The influence of the crystal synthesis method on the crystallographic structure of caffeine–citric acid co­crystals was analyzed thanks to the synthesis of a new polymorphic form of the cocrystal. In order to com­pare the new form to the already known forms, the crystal structure of the new cocrystal (C8H10N4O2·C6H8O7) was solved by powder X-ray diffraction thanks to synchrotron experiments. The structure determination was performed using `GALLOP', a recently developed hybrid approach based on a local optimization with a particle swarm optimizer, particularly powerful when applied to the structure resolution of materials of pharmaceutical inter­est, com­pared to classical Monte-Carlo simulated annealing. The final structure was obtained through Rietveld refinement, and first-principles density functional theory (DFT) calculations were used to locate the H atoms. The symmetry is triclinic with the space group Poverline{1} and contains one mol­ecule of caffeine and one mol­ecule of citric acid per asymmetric unit. The crystallographic structure of this cocrystal involves different hydrogen-bond associations com­pared to the already known structures. The analysis of these hydrogen bonds indicates that the cocrystal obtained here is less stable than the co­crystals already identified in the literature. This analysis is confirmed by the determination of the melting point of this cocrystal, which is lower than that of the previously known co­crystals.

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.

By studying the structures of (μ-1,4,10,13-tetra­thia-7,16-di­aza­cyclo­octa­deca­ne)bis[iodidopalladium(II)] diiodide penta­(diiodine), [Pd2I2(C12H26N2S4)](I)2·5I2 or [Pd2I2([18]aneN2S4)](I)2·(I2)5, and 4,7,13,16,21,24-hexa­oxa-1,10-diazo­niabi­cyclo­[8.8.8]hexa­cosane triiodide iodide hemi­penta­(diiodine) di­chloro­methane mono­solvate, C18H38N2O62+·I3−·I−·2.5I2·CH2Cl2 or [H2([2.2.2]cryptand)](I3)(I)(I2)2.5·CH2Cl2, we confirm the structural variety of extended polyiodides achievable upon changing the shape, charge and dimensions of the cation template, by altering the synthetic strategy adopted and/or the experimental conditions. Although it is still often difficult to characterize discrete [I2m+n]n− polyiodides higher than I3− on the basis of structural parameters, such as I—I bond distances, FT–Raman spectroscopy appears to identify them as aggregates of I2, I− and (symmetric or slightly asymmetric) I3− building blocks linked by I⋯I inter­actions of varying strengths. However, because FT–Raman spectroscopy carries no information about the topological features of extended polyiodides, the two techniques should therefore be applied in combination to enhance the analysis of this kind of compounds.

The synthesis and structural characterization of three families of coordination com­plexes synthesized from 4'-phenyl-2,2':6',2''-terpyridine (8, Ph-TPY), 4'-(4-chloro­phen­yl)-2,2':6',2''-terpyridine (9, ClPh-TPY) and 4'-(4-meth­oxy­phen­yl)-2,2':6',2''-terpyridine (10, MeOPh-TPY) ligands with the divalent metals Co2+, Fe2+, Mn2+ and Ni2+ are reported. The com­pounds were synthesized from a 1:2 mixture of the metal and ligand, resulting in a series of com­plexes with the general formula [M(R-TPY)2](ClO4)2 (where M = Co2+, Fe2+, Mn2+ and Ni2+, and R-TPY = Ph-TPY, ClPh-TPY and MeOPh-TPY). The general formula and structural and supra­molecular features were determinated by single-crystal X-ray diffraction for bis­(4'-phenyl-2,2':6',2''-terpyridine)­nickel(II) bis­(per­chlo­rate), [Ni(C21H15N3)2](ClO4)2 or [Ni(Ph-TPY)2](ClO4)2, bis­[4'-(4-meth­oxy­phen­yl)-2,2':6',2''-terpyridine]­manganese(II) bis­(per­chlo­rate), [Mn(C22H17N3O)2](ClO4)2 or [Mn(MeOPh-TPY)2](ClO4)2, and bis­(4'-phenyl-2,2':6',2''-ter­py­ridine)­manganese(II) bis­(per­chlo­rate), [Mn(C21H15N3)2](ClO4)2 or [Mn(Ph-TPY)2](ClO4)2. In all three cases, the com­plexes present distorted octa­hedral coordination polyhedra and the crystal packing is determined mainly by weak C—H⋯π inter­actions. All the com­pounds (except for the Ni derivatives, for which FT–IR, UV–Vis and thermal analysis are reported) were fully characterized by spectroscopic (FT–IR, UV–Vis and NMR spectroscopy) and thermal (TGA–DSC, thermogravimetric analysis–differential scanning calorimetry) methods.

The well-known copper car­box­yl­ate dimer, with four car­box­yl­ate ligands ex­ten­ding outwards towards the corners of a square, has been employed to generate a series of crystalline com­pounds. In particular, this work centres on the use of the 4-hy­droxy­benzoate anion (Hhba−) and its deprotonated phe­nol­ate form 4-oxidobenzoate (hba2−) to obtain complexes with the general formula [Cu2(Hhba)4–x(hba)xL2–y]x−, where L is an axial coligand (including solvent mol­ecules), x = 0, 1 or 2, and y = 0 or 1. In some cases, short hy­dro­gen bonds result in complexes which may be represented as [Cu2(Hhba)2(H0.5hba)2L2]−. The main focus of the investigation is on the formation of a variety of extended networks through hy­dro­gen bonding and, in some crystals, coordinate bonds when bridging coligands (L) are employed. Crystals of [Cu2(Hhba)4(di­ox­ane)2]·4(di­ox­ane) consist of the expected Cu dimer with the Hhba− anions forming hy­dro­gen bonds to 1,4-di­ox­ane mol­ecules which block network formation. In the case of crystals of com­position [Et4N][Cu2(Hhba)2(H0.5hba)2(CH3OH)(H2O)]·2(di­ox­ane), Li[Cu2(Hhba)2(H0.5hba)2(H2O)2]·3(di­ox­ane)·4H2O and [Cu2(Hhba)2(H0.5hba)2(H0.5DABCO)2]·3CH3OH (DABCO is 1,4-di­aza­bicyclo­[2.2.2]octa­ne), square-grid hy­dro­gen-bonded networks are generated in which the complex serves as one type of 4-con­necting node, whilst a second 4-con­necting node is a hy­dro­gen-bonding motif assembled from four phenol/phenolate groups. Another two-dimensional (2D) network based upon a related square-grid structure is formed in the case of [Et4N]2[Cu2(Hhba)2(hba)2(di­ox­ane)2][Cu2(Hhba)4(di­ox­ane)(H2O)]·CH3OH. In [Cu2(Hhba)4(H2O)2]·2(Et4NNO3), a square-grid structure is again apparent, but, in this case, a pair of nitrate anions, along with four phenolic groups and a pair of water mol­ecules, combine to form a second type of 4-con­necting node. When 1,8-bis­(di­methyl­amino)­naphthalene (bdn, `proton sponge') is used as a base, another square-grid network is generated, i.e. [Hbdn]2[Cu2(Hhba)2(hba)2(H2O)2]·3(di­ox­ane)·H2O, but with only the copper dimer complex serving as a 4-con­necting node. Complex three-dimensional networks are formed in [Cu2(Hhba)4(O-bipy)]·H2O and [Cu2(Hhba)4(O-bipy)2]·2(di­ox­ane), where the potentially bridging 4,4'-bi­pyridine N,N'-dioxide (O-bipy) ligand is employed. Rare cases of mixed car­box­yl­ate copper dimer complexes were obtained in the cases of [Cu2(Hhba)3(OAc)(di­ox­ane)]·3.5(di­ox­ane) and [Cu2(Hhba)2(OAc)2(DABCO)2]·10(di­ox­ane), with each structure possessing a 2D network structure. The final com­pound re­por­ted is a simple hy­dro­gen-bonded chain of com­position (H0.5DABCO)(H1.5hba), formed from the reaction of H2hba and DABCO.

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.

3D electron diffraction (3D ED), or microcrystal electron diffraction (MicroED), has become an alternative technique for determining the high-resolution crystal structures of compounds from sub-micron-sized crystals. Here, we considered l-alanine, α-glycine and urea, which are known to form good-quality crystals, and collected high-resolution 3D ED data on our in-house TEM instrument. In this study, we present a comparison of independent atom model (IAM) and transferable aspherical atom model (TAAM) kinematical refinement against experimental and simulated data. TAAM refinement on both experimental and simulated data clearly improves the model fitting statistics (R factors and residual electrostatic potential) compared to IAM refinement. This shows that TAAM better represents the experimental electrostatic potential of organic crystals than IAM. Furthermore, we compared the geometrical parameters and atomic displacement parameters (ADPs) resulting from the experimental refinements with the simulated refinements, with the periodic density functional theory (DFT) calculations and with published X-ray and neutron crystal structures. The TAAM refinements on the 3D ED data did not improve the accuracy of the bond lengths between the non-H atoms. The experimental 3D ED data provided more accurate H-atom positions than the IAM refinements on the X-ray diffraction data. The IAM refinements against 3D ED data had a tendency to lead to slightly longer X—H bond lengths than TAAM, but the difference was statistically insignificant. Atomic displacement parameters were too large by tens of percent for l-alanine and α-glycine. Most probably, other unmodelled effects were causing this behaviour, such as radiation damage or dynamical scattering.

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.

Starting from [Mn(C5H4Br)(PPh3)(CO)2] (1a), the cymantrenyl thio­ethers [Mn(C5H4SMe)(PPh3)(CO)2] (1b) and [Mn{C5H4–nBr(SMe)n}(PPh3)(CO)2] (n = 1 for com­pound 2, n = 2 for 3 and n = 3 for 4) were obtained, using either n-butyllithium (n-BuLi), lithium diiso­propyl­amide (LDA) or lithium tetra­methyl­piperidide (LiTMP) as base, followed by electrophilic quenching with MeSSMe. Stepwise consecutive reaction of [Mn(C5Br5)(PPh3)(CO)2] with n-BuLi and MeSSMe led finally to [Mn{C5(SMe)5}(PPh3)(CO)2] (11), only the fifth com­plex to be reported containing a perthiol­ated cyclo­penta­dienyl ring. The mol­ecular and crystal structures of 1b, 3, 4 and 11 were determined and were studied for the occurrence of S⋯S and S⋯Br inter­actions. It turned out that although some inter­actions of this type occurred, they were of minor importance for the arrangement of the mol­ecules in the crystal.

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.

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 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.

It is well known that Hirshfeld surfaces provide an easy and straightforward way of analysing inter­molecular inter­actions in the crystal environment. The use of atomic Hirshfeld surfaces has also demonstrated that such surfaces carry information related to chemical bonds which allow a deeper evaluation of the structures. Here we briefly summarize the approach of atomic Hirshfeld surfaces while further evaluating the kind of information that can be retrieved from them. We show that the analysis of the metal-centre Hirshfeld surfaces from structures refined via Hirshfeld Atom Refinement (HAR) allow accurate evaluation of contacts of type M⋯H, and that such contacts can be related to the overall shape of the surfaces. The com­pounds analysed were tetra­aqua­bis­(3-carb­oxy­propionato)metal(II), [M(C4H3O4)2(H2O)4], for metal(II)/M = manganese/Mn, cobalt/Co, nickel/Ni and zinc/Zn. We also evaluate the sensitivity of the surfaces by an investigation of seemingly flat surfaces through analysis of the curvature functions in the direction of C—C bonds. The obtained values not only demonstrate variations in curvature but also show a correlation with the hybridization of the C atoms involved in the bond.

The activation of C—C bonds by transition-metal com­plexes is of continuing inter­est and aceto­nitrile (MeCN) has attracted attention as a cyanide source with com­paratively low toxicity for organic cyanation reactions. A di­iron end-on μ-η1:η1-CN-bridged com­plex was obtained from a crystallization experiment of an open-chain iron–NHC com­plex, namely, μ-cyanido-κ2C:N-bis­{[(aceto­nitrile-κN)[3,3'-bis­(pyridin-2-yl)-1,1'-(methyl­idene)bis­(benzimidazol-2-yl­idene)]iron(II)} tris­(hexa­fluoro­phos­phate), [Fe2(CN)(C2H3N)2(C25H18N6)2](PF6)3. The cyanide appears to originate from the MeCN solvent by C—C bond cleavage or through carbon–hy­dro­gen oxidation.

Three 2,4-di­aryl­pyrroles were synthesized starting from 4-nitro­butano­nes and the crystal structures of two derivatives were analysed. These are 4-(4-meth­oxy­phen­yl)-2-(thio­phen-2-yl)-1H-pyrrole, C15H13NOS, and 3-(4-bromo­phen­yl)-2-nitroso-5-phenyl-1H-pyrrole, C16H11BrN2O. Although pyrroles without sub­stituents at the α-position with respect to the N atom are very air sensitive and tend to polymerize, we succeeded in growing an adequate crystal for X-ray diffraction analysis. Further derivatization using sodium nitrite afforded a nitrosyl pyrrole derivative, which crystallized in the triclinic space group Poverline{1} with Z = 6. Thus, herein we report the first crystal structure of a nitrosyl pyrrole. Inter­estingly, the co-operative hydrogen bonds in this NO-substituted pyrrole lead to a trimeric structure with bifurcated halogen bonds at the ends, forming a two-dimensional (2D) layer with inter­stitial voids having a radius of 5 Å, similar to some reported macrocyclic porphyrins.

Treating the amide acetanilide (N-phenyl­acetamide, C8H9NO) with aqueous strong acids allowed the structures of five hemi-protonated salt forms of acetanilide to be elucidated. N-(1-Hy­droxy­ethyl­idene)anilinium chloride–N-phenyl­acetamide (1/1), [(C8H9NO)2H][Cl], and the bromide, [(C8H9NO)2H][Br], triiodide, [(C8H9NO)2H][I3], tetra­fluoro­borate, [(C8H9NO)2H][BF4], and di­iodo­bromide hemi(diiodine), [(C8H9NO)2H][I2Br]·0.5I2, analogues all feature centrosymmetric dimeric units linked by O—H⋯O hy­dro­gen bonds that extend into one-dimensional hy­dro­gen-bonded chains through N—H⋯X inter­actions, where X is the halide atom of the anion. Protonation occurs at the amide O atom and results in systematic lengthening of the C=O bond and a corresponding shortening of the C—N bond. The size of these geometric changes is similar to those found for hemi-protonated paracetamol structures, but less than those in fully protonated paracetamol structures. The bond angles of the amide fragments are also found to change on protonation, but these angular changes are also influenced by conformation, namely, whether the amide group is coplanar with the phenyl ring or twisted out of plane.

Three structurally diverse 5-phenyl­tetra­zolato (Tz) Ti, Zr, and Ta com­plexes, namely, (C2H8N)[Ti2(C7H5N4)5(C2H6N)4]·1.45C6H6 or (Me2NH2)[Ti2(NMe2)4(2,3-μ-Tz)3(2-η1-Tz)2]·1.45C6H6, (1·1.45C6H6), [Zr2(C7H5N4)6(C2H6N)2(C2H7N)2]·1.12C6H6·0.382CH2Cl2 or [Zr2(Me2NH)2(NMe2)2(2,3-μ-Tz)3(2-η1-Tz)2(1,2-η2-Tz)]·1.12C6H6·0.38CH2Cl2 (2·1.12C6H6·0.38CH2Cl2), and (C2H8N)2[Ta2(C7H5N4)8(C2H6N)2O]·0.25C7H8 or (Me2NH2)2[Ta2(NMe2)2(2,3-μ-Tz)2(2-η1-Tz)6O]·0.25C7H8 (3·0.25C7H8), where TzH is 5-phenyl-1H-tetra­zole, have been synthesized and structurally characterized. All three com­plexes are dinuclear; the Ti center in 1 is six-coordinate, whereas the Zr and Ta atoms in 2 and 3 are seven-coordinate. The coordination environments of the Ti centers in 1 are similar, and so are the ligations of the Ta centers in 3. In contrast, the two Zr centers in 2 bear a different number of ligands, one of which is a bidentate η2-5-phenyl­tetra­zolato ligand that has not been observed previously for d-block elements. The di­methyl­amido ligand, present in the starting materials, remained un­changed, or was converted to di­methyl­amine and di­methyl­ammonium during the synthesis. Di­methyl­amine coordinates as a neutral ligand, whereas di­methyl­ammonium is retained as a hy­dro­gen-bonded entity bridging Tz ligands.

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.

The synthesis and structural characterization of four novel supra­molecular hy­dro­gen-bonded arrangements based on self-assembly from mol­ecular `[Cu(2,2'-bi­imid­az­ole)]' modules and malonate anions are pre­sent­ed, namely, tetra­kis­(2,2'-bi­imid­az­ole)di-μ-chlorido-dimal­on­atotricopper(II) penta­hydrate, [Cu3(C3H2O4)2Cl2(C6H6N4)4]·5H2O or [Cu(H2biim)2(μ-Cl)Cu0.5(mal)]2·5H2O, aqua­(2,2'-bi­imid­az­ole)­mal­on­atocopper(II) dihydrate, [Cu(C3H2O4)(C6H6N4)(H2O)]·2H2O or [Cu(H2biim)(mal)(H2O)]·2H2O, bis­[aqua­bis­(2,2'-bi­imid­az­ole)­cop­per(II)] di­mal­on­atodi­perchloratocopper(II) 2.2-hydrate, [Cu(C6H6N4)2(H2O)]2[Cu(C3H2O4)(ClO4)2]·2.2H2O or [Cu(H2biim)2(H2O)]2[Cu(mal)2(ClO4)2]·2.2H2O, and bis­(2,2'-bi­imid­az­ole)­copper(II) bis­[bis­(2,2'-bi­imid­az­ole)(2-carb­oxy­acetato)mal­on­atocopper(II)] tridecahydrate, [Cu(C6H6N4)2][Cu(C3H2O4)(C3H3O4)(C6H6N4)2]·13H2O or [Cu(H2biim)2][Cu(H2biim)2(Hmal)(mal)]2·13H2O. These as­sem­blies are characterized by self-com­plementary donor–acceptor mol­ecular inter­actions, demonstrating a recurrent and distinctive pattern of hy­dro­gen-bonding preferences among the carboxyl­ate, carb­oxy­lic acid and N—H groups of the coordinated 2,2'-bi­imid­az­ole and malonate ligands. Additionally, co­or­din­ation of the carboxyl­ate group with the metallic centre helps sustain re­mark­able supra­molecular assemblies, such as layers, helices, double helix columns or 3D channeled architectures, including mixed-metal com­plexes, into a single structure.

The crystal structure of the salt calcium (2R,3R)-tar­trate tetra­hydrate {sys­tem­atic name: poly[[di­aqua­[μ4-(2R,3R)-2,3-di­hydroxy­butane­dioato]calcium(II)] di­hydrate]}, {[Ca(C4H8O8)(H2O)2]·2H2O}n, is reported. The absolute configuration of the crystal was established unambiguously using anomalous dispersion effects in the diffraction patterns. High-quality data also allowed the location and free refinement of all the H atoms, and therefore to a careful analysis of the hy­dro­gen-bond inter­actions.

The technique of time-resolved macromolecular crystallography (TR-MX) has recently been rejuvenated at synchrotrons, resulting in the design of dedicated beamlines. Using pump–probe schemes, this should make the mechanistic study of photoactive proteins and other suitable systems possible with time resolutions down to microseconds. In order to identify relevant time delays, time-resolved spectroscopic experiments directly performed on protein crystals are often desirable. To this end, an instrument has been built at the icOS Lab (in crystallo Optical Spectroscopy Laboratory) at the European Synchrotron Radiation Facility using reflective focusing objectives with a tuneable nanosecond laser as a pump and a microsecond xenon flash lamp as a probe, called the TR-icOS (time-resolved icOS) setup. Using this instrument, pump–probe spectra can rapidly be recorded from single crystals with time delays ranging from a few microseconds to seconds and beyond. This can be repeated at various laser pulse energies to track the potential presence of artefacts arising from two-photon absorption, which amounts to a power titration of a photoreaction. This approach has been applied to monitor the rise and decay of the M state in the photocycle of crystallized bacteriorhodopsin and showed that the photocycle is increasingly altered with laser pulses of peak fluence greater than 100 mJ cm−2, providing experimental laser and delay parameters for a successful TR-MX experiment.

The use of artificial intelligence to process diffraction images is challenged by the need to assemble large and precisely designed training data sets. To address this, a codebase called Resonet was developed for synthesizing diffraction data and training residual neural networks on these data. Here, two per-pattern capabilities of Resonet are demonstrated: (i) interpretation of crystal resolution and (ii) identification of overlapping lattices. Resonet was tested across a compilation of diffraction images from synchrotron experiments and X-ray free-electron laser experiments. Crucially, these models readily execute on graphics processing units and can thus significantly outperform conventional algorithms. While Resonet is currently utilized to provide real-time feedback for macromolecular crystallography users at the Stanford Synchrotron Radiation Lightsource, its simple Python-based interface makes it easy to embed in other processing frameworks. This work highlights the utility of physics-based simulation for training deep neural networks and lays the groundwork for the development of additional models to enhance diffraction collection and analysis.

This article describes the High-Pressure Freezing Laboratory for Macromolecular Crystallography (HPMX) at the ESRF, and highlights new and complementary research opportunities that can be explored using this facility. The laboratory is dedicated to investigating interactions between macromolecules and gases in crystallo, and finds applications in many fields of research, including fundamental biology, biochemistry, and environmental and medical science. At present, the HPMX laboratory offers the use of different high-pressure cells adapted for helium, argon, krypton, xenon, nitrogen, oxygen, carbon dioxide and methane. Important scientific applications of high pressure to macromolecules at the HPMX include noble-gas derivatization of crystals to detect and map the internal architecture of proteins (pockets, tunnels and channels) that allows the storage and diffusion of ligands or substrates/products, the investigation of the catalytic mechanisms of gas-employing enzymes (using oxygen, carbon dioxide or methane as substrates) to possibly decipher intermediates, and studies of the conformational fluctuations or structure modifications that are necessary for proteins to function. Additionally, cryo-cooling protein crystals under high pressure (helium or argon at 2000 bar) enables the addition of cryo-protectant to be avoided and noble gases can be employed to produce derivatives for structure resolution. The high-pressure systems are designed to process crystals along a well defined pathway in the phase diagram (pressure–temperature) of the gas to cryo-cool the samples according to the three-step `soak-and-freeze method'. Firstly, crystals are soaked in a pressurized pure gas atmosphere (at 294 K) to introduce the gas and facilitate its inter­actions within the macromolecules. Samples are then flash-cooled (at 100 K) while still under pressure to cryo-trap macromolecule–gas complexation states or pressure-induced protein modifications. Finally, the samples are recovered after depressurization at cryo-temperatures. The final section of this publication presents a selection of different typical high-pressure experiments carried out at the HPMX, showing that this technique has already answered a wide range of scientific questions. It is shown that the use of different gases and pressure conditions can be used to probe various effects, such as mapping the functional internal architectures of enzymes (tunnels in the haloalkane dehalogenase DhaA) and allosteric sites on membrane-protein surfaces, the interaction of non-inert gases with proteins (oxygen in the hydrogenase ReMBH) and pressure-induced structural changes of proteins (tetramer dissociation in urate oxidase). The technique is versatile and the provision of pressure cells and their application at the HPMX is gradually being extended to address new scientific questions.

Cryo-electron microscopy (cryo-EM) has witnessed radical progress in the past decade, driven by developments in hardware and software. While current software packages include processing pipelines that simplify the image-processing workflow, they do not prioritize the in-depth analysis of crucial metadata, limiting troubleshooting for challenging data sets. The widely used RELION software package lacks a graphical native representation of the underlying metadata. Here, two web-based tools are introduced: relion_live.py, which offers real-time feedback on data collection, aiding swift decision-making during data acquisition, and relion_analyse.py, a graphical interface to represent RELION projects by plotting essential metadata including interactive data filtration and analysis. A useful script for estimating ice thickness and data quality during movie pre-processing is also presented. These tools empower researchers to analyse data efficiently and allow informed decisions during data collection and processing.

Over the last decade, the development of time-resolved serial crystallography (TR-SX) at X-ray free-electron lasers (XFELs) and synchrotrons has allowed researchers to study phenomena occurring in proteins on the femtosecond-to-minute timescale, taking advantage of many technical and methodological breakthroughs. Protein crystals of various sizes are presented to the X-ray beam in either a static or a moving medium. Photoactive proteins were naturally the initial systems to be studied in TR-SX experiments using pump–probe schemes, where the pump is a pulse of visible light. Other reaction initiations through small-molecule diffusion are gaining momentum. Here, selected examples of XFEL and synchrotron time-resolved crystallography studies will be used to highlight the specificities of the various instruments and methods with respect to time resolution, and are compared with cryo-trapping studies.

Catalase is an antioxidant enzyme that breaks down hydrogen peroxide (H2O2) into molecular oxygen and water. In all monofunctional catalases the pathway that H2O2 takes to the catalytic centre is via the `main channel'. However, the structure of this channel differs in large-subunit and small-subunit catalases. In large-subunit catalases the channel is 15 Å longer and consists of two distinct parts, including a hydrophobic lower region near the heme and a hydrophilic upper region where multiple H2O2 routes are possible. Conserved glutamic acid and threonine residues are located near the intersection of these two regions. Mutations of these two residues in the Scytalidium thermophilum catalase had no significant effect on catalase activity. However, the secondary phenol oxidase activity was markedly altered, with kcat and kcat/Km values that were significantly increased in the five variants E484A, E484I, T188D, T188I and T188F. These variants also showed a lower affinity for inhibitors of oxidase activity than the wild-type enzyme and a higher affinity for phenolic substrates. Oxidation of heme b to heme d did not occur in most of the studied variants. Structural changes in solvent-chain integrity and channel architecture were also observed. In summary, modification of the main-channel gate glutamic acid and threonine residues has a greater influence on the secondary activity of the catalase enzyme, and the oxidation of heme b to heme d is predominantly inhibited by their conversion to aliphatic and aromatic residues.

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.

Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the first component of the mitochondrial respiratory chain. In recent years, high-resolution cryo-EM studies of complex I from various species have greatly enhanced the understanding of the structure and function of this important membrane-protein complex. Less well studied is the structural basis of complex I biogenesis. The assembly of this complex of more than 40 subunits, encoded by nuclear or mitochondrial DNA, is an intricate process that requires at least 20 different assembly factors in humans. These are proteins that are transiently associated with building blocks of the complex and are involved in the assembly process, but are not part of mature complex I. Although the assembly pathways have been studied extensively, there is limited information on the structure and molecular function of the assembly factors. Here, the insights that have been gained into the assembly process using cryo-EM are reviewed.

Electron cryo-microscopy image-processing workflows are typically composed of elements that may, broadly speaking, be categorized as high-throughput workloads which transition to high-performance workloads as preprocessed data are aggregated. The high-throughput elements are of particular importance in the context of live processing, where an optimal response is highly coupled to the temporal profile of the data collection. In other words, each movie should be processed as quickly as possible at the earliest opportunity. The high level of disconnected parallelization in the high-throughput problem directly allows a completely scalable solution across a distributed computer system, with the only technical obstacle being an efficient and reliable implementation. The cloud computing frameworks primarily developed for the deployment of high-availability web applications provide an environment with a number of appealing features for such high-throughput processing tasks. Here, an implementation of an early-stage processing pipeline for electron cryotomography experiments using a service-based architecture deployed on a Kubernetes cluster is discussed in order to demonstrate the benefits of this approach and how it may be extended to scenarios of considerably increased complexity.

Mevalonate kinase is central to the isoprenoid biosynthesis pathway. Here, high-resolution X-ray crystal structures of two mevalonate kinases are presented: a eukaryotic protein from Ramazzottius varieornatus and an archaeal protein from Methanococcoides burtonii. Both enzymes possess the highly conserved motifs of the GHMP enzyme superfamily, with notable differences between the two enzymes in the N-terminal part of the structures. Biochemical characterization of the two enzymes revealed major differences in their sensitivity to geranyl pyrophosphate and farnesyl pyrophosphate, and in their thermal stabilities. This work adds to the understanding of the structural basis of enzyme inhibition and thermostability in mevalonate kinases.

The validation of structural models obtained by macromolecular X-ray crystallography against experimental diffraction data, whether before deposition into the PDB or after, is typically carried out exclusively against the merged data that are eventually archived along with the atomic coordinates. It is shown here that the availability of unmerged reflection data enables valuable additional analyses to be performed that yield improvements in the final models, and tools are presented to implement them, together with examples of the results to which they give access. The first example is the automatic identification and removal of image ranges affected by loss of crystal centering or by excessive decay of the diffraction pattern as a result of radiation damage. The second example is the `reflection-auditing' process, whereby individual merged data items showing especially poor agreement with model predictions during refinement are investigated thanks to the specific metadata (such as image number and detector position) that are available for the corresponding unmerged data, potentially revealing previously undiagnosed instrumental, experimental or processing problems. The third example is the calculation of so-called F(early) − F(late) maps from carefully selected subsets of unmerged amplitude data, which can not only highlight the location and extent of radiation damage but can also provide guidance towards suitable fine-grained parametrizations to model the localized effects of such damage.

The widespread adoption of cryoEM technologies for structural biology has pushed the discipline to new frontiers. A significant worldwide effort has refined the single-particle analysis (SPA) workflow into a reasonably standardized procedure. Significant investments of development time have been made, particularly in sample preparation, microscope data-collection efficiency, pipeline analyses and data archiving. The widespread adoption of specific commercial microscopes, software for controlling them and best practices developed at facilities worldwide has also begun to establish a degree of standardization to data structures coming from the SPA workflow. There is opportunity to capitalize on this moment in the maturation of the field, to capture metadata from SPA experiments and correlate the metadata with experimental outcomes, which is presented here in a set of programs called EMinsight. This tool aims to prototype the framework and types of analyses that could lead to new insights into optimal microscope configurations as well as to define methods for metadata capture to assist with the archiving of cryoEM SPA data. It is also envisaged that this tool will be useful to microscope operators and facilities looking to rapidly generate reports on SPA data-collection and screening sessions.

The axoneme, a microtubule-based array at the center of every cilium, has been the subject of structural investigations for decades, but only recent advances in cryo-EM and cryo-ET have allowed a molecular-level interpretation of the entire complex to be achieved. The unique properties of the nine doublet microtubules and central pair of singlet microtubules that form the axoneme, including the highly decorated tubulin lattice and the docking of massive axonemal complexes, provide opportunities and challenges for sample preparation, 3D reconstruction and atomic modeling. Here, the approaches used for cryo-EM and cryo-ET of axonemes are reviewed, while highlighting the unique opportunities provided by the latest generation of AI-guided tools that are transforming structural biology.

Data acquisition and processing for cryo-electron tomography can be a significant bottleneck for users. To simplify and streamline the cryo-ET workflow, Tomo Live, an on-the-fly solution that automates the alignment and reconstruction of tilt-series data, enabling real-time data-quality assessment, has been developed. Through the integration of Tomo Live into the data-acquisition workflow for cryo-ET, motion correction is performed directly after each of the acquired tilt angles. Immediately after the tilt-series acquisition has completed, an unattended tilt-series alignment and reconstruction into a 3D volume is performed. The results are displayed in real time in a dedicated remote web platform that runs on the microscope hardware. Through this web platform, users can review the acquired data (aligned stack and 3D volume) and several quality metrics that are obtained during the alignment and reconstruction process. These quality metrics can be used for fast feedback for subsequent acquisitions to save time. Parameters such as Alignment Accuracy, Deleted Tilts and Tilt Axis Correction Angle are visualized as graphs and can be used as filters to export only the best tomograms (raw data, reconstruction and intermediate data) for further processing. Here, the Tomo Live algorithms and workflow are described and representative results on several biological samples are presented. The Tomo Live workflow is accessible to both expert and non-expert users, making it a valuable tool for the continued advancement of structural biology, cell biology and histology.

Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before being able to inspect a biological sample in an electron microscope, it needs to be deposited in a thin layer on a grid and rapidly frozen. The VitroJet was designed with this aim, as well as avoiding the delicate manual handling and transfer steps that occur during the conventional grid-preparation process. Since its creation, numerous technical developments have resulted in a device that is now widely utilized in multiple laboratories worldwide. It features plasma treatment, low-volume sample deposition through pin printing, optical ice-thickness measurement and cryofixation of pre-clipped Autogrids through jet vitrification. This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow. A wide variety of applications are shown: membrane proteins, nucleosomes, fatty-acid synthase, Tobacco mosaic virus, lipid nanoparticles, tick-borne encephalitis viruses and bacteriophages. These case studies illustrate the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.

A considerable bottleneck in serial crystallography at XFEL and synchrotron sources is the efficient production of large quantities of homogenous, well diffracting microcrystals. Efficient high-throughput screening of batch-grown microcrystals and the determination of ground-state structures from different conditions is thus of considerable value in the early stages of a project. Here, a highly sample-efficient methodology to measure serial crystallography data from microcrystals by raster scanning within standard in situ 96-well crystallization plates is described. Structures were determined from very small quantities of microcrystal suspension and the results were compared with those from other sample-delivery methods. The analysis of a two-dimensional batch crystallization screen using this method is also described as a useful guide for further optimization and the selection of appropriate conditions for scaling up microcrystallization.

Lanthanide ions have ideal chemical properties for catalysis, such as hard Lewis acidity, fast ligand-exchange kinetics, high coordination-number preferences and low geometric requirements for coordination. As a result, many small-molecule lanthanide catalysts have been described in the literature. Yet, despite the ability of enzymes to catalyse highly stereoselective reactions under gentle conditions, very few lanthanoenzymes have been investigated. In this work, the mononuclear binding of europium(III) and gadolinium(III) to the active site of a mutant of the model enzyme phosphotriesterase are described using X-ray crystallography at 1.78 and 1.61 Å resolution, respectively. It is also shown that despite coordinating a single non-natural metal cation, the PTE-R18 mutant is still able to maintain esterase activity.

Diffuse scattering is a promising method to gain additional insight into protein dynamics from macromolecular crystallography experiments. Bragg intensities yield the average electron density, while the diffuse scattering can be processed to obtain a three-dimensional reciprocal-space map that is further analyzed to determine correlated motion. To make diffuse scattering techniques more accessible, software for data processing called mdx2 has been created that is both convenient to use and simple to extend and modify. mdx2 is written in Python, and it interfaces with DIALS to implement self-contained data-reduction workflows. Data are stored in NeXus format for software interchange and convenient visualization. mdx2 can be run on the command line or imported as a package, for instance to encapsulate a complete workflow in a Jupyter notebook for reproducible computing and education. Here, mdx2 version 1.0 is described, a new release incorporating state-of-the-art techniques for data reduction. The implementation of a complete multi-crystal scaling and merging workflow is described, and the methods are tested using a high-redundancy data set from cubic insulin. It is shown that redundancy can be leveraged during scaling to correct systematic errors and obtain accurate and reproducible measurements of weak diffuse signals.

The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening (FFCS) software suite. This framework is further enriched by the option to utilize the Smart Digital User (SDU) software for automated data collection across the PXI, PXII and PXIII beamlines. In this work, the newly developed HEIDI webpage (https://heidi.psi.ch) is introduced: a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments. The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state transfer application programming interface (REST API). The migration of the local FFCS MongoDB instance to the cloud is highlighted and detailed. This transition ensures secure, encrypted and consistently accessible data through a robust and reliable REST API tailored for the FFCS software suite. Collectively, these advancements not only significantly elevate the user experience, but also pave the way for future expansions and improvements in the capabilities of the system.

Cryo-electron tomography (cryo-ET) enables molecular-resolution 3D imaging of complex biological specimens such as viral particles, cellular sections and, in some cases, whole cells. This enables the structural characterization of molecules in their near-native environments, without the need for purification or separation, thereby preserving biological information such as conformational states and spatial relationships between different molecular species. Subtomogram averaging is an image-processing workflow that allows users to leverage cryo-ET data to identify and localize target molecules, determine high-resolution structures of repeating molecular species and classify different conformational states. Here, STOPGAP, an open-source package for subtomogram averaging that is designed to provide users with fine control over each of these steps, is described. In providing detailed descriptions of the image-processing algorithms that STOPGAP uses, this manuscript is also intended to serve as a technical resource to users as well as for further community-driven software development.

Metalloproteins are ubiquitous in all living organisms and take part in a very wide range of biological processes. For this reason, their experimental characterization is crucial to obtain improved knowledge of their structure and biological functions. The three-dimensional structure represents highly relevant information since it provides insight into the interaction between the metal ion(s) and the protein fold. Such interactions determine the chemical reactivity of the bound metal. The available PDB structures can contain errors due to experimental factors such as poor resolution and radiation damage. A lack of use of distance restraints during the refinement and validation process also impacts the structure quality. Here, the aim was to obtain a thorough overview of the distribution of the distances between metal ions and their donor atoms through the statistical analysis of a data set based on more than 115 000 metal-binding sites in proteins. This analysis not only produced reference data that can be used by experimentalists to support the structure-determination process, for example as refinement restraints, but also resulted in an improved insight into how protein coordination occurs for different metals and the nature of their binding interactions. In particular, the features of carboxylate coordination were inspected, which is the only type of interaction that is commonly present for nearly all metals.

Radiation damage remains one of the major impediments to accurate structure solution in macromolecular crystallography. The artefacts of radiation damage can manifest as structural changes that result in incorrect biological interpretations being drawn from a model, they can reduce the resolution to which data can be collected and they can even prevent structure solution entirely. In this article, we discuss how to identify and mitigate against the effects of radiation damage at each stage in the macromolecular crystal structure-solution pipeline.

Type VII secretion (T7S) systems, also referred to as ESAT-6 secretion (ESX) systems, are molecular machines that have gained great attention due to their implications in cell homeostasis and in host–pathogen interactions in mycobacteria. The latter include important human pathogens such as Mycobacterium tuberculosis (Mtb), the etiological cause of human tuberculosis, which constitutes a pandemic accounting for more than one million deaths every year. The ESX-5 system is exclusively found in slow-growing pathogenic mycobacteria, where it mediates the secretion of a large family of virulence factors: the PE and PPE proteins. The secretion driving force is provided by EccC5, a multidomain ATPase that operates using four globular cytosolic domains: an N-terminal domain of unknown function (EccC5DUF) and three FtsK/SpoIIIE ATPase domains. Recent structural and functional studies of ESX-3 and ESX-5 systems have revealed EccCDUF to be an ATPase-like fold domain with potential ATPase activity, the functionality of which is essential for secretion. Here, the crystal structure of the MtbEccC5DUF domain is reported at 2.05 Å resolution, which reveals a nucleotide-free structure with degenerated cis-acting and trans-acting elements involved in ATP binding and hydrolysis. This crystallographic study, together with a biophysical assessment of the interaction of MtbEccC5DUF with ATP/Mg2+, supports the absence of ATPase activity proposed for this domain. It is shown that this degeneration is also present in DUF domains from other ESX and ESX-like systems, which are likely to exhibit poor or null ATPase activity. Moreover, based on an in silico model of the N-terminal region of MtbEccC5DUF, it is hypothesized that MtbEccC5DUF is a degenerated ATPase domain that may have retained the ability to hexamerize. These observations draw attention to DUF domains as structural elements with potential implications in the opening and closure of the membrane pore during the secretion process via their involvement in inter-protomer interactions.

The detection of specific biological macromolecules in cryogenic electron tomography data is frequently approached by applying cross-correlation-based 3D template matching. To reduce computational cost and noise, high binning is used to aggregate voxels before template matching. This remains a prevalent practice in both practical applications and methods development. Here, the relation between template size, shape and angular sampling is systematically evaluated to identify ribosomes in a ground-truth annotated data set. It is shown that at the commonly used binning, a detailed subtomogram average, a sphere and a heart emoji result in near-identical performance. These findings indicate that with current template-matching practices macromolecules can only be detected with high precision if their shape and size are sufficiently different from the background. Using theoretical considerations, the experimental results are rationalized and it is discussed why primarily low-frequency information remains at high binning and that template matching fails to be accurate because similarly shaped and sized macromolecules have similar low-frequency spectra. These challenges are discussed and potential enhancements for future template-matching methodologies are proposed.

Fragment-based drug design using X-ray crystallography is a powerful technique to enable the development of new lead compounds, or probe molecules, against biological targets. This study addresses the need to determine fragment binding orientations for low-occupancy fragments with incomplete electron density, an essential step before further development of the molecule. Halogen atoms play multiple roles in drug discovery due to their unique combination of electronegativity, steric effects and hydrophobic properties. Fragments incorporating halogen atoms serve as promising starting points in hit-to-lead development as they often establish halogen bonds with target proteins, potentially enhancing binding affinity and selectivity, as well as counteracting drug resistance. Here, the aim was to unambiguously identify the binding orientations of fragment hits for SARS-CoV-2 nonstructural protein 1 (nsp1) which contain a combination of sulfur and/or chlorine, bromine and iodine substituents. The binding orientations of carefully selected nsp1 analogue hits were focused on by employing their anomalous scattering combined with Pan-Dataset Density Analysis (PanDDA). Anomalous difference Fourier maps derived from the diffraction data collected at both standard and long-wavelength X-rays were compared. The discrepancies observed in the maps of iodine-containing fragments collected at different energies were attributed to site-specific radiation-damage stemming from the strong X-ray absorption of I atoms, which is likely to cause cleavage of the C—I bond. A reliable and effective data-collection strategy to unambiguously determine the binding orientations of low-occupancy fragments containing sulfur and/or halogen atoms while mitigating radiation damage is presented.