acknowledged as an attractive antitubercular target, the structural findings presented in this work may contribute towards future antituberculosis drug development.

When solving a structure of a protein from single-wavelength anomalous diffraction X-ray data, the initial phases obtained by phasing from an anomalously scattering substructure usually need to be improved by an iterated electron-density modification. In this manuscript, the use of convolutional neural networks (CNNs) for segmentation of the initial experimental phasing electron-density maps is proposed. The results reported demonstrate that a CNN with U-net architecture, trained on several thousands of electron-density maps generated mainly using X-ray data from the Protein Data Bank in a supervised learning, can improve current density-modification methods.

The Azotobacter vinelandii FeSII protein forms an oxygen-resistant complex with the nitrogenase MoFe and Fe proteins. FeSII is an adrenodoxin-type ferredoxin that forms a dimer in solution. Previously, the crystal structure was solved [Schlesier et al. (2016), J. Am. Chem. Soc. 138, 239–247] with five copies in the asymmetric unit. One copy is a normal adrenodoxin domain that forms a dimer with its crystallographic symmetry mate. The other four copies are in an `open' conformation with a loop flipped out exposing the 2Fe–2S cluster. The open and closed conformations were interpreted as oxidized and reduced, respectively, and the large conformational change in the open configuration allowed binding to nitrogenase. Here, the structure of FeSII was independently solved in the same crystal form. The positioning of the atoms in the unit cell is similar to the earlier report. However, the interpretation of the structure is different. The `open' conformation is interpreted as the product of a crystallization-induced domain swap. The 2Fe–2S cluster is not exposed to solvent, but in the crystal its interacting helix is replaced by the same helix residues from a crystal symmetry mate. The domain swap is complicated, as it is unusual in being in the middle of the protein rather than at a terminus, and it creates arrangements of molecules that can be interpreted in multiple ways. It is also cautioned that crystal structures should be interpreted in terms of the contents of the entire crystal rather than of one asymmetric unit.

Eukaryotic TIR (Toll/interleukin-1 receptor protein) domains signal via TIR–TIR interactions, either by self-association or by interaction with other TIR domains. In mammals, TIR domains are found in Toll-like receptors (TLRs) and cytoplasmic adaptor proteins involved in pro-inflammatory signaling. Previous work revealed that the MAL TIR domain (MALTIR) nucleates the assembly of MyD88TIR into crystalline arrays in vitro. A microcrystal electron diffraction (MicroED) structure of the MyD88TIR assembly has previously been solved, revealing a two-stranded higher-order assembly of TIR domains. In this work, it is demonstrated that the TIR domain of TLR2, which is reported to signal as a heterodimer with either TLR1 or TLR6, induces the formation of crystalline higher-order assemblies of MyD88TIR in vitro, whereas TLR1TIR and TLR6TIR do not. Using an improved data-collection protocol, the MicroED structure of TLR2TIR-induced MyD88TIR microcrystals was determined at a higher resolution (2.85 Å) and with higher completeness (89%) compared with the previous structure of the MALTIR-induced MyD88TIR assembly. Both assemblies exhibit conformational differences in several areas that are important for signaling (for example the BB loop and CD loop) compared with their monomeric structures. These data suggest that TLR2TIR and MALTIR interact with MyD88 in an analogous manner during signaling, nucleating MyD88TIR assemblies uni­directionally.

Pollution from plastics is a global problem that threatens the biosphere for a host of reasons, including the time scale that it takes for most plastics to degrade. Biodegradation is an ideal solution for remediating bioplastic waste as it does not require the high temperatures necessary for thermal degradation and does not introduce additional pollutants into the environment. Numerous organisms can scavenge for bioplastics, such as polylactic acid (PLA) or poly-(R)-hydroxybutyrate (PHB), which they can use as an energy source. Recently, a promiscuous PHBase from the thermophilic soil bacterium Lihuaxuella thermophila (LtPHBase) was identified. LtPHBase can accommodate many substrates, including PHB granules and films and PHB block copolymers, as well as the unrelated polymers polylactic acid (PLA) and polycaprolactone (PCL). LtPHBase uses the expected Ser–His–Asp catalytic triad for hydrolysis at an optimal enzyme activity near 70°C. Here, the 1.75 Å resolution crystal structure of apo LtPHBase is presented and its chemical stability is profiled. Knowledge of its substrate preferences was extended to different-sized PHB granules. It is shown that LtPHBase is highly resistant to unfolding, with barriers typical for thermophilic enzymes, and shows a preference for low-molecular-mass PHB granules. These insights have implications for the long-term potential of LtPHBase as an industrial PHB hydrolase and shed light on the evolutionary role that this enzyme plays in bacterial metabolism.

Serial electron diffraction (SerialED), which applies a snapshot data acquisition strategy for each crystal, was introduced to tackle the problem of radiation damage in the structure determination of beam-sensitive materials by three-dimensional electron diffraction (3DED). The snapshot data acquisition in SerialED can be realized using both transmission and scanning transmission electron microscopes (TEM/STEM). However, the current SerialED workflow based on STEM setups requires special external devices and software, which limits broader adoption. Here, we present a simplified experimental implementation of STEM-based SerialED on Thermo Fisher Scientific STEMs using common proprietary software interfaced through Python scripts to automate data collection. Specifically, we utilize TEM Imaging and Analysis (TIA) scripting and TEM scripting to access the STEM functionalities of the microscope, and DigitalMicrograph scripting to control the camera for snapshot data acquisition. Data analysis adapts the existing workflow using the software CrystFEL, which was developed for serial X-ray crystallography. Our workflow for STEM SerialED can be used on any Gatan or Thermo Fisher Scientific camera. We apply this workflow to collect high-resolution STEM SerialED data from two aluminosilicate zeolites, zeolite Y and ZSM-25. We demonstrate, for the first time, ab initio structure determination through direct methods using STEM SerialED data. Zeolite Y is relatively stable under the electron beam, and STEM SerialED data extend to 0.60 Å. We show that the structural model obtained using STEM SerialED data merged from 358 crystals is nearly identical to that using continuous rotation electron diffraction data from one crystal. This demonstrates that accurate structures can be obtained from STEM SerialED. Zeolite ZSM-25 is very beam-sensitive and has a complex structure. We show that STEM SerialED greatly improves the data resolution of ZSM-25, compared with serial rotation electron diffraction (SerialRED), from 1.50 to 0.90 Å. This allows, for the first time, the use of standard phasing methods, such as direct methods, for the ab initio structure determination of ZSM-25.

Single-particle imaging using X-ray free-electron lasers (XFELs) is a promising technique for observing nanoscale biological samples under near-physiological conditions. However, as the sample's orientation in each diffraction pattern is unknown, advanced algorithms are required to reconstruct the 3D diffraction intensity volume and subsequently the sample's density model. While most approaches perform 3D reconstruction via determining the orientation of each diffraction pattern, a correlation-based approach utilizes the averaged spatial correlations of diffraction intensities over all patterns, making it well suited for processing experimental data with a poor signal-to-noise ratio of individual patterns. Here, a method is proposed to determine the 3D structure of a sample by analyzing the double, triple and quadruple spatial correlations in diffraction patterns. This ab initio method can reconstruct the basic shape of an irregular unsymmetric 3D sample without requiring any prior knowledge of the sample. The impact of background and noise on correlations is investigated and corrected to ensure the success of reconstruction under simulated experimental conditions. Additionally, the feasibility of using the correlation-based approach to process incomplete partial diffraction patterns is demonstrated. The proposed method is a variable addition to existing algorithms for 3D reconstruction and will further promote the development and adoption of XFEL single-particle imaging techniques.

Highly accurate protein structure prediction can generate accurate models of protein and protein–protein complexes in X-ray crystallography. However, the question of how to make more effective use of predicted models for completing structure analysis, and which strategies should be employed for the more challenging cases such as multi-helical structures, multimeric structures and extremely large structures, both in the model preparation and in the completion steps, remains open for discussion. In this paper, a new strategy is proposed based on the framework of direct methods and dual-space iteration, which can greatly simplify the pre-processing steps of predicted models both in normal and in challenging cases. Following this strategy, full-length models or the conservative structural domains could be used directly as the starting model, and the phase error and the model bias between the starting model and the real structure would be modified in the direct-methods-based dual-space iteration. Many challenging cases (from CASP14) have been tested for the general applicability of this constructive strategy, and almost complete models have been generated with reasonable statistics. The hybrid strategy therefore provides a meaningful scheme for X-ray structure determination using a predicted model as the starting point.

Vancomycin is a glycopeptide antibiotic that for decades has been a mainstay of treatment for persistent bacterial infections. However, the spread of antibiotic resistance threatens its continued utility. In particular, vancomycin-resistant enterococci (VRE) have become a pressing clinical challenge. Vancomycin acts by binding and sequestering the intermediate Lipid II in cell-wall biosynthesis, specifically recognizing a d-alanine-d-alanine dipeptide motif within the Lipid II molecule. VRE achieve resistance by remodeling this motif to either d-alanine-d-lactate or d-alanine-d-serine; the former substitution essentially abolishes recognition by vancomycin of Lipid II, whereas the latter reduces the affinity of the antibiotic by roughly one order of magnitude. The complex of vancomycin bound to d-alanine-d-serine has been crystallized, and its 1.20 Å X-ray crystal structure is presented here. This structure reveals that the d-alanine-d-serine ligand is bound in essentially the same position and same pose as the native d-alanine-d-alanine ligand. The serine-containing ligand appears to be slightly too large to be comfortably accommodated in this way, suggesting one possible contribution to the reduced binding affinity. In addition, two flexible hydroxyl groups – one from the serine side chain of the ligand, and the other from a glucose sugar on the antibiotic – are locked into single conformations in the complex, which is likely to contribute an unfavorable entropic component to the recognition of the serine-containing ligand.

An X-ray absorption spectroscopy (XAS) electrochemical cell was used to collect high-quality XAS measurements of N-truncated Cu:amyloid-β (Cu:Aβ) samples under near-physiological conditions. N-truncated Cu:Aβ peptide complexes contribute to oxidative stress and neurotoxicity in Alzheimer's patients' brains. However, the redox properties of copper in different Aβ peptide sequences are inconsistent. Therefore, the geometry of binding sites for the copper binding in Aβ4–8/12/16 was determined using novel advanced extended X-ray absorption fine structure (EXAFS) analysis. This enables these peptides to perform redox cycles in a manner that might produce toxicity in human brains. Fluorescence XAS measurements were corrected for systematic errors including defective-pixel data, monochromator glitches and dispersion of pixel spectra. Experimental uncertainties at each data point were measured explicitly from the point-wise variance of corrected pixel measurements. The copper-binding environments of Aβ4–8/12/16 were precisely determined by fitting XAS measurements with propagated experimental uncertainties, advanced analysis and hypothesis testing, providing a mechanism to pursue many similarly complex questions in bioscience. The low-temperature XAS measurements here determine that CuII is bound to the first amino acids in the high-affinity amino-terminal copper and nickel (ATCUN) binding motif with an oxygen in a tetragonal pyramid geometry in the Aβ4–8/12/16 peptides. Room-temperature XAS electrochemical-cell measurements observe metal reduction in the Aβ4–16 peptide. Robust investigations of XAS provide structural details of CuII binding with a very different bis-His motif and a water oxygen in a quasi-tetrahedral geometry. Oxidized XAS measurements of Aβ4–12/16 imply that both CuII and CuIII are accommodated in an ATCUN-like binding site. Hypotheses for these CuI, CuII and CuIII geometries were proven and disproven using the novel data and statistical analysis including F tests. Structural parameters were determined with an accuracy some tenfold better than literature claims of past work. A new protocol was also developed using EXAFS data analysis for monitoring radiation damage. This gives a template for advanced analysis of complex biosystems.

Bacterial ABC toxin complexes (Tcs) comprise three core proteins: TcA, TcB and TcC. The TcA protein forms a pentameric assembly that attaches to the surface of target cells and penetrates the cell membrane. The TcB and TcC proteins assemble as a heterodimeric TcB–TcC subcomplex that makes a hollow shell. This TcB–TcC subcomplex self-cleaves and encapsulates within the shell a cytotoxic `cargo' encoded by the C-terminal region of the TcC protein. Here, we describe the structure of a previously uncharacterized TcC protein from Yersinia entomophaga, encoded by a gene at a distant genomic location from the genes encoding the rest of the toxin complex, in complex with the TcB protein. When encapsulated within the TcB–TcC shell, the C-terminal toxin adopts an unfolded and disordered state, with limited areas of local order stabilized by the chaperone-like inner surface of the shell. We also determined the structure of the toxin cargo alone and show that when not encapsulated within the shell, it adopts an ADP-ribosyltransferase fold most similar to the catalytic domain of the SpvB toxin from Salmonella typhimurium. Our structural analysis points to a likely mechanism whereby the toxin acts directly on actin, modifying it in a way that prevents normal polymerization.

Human peptidylarginine deiminase isoform VI (PAD6), which is predominantly limited to cytoplasmic lattices in the mammalian oocytes in ovarian tissue, is essential for female fertility. It belongs to the peptidylarginine deiminase (PAD) enzyme family that catalyzes the conversion of arginine residues to citrulline in proteins. In contrast to other members of the family, recombinant PAD6 was previously found to be catalytically inactive. We sought to provide structural insight into the human homologue to shed light on this observation. We report here the first crystal structure of PAD6, determined at 1.7 Å resolution. PAD6 follows the same domain organization as other structurally known PAD isoenzymes. Further structural analysis and size-exclusion chromatography show that PAD6 behaves as a homodimer similar to PAD4. Differential scanning fluorimetry suggests that PAD6 does not coordinate Ca2+ which agrees with acidic residues found to coordinate Ca2+ in other PAD homologs not being conserved in PAD6. The crystal structure of PAD6 shows similarities with the inactive state of apo PAD2, in which the active site conformation is unsuitable for catalytic citrullination. The putative active site of PAD6 adopts a non-productive conformation that would not allow protein–substrate binding due to steric hindrance with rigid secondary structure elements. This observation is further supported by the lack of activity on the histone H3 and cytokeratin 5 substrates. These findings suggest a different mechanism for enzymatic activation compared with other PADs; alternatively, PAD6 may exert a non-enzymatic function in the cytoplasmic lattice of oocytes and early embryos.

The Protein Data Bank (PDB) was established as the first open-access digital data resource in biology and medicine in 1971 with seven X-ray crystal structures of proteins. Today, the PDB houses >210 000 experimentally determined, atomic level, 3D structures of proteins and nucleic acids as well as their complexes with one another and small molecules (e.g. approved drugs, enzyme cofactors). These data provide insights into fundamental biology, biomedicine, bioenergy and biotechnology. They proved particularly important for understanding the SARS-CoV-2 global pandemic. The US-funded Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) and other members of the Worldwide Protein Data Bank (wwPDB) partnership jointly manage the PDB archive and support >60 000 `data depositors' (structural biologists) around the world. wwPDB ensures the quality and integrity of the data in the ever-expanding PDB archive and supports global open access without limitations on data usage. The RCSB PDB research-focused web portal at https://www.rcsb.org/ (RCSB.org) supports millions of users worldwide, representing a broad range of expertise and interests. In addition to retrieving 3D structure data, PDB `data consumers' access comparative data and external annotations, such as information about disease-causing point mutations and genetic variations. RCSB.org also provides access to >1 000 000 computed structure models (CSMs) generated using artificial intelligence/machine-learning methods. To avoid doubt, the provenance and reliability of experimentally determined PDB structures and CSMs are identified. Related training materials are available to support users in their RCSB.org explorations.

Categorization underlies understanding. Conceptualizing solid-state structures of organic molecules with `archetype crystal structures' bridges established categories of disorder, polymorphism and solid solutions and is herein extended to special position and high-Z' structures. The concept was developed in the context of disorder modelling [Dittrich, B. (2021). IUCrJ, 8, 305–318] and relies on adding quantum chemical energy differences between disorder components to other criteria as an explanation as to why disorder – and disappearing disorder – occurs in an average structure. Part of the concept is that disorder, as probed by diffraction, affects entire molecules, rather than just the parts of a molecule with differing conformations, and the finding that an R·T energy difference between disorder archetypes is usually not exceeded. An illustrative example combining disorder and special positions is the crystal structure of oestradiol hemihydrate analysed here, where its space-group/subgroup relationship is required to explain its disorder of hydrogen-bonded hydrogen atoms. In addition, we show how high-Z' structures can also be analysed energetically and understood via archetypes: high-Z' structures occur when an energy gain from combining different rather than overall alike conformations in a crystal significantly exceeds R·T, and this finding is discussed in the context of earlier explanations in the literature. Twinning is not related to archetype structures since it involves macroscopic domains of the same crystal structure. Archetype crystal structures are distinguished from crystal structure prediction trial structures in that an experimental reference structure is required for them. Categorization into archetype structures also has practical relevance, leading to a new practice of disorder modelling in experimental least-squares refinement alluded to in the above-mentioned publication.

Appreciating that the role of the solute–solvent and other outer-sphere interactions is essential for understanding chemistry and chemical dynamics in solution, experimental approaches are needed to address the structural consequences of these interactions, complementing condensed-matter simulations and coarse-grained theories. High-energy X-ray scattering (HEXS) combined with pair distribution function analysis presents the opportunity to probe these structures directly and to develop quantitative, atomistic models of molecular systems in situ in the solution phase. However, at concentrations relevant to solution-phase chemistry, the total scattering signal is dominated by the bulk solvent, prompting researchers to adopt a differential approach to eliminate this unwanted background. Though similar approaches are well established in quantitative structural studies of macromolecules in solution by small- and wide-angle X-ray scattering (SAXS/WAXS), analogous studies in the HEXS regime—where sub-ångström spatial resolution is achieved—remain underdeveloped, in part due to the lack of a rigorous theoretical description of the experiment. To address this, herein we develop a framework for differential solution scattering experiments conducted at high energies, which includes concepts of the solvent-excluded volume introduced to describe SAXS/WAXS data, as well as concepts from the time-resolved X-ray scattering community. Our theory is supported by numerical simulations and experiment and paves the way for establishing quantitative methods to determine the atomic structures of small molecules in solution with resolution approaching that of crystallography.

A new polymorph of 1,3-diacetylpyrene has been obtained from its melt and thoroughly characterized using single-crystal X-ray diffraction, steady-state UV–Vis spectroscopy and periodic density functional theory calculations. Experimental studies covered the temperature range from 90 to 390 K and the pressure range from atmospheric to 4.08 GPa. Optimal sample placement in a diamond anvil cell according to our previously presented methodology ensured over 80% data coverage up to 0.8 Å for a monoclinic sample. Unrestrained Hirshfeld atom refinement of the high-pressure crystal structures was successful and anharmonic behavior of carbonyl oxygen atoms was observed. Unlike the previously characterized polymorph, the structure of 2°AP-β is based on infinite π-stacks of antiparallel 2°AP molecules. 2°AP-β displays piezochromism and piezofluorochromism which are directly related to the variation in interplanar distances within the π-stacking. The importance of weak intermolecular interactions is reflected in the substantial negative thermal expansion coefficient of −55.8 (57) MK−1 in the direction of C—H⋯O interactions.

Although COF-300 is often used as an example to study the synthesis and structure of (3D) covalent organic frameworks (COFs), knowledge of the underlying synthetic processes is still fragmented. Here, an optimized synthetic procedure based on a combination of linker protection and modulation was applied. Using this approach, the influence of time and temperature on the synthesis of COF-300 was studied. Synthesis times that were too short produced materials with limited crystallinity and porosity, lacking the typical pore flexibility associated with COF-300. On the other hand, synthesis times that were too long could be characterized by loss of crystallinity and pore order by degradation of the tetrakis(4-aminophenyl)methane (TAM) linker used. The presence of the degradation product was confirmed by visual inspection, Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). As TAM is by far the most popular linker for the synthesis of 3D COFs, this degradation process might be one of the reasons why the development of 3D COFs is still lagging compared with 2D COFs. However, COF crystals obtained via an optimized procedure could be structurally probed using 3D electron diffraction (3DED). The 3DED analysis resulted in a full structure determination of COF-300 at atomic resolution with satisfying data parameters. Comparison of our 3DED-derived structural model with previously reported single-crystal X-ray diffraction data for this material, as well as parameters derived from the Cambridge Structural Database, demonstrates the high accuracy of the 3DED method for structure determination. This validation might accelerate the exploitation of 3DED as a structure determination technique for COFs and other porous materials.

Crystallography is a quintessential method for determining the atomic structure of crystals. The most common implementation of crystallography uses single crystals that must be of sufficient size, typically tens of micrometres or larger, depending on the complexity of the crystal structure. The emergence of serial data-collection methods in crystallography, particularly for time-resolved experiments, opens up opportunities to develop new routes to structure determination for nanocrystals and ensembles of crystals. Fluctuation X-ray scattering is a correlation-based approach for single-particle imaging from ensembles of identical particles, but has yet to be applied to crystal structure determination. Here, an iterative algorithm is presented that recovers crystal structure-factor intensities from fluctuation X-ray scattering correlations. The capabilities of this algorithm are demonstrated by recovering the structure of three small-molecule crystals and a protein crystal from simulated fluctuation X-ray scattering correlations. This method could facilitate the recovery of structure-factor intensities from crystals in serial crystallography experiments and relax sample requirements for crystallography experiments.

Carbonic anhydrase (CA) was among the first proteins whose X-ray crystal structure was solved to atomic resolution. CA proteins have essentially the same fold and similar active centers that differ in only several amino acids. Primary sulfonamides are well defined, strong and specific binders of CA. However, minor variations in chemical structure can significantly alter their binding properties. Over 1000 sulfonamides have been designed, synthesized and evaluated to understand the correlations between the structure and thermodynamics of their binding to the human CA isozyme family. Compound binding was determined by several binding assays: fluorescence-based thermal shift assay, stopped-flow enzyme activity inhibition assay, isothermal titration calorimetry and competition assay for enzyme expressed on cancer cell surfaces. All assays have advantages and limitations but are necessary for deeper characterization of these protein–ligand interactions. Here, the concept and importance of intrinsic binding thermodynamics is emphasized and the role of structure–thermodynamics correlations for the novel inhibitors of CA IX is discussed – an isozyme that is overexpressed in solid hypoxic tumors, and thus these inhibitors may serve as anticancer drugs. The abundant structural and thermodynamic data are assembled into the Protein–Ligand Binding Database to understand general protein–ligand recognition principles that could be used in drug discovery.

The success of experimental phasing in macromolecular crystallography relies primarily on the accurate locations of heavy atoms bound to the target crystal. To improve the process of substructure determination, a modified phase-retrieval algorithm built on the framework of the relaxed alternating averaged reflection (RAAR) algorithm has been developed. Importantly, the proposed algorithm features a combination of the π-half phase perturbation for weak reflections and enforces the direct-method-based tangent formula for strong reflections in reciprocal space. The proposed algorithm is extensively demonstrated on a total of 100 single-wavelength anomalous diffraction (SAD) experimental datasets, comprising both protein and nucleic acid structures of different qualities. Compared with the standard RAAR algorithm, the modified phase-retrieval algorithm exhibits significantly improved effectiveness and accuracy in SAD substructure determination, highlighting the importance of additional constraints for algorithmic performance. Furthermore, the proposed algorithm can be performed without human intervention under most conditions owing to the self-adaptive property of the input parameters, thus making it convenient to be integrated into the structural determination pipeline. In conjunction with the IPCAS software suite, we demonstrated experimentally that automatic de novo structure determination is possible on the basis of our proposed algorithm.

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.

Single-particle cryo-electron microscopy (cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution, at which individual atoms, including hydrogen atoms, can be resolved. In this study, we used the enzyme involved in the penultimate step of riboflavin biosynthesis as a test specimen to benchmark a recently installed microscope and determine if other protein complexes could reach a resolution of 1.5 Å or better, which so far has only been achieved for the iron carrier ferritin. Using state-of-the-art microscope and detector hardware as well as the latest software techniques to overcome microscope and sample limitations, a 1.42 Å map of Aquifex aeolicus lumazine synthase (AaLS) was obtained from a 48 h microscope session. In addition to water molecules and ligands involved in the function of AaLS, we can observe positive density for ∼50% of the hydrogen atoms. A small improvement in the resolution was achieved by Ewald sphere correction which was expected to limit the resolution to ∼1.5 Å for a molecule of this diameter. Our study confirms that other protein complexes can be solved to near-atomic resolution. Future improvements in specimen preparation and protein complex stabilization may allow more flexible macromolecules to reach this level of resolution and should become a priority of study in the field.

The aberrant fibrillization of huntingtin exon 1 (Httex1) characterized by an expanded polyglutamine (polyQ) tract is a defining feature of Huntington's disease, a neurodegenerative disorder. Recent investigations underscore the involvement of a small EDRK-rich factor 1a (SERF1a) in promoting Httex1 fibrillization through interactions with its N terminus. By establishing an integrated approach with size-exclusion-column-based small- and wide-angle X-ray scattering (SEC-SWAXS), NMR, and molecular simulations using Rosetta, the analysis here reveals a tight binding of two NT17 fragments of Httex1 (comprising the initial 17 amino acids at the N terminus) to the N-terminal region of SERF1a. In contrast, examination of the complex structure of SERF1a with a coiled NT17-polyQ peptide (33 amino acids in total) indicates sparse contacts of the NT17 and polyQ segments with the N-terminal side of SERF1a. Furthermore, the integrated SEC-SWAXS and molecular-simulation analysis suggests that the coiled NT17 segment can transform into a helical conformation when associated with a polyQ segment exhibiting high helical content. Intriguingly, NT17-polyQ peptides with enhanced secondary structures display diminished interactions with SERF1a. This insight into the conformation-dependent binding of NT17 provides clues to a catalytic association mechanism underlying SERF1a's facilitation of Httext1 fibrillization.

This study examines various methods for modelling the electron density and, thus, the electrostatic potential of an organometallic complex for use in crystal structure refinement against 3D electron diffraction (ED) data. It focuses on modelling the scattering factors of iron(III), considering the electron density distribution specific for coordination with organic linkers. We refined the structural model of the metal–organic complex, iron(III) acetyl­acetonate (FeAcAc), using both the independent atom model (IAM) and the transferable aspherical atom model (TAAM). TAAM refinement initially employed multipolar parameters from the MATTS databank for acetyl­acetonate, while iron was modelled with a spherical and neutral approach (TAAM ligand). Later, custom-made TAAM scattering factors for Fe—O coordination were derived from DFT calculations [TAAM-ligand-Fe(III)]. Our findings show that, in this compound, the TAAM scattering factor corresponding to Fe3+ has a lower scattering amplitude than the Fe3+ charged scattering factor described by IAM. When using scattering factors corresponding to the oxidation state of iron, IAM inaccurately represents electrostatic potential maps and overestimates the scattering potential of the iron. In addition, TAAM significantly improved the fitting of the model to the data, shown by improved R1 values, goodness-of-fit (GooF) and reduced noise in the Fourier difference map (based on the residual distribution analysis). For 3D ED, R1 values improved from 19.36% (IAM) to 17.44% (TAAM-ligand) and 17.49% (TAAM-ligand-Fe3+), and for single-crystal X-ray diffraction (SCXRD) from 3.82 to 2.03% and 1.98%, respectively. For 3D ED, the most significant R1 reductions occurred in the low-resolution region (8.65–2.00 Å), dropping from 20.19% (IAM) to 14.67% and 14.89% for TAAM-ligand and TAAM-ligand-Fe(III), respectively, with less improvement in high-resolution ranges (2.00–0.85 Å). This indicates that the major enhancements are due to better scattering modelling in low-resolution zones. Furthermore, when using TAAM instead of IAM, there was a noticeable improvement in the shape of the thermal ellipsoids, which more closely resembled those of an SCXRD-refined model. This study demonstrates the applicability of more sophisticated scattering factors to improve the refinement of metal–organic complexes against 3D ED data, suggesting the need for more accurate modelling methods and highlighting the potential of TAAM in examining the charge distribution of large molecular structures using 3D ED.

Reaching beyond the commonly used spherical atomic electron density model allows one to greatly improve the accuracy of hydrogen atom structural param­eters derived from X-ray data. However, the effects of atomic asphericity are less explored for electron diffraction data. In this work, Hirshfeld atom refinement (HAR), a method that uses an accurate description of electron density by quantum mechanical calculation for a system of interest, was applied for the first time to the kinematical refinement of electron diffraction data. This approach was applied here to derive the structure of ordinary hexagonal ice (Ih). The effect of introducing HAR is much less noticeable than in the case of X-ray refinement and it is largely overshadowed by dynamical scattering effects. It led to only a slight change in the O—H bond lengths (shortening by 0.01 Å) compared with the independent atom model (IAM). The average absolute differences in O—H bond lengths between the kinematical refinements and the reference neutron structure were much larger: 0.044 for IAM and 0.046 Å for HAR. The refinement results changed considerably when dynamical scattering effects were modelled – with extinction correction or with dynamical refinement. The latter led to an improvement of the O—H bond length accuracy to 0.021 Å on average (with IAM refinement). Though there is a potential for deriving more accurate structures using HAR for electron diffraction, modelling of dynamical scattering effects seems to be a necessary step to achieve this. However, at present there is no software to support both HAR and dynamical refinement.

The antipsychotic drug olanzapine is well known for its complex polymorphism. Although widely investigated, the crystal structure of one of its anhydrous polymorphs, form III, is still unknown. Its appearance, always in concomitance with forms II and I, and the impossibility of isolating it from that mixture, have prevented its structure determination so far. The scenario has changed with the emerging field of 3D electron diffraction (3D ED) and its great advantages in the characterization of polyphasic mixtures of nanosized crystals. In this study, we show how the application of 3D ED allows the ab initio structure determination and dynamical refinement of this elusive crystal structure that remained unknown for more than 20 years. Olanzapine form III is monoclinic and shows a similar but shifted packing with respect to form II. It is remarkably different from the lowest-energy structures predicted by the energy-minimization algorithms of crystal structure prediction.

A pressure-induced triclinic-to-monoclinic phase transition has been caught `in the act' over a wider series of high-pressure synchrotron diffraction experiments conducted on a large, photoluminescent organo-gold(I) compound. Here, we describe the mechanism of this single-crystal-to-single-crystal phase transition, the onset of which occurs at ∼0.6 GPa, and we report a high-quality structure of the new monoclinic phase, refined using aspherical atomic scattering factors. Our case illustrates how conducting a fast series of diffraction experiments, enabled by modern equipment at synchrotron facilities, can lead to overestimation of the actual pressure of a phase transition due to slow transformation kinetics.

Ultrahigh-resolution structures provide unprecedented details about protein dynamics, hydrogen bonding and solvent networks. The reported 0.70 Å, room-temperature crystal structure of crambin is the highest-resolution ambient-temperature structure of a protein achieved to date. Sufficient data were collected to enable unrestrained refinement of the protein and associated solvent networks using SHELXL. Dynamic solvent networks resulting from alternative side-chain conformations and shifts in water positions are revealed, demonstrating that polypeptide flexibility and formation of clathrate-type structures at hydro­phobic surfaces are the key features endowing crambin crystals with extraordinary diffraction power.

Biological materials have outstanding properties. With ease, challenging mechanical, optical or electrical properties are realised from comparatively `humble' building blocks. The key strategy to realise these properties is through extensive hierarchical structuring of the material from the millimetre to the nanometre scale in 3D. Though hierarchical structuring in biological materials has long been recognized, the 3D characterization of such structures remains a challenge. To understand the behaviour of materials, multimodal and multi-scale characterization approaches are needed. In this review, we outline current X-ray analysis approaches using the structures of bone and shells as examples. We show how recent advances have aided our understanding of hierarchical structures and their functions, and how these could be exploited for future research directions. We also discuss current roadblocks including radiation damage, data quantity and sample preparation, as well as strategies to address them.

3D electron diffraction (3DED) is increasingly employed to determine molec­ular and crystal structures from micro-crystals. Indomethacin is a well known, marketed, small-molecule non-steroidal anti-inflammatory drug with eight known polymorphic forms, of which four structures have been elucidated to date. Using 3DED, we determined the structure of a new ninth polymorph, σ, found within an amorphous solid dispersion, a product formulation sometimes used for active pharmaceutical ingredients with poor aqueous solubility. Subsequently, we found that σ indomethacin can be produced from direct solvent evaporation using di­chloro­methane. These results demonstrate the relevance of 3DED within drug development to directly probe product formulations.

MltG, a membrane-bound lytic transglycosyl­ase, has roles in terminating glycan polymerization in peptidoglycan and incorporating glycan chains into the cell wall, making it significant in bacterial cell-wall biosynthesis and remodeling. This study provides the first reported MltG structure from Mycobacterium abscessus (maMltG), a superbug that has high antibiotic resistance. Our structural and biochemical analyses revealed that MltG has a flexible peptidoglycan-binding domain and exists as a monomer in solution. Further, the putative active site of maMltG was disclosed using structural analysis and sequence comparison. Overall, this study contributes to our understanding of the transglycosyl­ation reaction of the MltG family, aiding the design of next-generation antibiotics targeting M. abscessus.