The crystal structure of a glycosyl­ated porphyrin (P_Gal2) system, C70H70N4O12, where two iso­propyl­idene protected galactose moieties are attached to the meso position of a substituted tetra­aryl porphyrin is reported. This structure reveals that the parent porphyrin is planar, with the galactose moieties positioned above and below the porphyrin macrocycle. This orientation likely prevents porphyrin–porphyrin H-type aggregation, potentially enhancing its efficiency as a photosensitizer in photodynamic therapy. Notable non-bonding C—H⋯O and C—H⋯π inter­actions among adjacent P_Gal2 systems are observed in this crystal network. Additionally, the tolyl groups of each porphyrin can engage in π–π inter­actions with the delocalized π-systems of neighboring porphyrins.

The title compound, C14H20NO5+·Cl−, was prepared as a racemate of R,R- and S,S-enanti­omers by reduction of the corresponding hy­droxy­imino­ketone. In the crystal, layers are formed via hydrogen bridges of four ammonium groups to chloride ions; these lamellae are connected via inter­digitated benzoic ester groups.

The crystal structure of the title compound, C14H12N2O2S, reveals two symmetrically independent mol­ecules within the asymmetric unit. Each mol­ecule contains a chromenone core attached to a 2-thio­phene ring, cyano, and amino groups. The 2-thio­phene ring of one of the two mol­ecules in the asymmetric unit was found to be disordered over two positions, with the major component having a site occupancy factor of 0.837 (2). The 2-thio­phene ring is nearly orthogonal to the fused 4H-pyran ring, with dihedral angles between the two sets of planes being 89.5 (5) and 89.63 (8)°. Inter­molecular hydrogen bonding, involving N—H⋯N and N—H⋯O inter­actions, creates two distinct motifs leading to the formation of a two-dimensional supra­molecular network along the crystallographic ac plane.

The crystal structure of 1,4-bis­(neopent­yloxy)pillar[5]arene, C95H140N2O10 (TbuP), featuring two encapsulated pyridine mol­ecules, reveals significant host–guest inter­actions. Inter­estingly, the pyridine guests are positioned near the neopent­yloxy substituents instead of the electron-rich aromatic core of the pillar[5]arene. This spatial arrangement suggests a preference for the pyridine mol­ecules to engage with the aliphatic regions of the host. Detailed analysis of the structural characteristics of this host–guest system (TbuP·2Py), as well as its packing pattern within the crystal network, is presented and discussed.

α-d-2'-De­oxy­ribonucleosides are products of the γ-irradiation of DNA under oxygen-free conditions and are constituents of anomeric DNA. They are not found as natural building blocks of canonical DNA. Reports on their conformational properties are limited. Herein, the single-crystal X-ray structure of α-d-2'-de­oxy­adenosine (α-dA), C10H13N5O3, and its conformational parameters were determined. In the crystalline state, α-dA forms two conformers in the asymmetric unit which are connected by hydro­gen bonds. The sugar moiety of each conformer is arranged in a `clamp'-like fashion with respect to the other conformer, forming hydro­gen bonds to its nucleobase and sugar residue. For both conformers, a syn conformation of the nucleobase with respect to the sugar moiety was found. This is contrary to the anti conformation usually preferred by α-nucleosides. The sugar conformation of both conformers is C2'-endo, and the 5'-hydroxyl groups are in a +sc orientation, probably due to the hydro­gen bonds formed by the conformers. The formation of the supra­molecular assembly of α-dA is controlled by hydro­gen bonding and stacking inter­actions, which was verified by a Hirshfeld and curvedness surface analysis. Chains of hydro­gen-bonded nucleobases extend parallel to the b direction and are linked to equivalent chains by hydro­gen bonds involving the sugar moieties to form a sheet. A com­parison of the solid-state structures of the anomeric 2'-de­oxy­adenosines revealed significant differences of their conformational parameters.

The crystal structure of a new 1:1 cocrystal of carbamazepine and S-naproxen (C15H12N2O·C14H14O3) was solved from powder X-ray diffraction (PXRD). The PXRD pattern was measured at the high-resolution beamline CRISTAL at synchrotron SOLEIL (France). The structure was solved using Monte Carlo simulated annealing, then refined with Rietveld refinement. The positions of the H atoms were obtained from density functional theory (DFT) ground-state calculations. The symmetry is ortho­rhom­bic with the space group P212121 (No. 19) and the following lattice parameters: a = 33.5486 (9), b = 26.4223 (6), c = 5.3651 (10) Å and V = 4755.83 (19) Å3.

X-ray and electron diffraction methods independently identify the S-enanti­omer of Berkecoumarin [systematic name: (S)-8-hy­droxy-3-(2-hy­droxy­prop­yl)-6-meth­oxy-2H-chromen-2-one]. Isolated from Berkeley Pit Lake Penicillium sp., Berkecoumarin is a natural product with a light-atom com­position (C13H14O5) that challenges in-house absolute structure determination by anomalous scattering. This study further demonstrates the utility of dynamical refinement of electron-diffraction data for absolute structure determination.

The coordination of hydro­tris­[3-(2-furyl)pyrazol-1-yl]borate (Tp2-Fu, C21H16BN6O3) to lan­tha­nide(III) ions is achieved for the first time with the com­plex [Ln(Tp2-Fu)2](BPh4)·xCH2Cl2 (1-Ln has Ln = Ce and x = 2; 1-Dy has Ln = Dy and x = 1). This was accom­plished via both hydrous (Ln = Ce) and anhydrous methods (Ln = Dy). When isolating the dysprosium analogue, the filtrate produced a second crop of crystals which were revealed to be the 1,2-borotropic-shifted product [Dy(κ4-Tp2-Fu)(κ5-Tp2-Fu*)](BPh4) (2) {Tp2-Fu* = hydro­bis­[3-(2-furyl)pyrazol-1-yl][5-(2-furyl)pyrazol-1-yl]borate}. We con­clude that the pres­ence of a strong Lewis acid and a sterically crowded coordination environment are contributing factors for the 1,2-borotropic shifting of scorpionate ligands in conjunction with the size of the conical angle with the scorpionate ligand.

The crystal structures of three mixed-valence copper cyanide alkanolamine polymers are presented, together with thermogravimetric analysis (TGA) and electron spin resonance (ESR) data. In all three structures, a CuII moiety on a crystallographic center of symmetry is coordinated by two alkanolamines and links two CuICN chains via cyanide bridging groups to form diperiodic sheets. The sheets are linked together by cuprophilic CuI–CuI inter­actions to form a three-dimensional network. In poly[bis­(μ-3-amino­propano­lato)tetra-μ-cyan­ido-dicopper(I)dicopper(II)], [Cu4(CN)4(C3H8NO)2]n, 1, propano­lamine bases have lost their hydroxyl H atoms and coordinate as chelates to two CuII atoms to form a dimeric CuII moiety bridged by the O atoms of the bases with CuII atoms in square-planar coordination. The ESR spectrum is very broad, indicating exchange between the two CuII centers. In poly[bis­(2-amino­pro­pan­ol)tetra-μ-cyanido-dicopper(I)copper(II)], [Cu3(CN)4(C3H9NO)2]n, 2, and poly[bis­(2-amino­ethanol)tetra-μ-cyanido-dicopper(I)copper(II)], [Cu3(CN)4(CH7NO)2]n, 3, a single CuII atom links the CuICN chains together via CN bridges. The chelating alkanolamines are not ionized, and the OH groups form rather long bonds in the axial positions of the octa­hedrally coordinated CuII atoms. The coordination geometries of CuII in 2 and 3 are almost identical, except that the Cu—O distances are longer in 2 than in 3, which may explain their somewhat different ESR spectra. Thermal decom­position in 2 and 3, but not in 1, begins with the loss of HCN(g), and this can be correlated with the presence of OH protons on the ligands in 2 and 3, which are not present in 1.

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.

We report on the latest advancements in Microcrystal Electron Diffraction (3D ED/MicroED), as discussed during a symposium at the National Center for CryoEM Access and Training housed at the New York Structural Biology Center. This snapshot describes cutting-edge developments in various facets of the field and identifies potential avenues for continued progress. Key sections discuss instrumentation access, research applications for small mol­ecules and biomacromolecules, data collection hardware and software, data reduction software, and finally reporting and validation. 3D ED/MicroED is still early in its wide adoption by the structural science community with ample opportunities for expansion, growth, and innovation.

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.

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.

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.

The title compound, 3-[(benzo-1,3-dioxol-5-yl)amino]-4-meth­oxy­cyclo­but-3-ene-1,2-dione, C12H9NO5 (3), is a precursor to an anti­mycobacterial squaramide. Block-shaped crystals of a monoclinic form (3-I, space group P21/c, Z = 8, Z' = 2) and needle-shaped crystals of a triclinic form (3-II, space group P-1, Z = 4, Z' = 2) were found to crystallize concomitantly. In both crystal forms, R22(10) dimers assemble through N—H⋯O=C hydrogen bonds. These dimers are formed from crystallographically unique mol­ecules in 3-I, but exhibit crystallographic Ci symmetry in 3-II. Twinning by pseudomerohedry was encountered in the crystals of 3-II. The conformations of 3 in the solid forms 3-I and 3-II are different from one another but are similar for the unique mol­ecules in each polymorph. Density functional theory (DFT) calculations on the free mol­ecule of 3 indicate that a nearly planar conformation is preferred.

Growing high-quality crystals remains a necessary part of crystallography and many other techniques. This article tabulates and describes several techniques and variations that will help individuals grow high-quality crystals in preparation for crystallographic techniques and other endeavors, such as form screening. The discussion is organized to focus on low-tech approaches available in any laboratory.

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.

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.

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.

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.

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.

Density modification is a standard step to provide a route for routine structure solution by any experimental phasing method, with single-wavelength or multi-wavelength anomalous diffraction being the most popular methods, as well as to extend fragments or incomplete models into a full solution. The effect of density modification on the starting maps from either source is illustrated in the case of SHELXE. The different modes in which the program can run are reviewed; these include less well known uses such as reading external phase values and weights or phase distributions encoded in Hendrickson–Lattman coefficients. Typically in SHELXE, initial phases are calculated from experimental data, from a partial model or map, or from a combination of both sources. The initial phase set is improved and extended by density modification and, if the resolution of the data and the type of structure permits, polyalanine tracing. As a feature to systematically eliminate model bias from phases derived from predicted models, the trace can be set to exclude the area occupied by the starting model. The trace now includes an extension into the gamma position or hydrophobic and aromatic side chains if a sequence is provided, which is performed in every tracing cycle. Once a correlation coefficient of over 30% between the structure factors calculated from such a trace and the native data indicates that the structure has been solved, the sequence is docked in all model-building cycles and side chains are fitted if the map supports it. The extensions to the tracing algorithm brought in to provide a complete model are discussed. The improvement in phasing performance is assessed using a set of tests.

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.

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.

Phenuiviridae nucleoprotein is the main structural and functional component of the viral cycle, protecting the viral RNA and mediating the essential replication/transcription processes. The nucleoprotein (N) binds the RNA using its globular core and polymerizes through the N-terminus, which is presented as a highly flexible arm, as demonstrated in this article. The nucleoprotein exists in an `open' or a `closed' conformation. In the case of the closed conformation the flexible N-terminal arm folds over the RNA-binding cleft, preventing RNA adsorption. In the open conformation the arm is extended in such a way that both RNA adsorption and N polymerization are possible. In this article, single-crystal X-ray diffraction and small-angle X-ray scattering were used to study the N protein of Toscana virus complexed with a single-chain camelid antibody (VHH) and it is shown that in the presence of the antibody the nucleoprotein is unable to achieve a functional assembly to form a ribonucleoprotein complex.

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.

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.

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.

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.

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 determination of the atomic resolution structure of biomacromolecules is essential for understanding details of their function. Traditionally, such a structure determination has been performed with crystallographic or nuclear resonance methods, but during the last decade, cryogenic transmission electron microscopy (cryo-TEM) has become an equally important tool. As the blotting and flash-freezing of the samples can induce conformational changes, external validation tools are required to ensure that the vitrified samples are representative of the solution. Although many validation tools have already been developed, most of them rely on fully resolved atomic models, which prevents early screening of the cryo-TEM maps. Here, a novel and automated method for performing such a validation utilizing small-angle X-ray scattering measurements, publicly available through the new software package AUSAXS, is introduced and implemented. The method has been tested on both simulated and experimental data, where it was shown to work remarkably well as a validation tool. The method provides a dummy atomic model derived from the EM map which best represents the solution structure.

The interpretation of cryo-EM maps often includes the docking of known or predicted structures of the components, which is particularly useful when the map resolution is worse than 4 Å. Although it can be effective to search the entire map to find the best placement of a component, the process can be slow when the maps are large. However, frequently there is a well-founded hypothesis about where particular components are located. In such cases, a local search using a map subvolume will be much faster because the search volume is smaller, and more sensitive because optimizing the search volume for the rotation-search step enhances the signal to noise. A Fourier-space likelihood-based local search approach, based on the previously published em_placement software, has been implemented in the new emplace_local program. Tests confirm that the local search approach enhances the speed and sensitivity of the computations. An interactive graphical interface in the ChimeraX molecular-graphics program provides a convenient way to set up and evaluate docking calculations, particularly in defining the part of the map into which the components should be placed.

Several proteins from plant pathogenesis-related family 10 (PR10) are highly abundant in the latex of opium poppy and have recently been shown to play diverse and important roles in the biosynthesis of benzylisoquinoline alkaloids (BIAs). The recent determination of the first crystal structures of PR10-10 showed how large conformational changes in a surface loop and adjacent β-strand are coupled to the binding of BIA compounds to the central hydrophobic binding pocket. A more detailed analysis of these conformational changes is now reported to further clarify how ligand binding is coupled to the formation and cleavage of an intermolecular disulfide bond that is only sterically allowed when the BIA binding pocket is empty. To decouple ligand binding from disulfide-bond formation, each of the two highly conserved cysteine residues (Cys59 and Cys155) in PR10-10 was replaced with serine using site-directed mutagenesis. Crystal structures of the Cys59Ser mutant were determined in the presence of papaverine and in the absence of exogenous BIA compounds. A crystal structure of the Cys155Ser mutant was also determined in the absence of exogenous BIA compounds. All three of these crystal structures reveal conformations similar to that of wild-type PR10-10 with bound BIA compounds. In the absence of exogenous BIA compounds, the Cys59Ser and Cys155Ser mutants appear to bind an unidentified ligand or mixture of ligands that was presumably introduced during expression of the proteins in Escherichia coli. The analysis of conformational changes triggered by the binding of BIA compounds suggests a molecular mechanism coupling ligand binding to the disruption of an intermolecular disulfide bond. This mechanism may be involved in the regulation of biosynthetic reactions in plants and possibly other organisms.

A group of three deep-learning tools, referred to collectively as CHiMP (Crystal Hits in My Plate), were created for analysis of micrographs of protein crystallization experiments at the Diamond Light Source (DLS) synchrotron, UK. The first tool, a classification network, assigns images into categories relating to experimental outcomes. The other two tools are networks that perform both object detection and instance segmentation, resulting in masks of individual crystals in the first case and masks of crystallization droplets in addition to crystals in the second case, allowing the positions and sizes of these entities to be recorded. The creation of these tools used transfer learning, where weights from a pre-trained deep-learning network were used as a starting point and repurposed by further training on a relatively small set of data. Two of the tools are now integrated at the VMXi macromolecular crystallography beamline at DLS, where they have the potential to absolve the need for any user input, both for monitoring crystallization experiments and for triggering in situ data collections. The third is being integrated into the XChem fragment-based drug-discovery screening platform, also at DLS, to allow the automatic targeting of acoustic compound dispensing into crystallization droplets.

The availability of highly accurate protein structure predictions from AlphaFold2 (AF2) and similar tools has hugely expanded the applicability of molecular replacement (MR) for crystal structure solution. Many structures can be solved routinely using raw models, structures processed to remove unreliable parts or models split into distinct structural units. There is therefore an open question around how many and which cases still require experimental phasing methods such as single-wavelength anomalous diffraction (SAD). Here, this question is addressed using a large set of PDB depositions that were solved by SAD. A large majority (87%) could be solved using unedited or minimally edited AF2 predictions. A further 18 (4%) yield straightforwardly to MR after splitting of the AF2 prediction using Slice'N'Dice, although different splitting methods succeeded on slightly different sets of cases. It is also found that further unique targets can be solved by alternative modelling approaches such as ESMFold (four cases), alternative MR approaches such as ARCIMBOLDO and AMPLE (two cases each), and multimeric model building with AlphaFold-Multimer or UniFold (three cases). Ultimately, only 12 cases, or 3% of the SAD-phased set, did not yield to any form of MR tested here, offering valuable hints as to the number and the characteristics of cases where experimental phasing remains essential for macromolecular structure solution.

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.

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.