Three-dimensional electron diffraction (3D ED), also known as microcrystal electron diffraction (microED), is an emerging method for determining structures from submicron-sized crystals. With the development of rapid and convenient data collection protocols, acquiring dozens of datasets in a single 3D ED/microED session has become routine. A fast and automated workflow for processing, scaling and merging a large number of 3D ED/microED datasets can significantly accelerate the structure determination process. Herein, we present an XDS-based pipeline with a graphical user interface for automated real-time and offline batch 3D ED/microED data processing (AutoLEI). We demonstrate the functionality and applications of the pipeline through four examples, using both offline and real-time data processing capabilities. The samples include small organic molecules, metal–organic frameworks (MOFs) and proteins, showcasing the versatility and efficiency of AutoLEI in various applications.

Multipolar scattering models, such as the transferable aspherical atom model, account for atomic chemical interactions and provide a more accurate representation of experimental data. However, the simpler independent atom model (IAM), which assumes non-interacting atoms, is the only model available in the most widely used macromolecular refinement programs. This is primarily because IAM offers a hard-to-beat combination of computational efficiency and modelling power at typical macromolecular resolutions. By contrast, more accurate multipolar modelling has historically been limited due to its computational cost and the absence of an interface between software capable of calculating structure factors and gradients based on multipolar models and software designed for macromolecular refinement. This work introduces pyDiSCaMB, a Python software package designed to integrate between the computational crystallography toolbox (cctbx) and the quantum crystallography library DiSCaMB (Densities in Structural Chemistry and Molecular Biology), thus enabling multipolar scattering models in Phenix's toolkit. The implementation, features and capabilities of pyDiSCaMB are presented, the runtimes for the calculation of structure factor and target gradients with respect to atomic parameters are explored, and Fourier images of electrostatic potential, electron density and deformation maps are computed as illustrative examples. The pyDiSCaMB library will make multipolar modelling widely available to the structural biology community, potentially transforming refinement and model-building for both crystallography and cryogenic electron microscopy (cryoEM).

Quantum refinement (QR) is an approach in which the empirical restraints used in standard structural refinement to ensure that the details of the structure, e.g. bond lengths and angles, make chemical sense are replaced by more accurate quantum mechanical calculations for a small but interesting part of the structure. QR has previously been used for X-ray and neutron crystallography, cryogenic electron microscopy, nuclear magnetic resonance, and extended X-ray absorption fine structure. Here, QR is used for the first time for X-ray free-electron laser (XFEL) crystallography and microcrystal electron diffraction (MicroED). As a test case, we use six structures of the R2a protein of ribonucleotide reductase, concentrating on the binuclear Fe₂ site in either the oxidized (Fe₂^III) or reduced (Fe₂^II) state, two each from single-crystal X-ray (SCX) crystallography, XFEL crystallography or MicroED. The results show that QR works well for data from all three radiation sources, even though scattering factors for neutral atoms had to be used for MicroED. QR corrects unrealistically short Fe—O distances in the reduced SCX structure and gives improved real-space Z scores for the reduced MicroED structure. The three methods give similar structures, apart from variation in the weak water ligands and in the binding of carboxylate groups (monodentate, bidentate or a mixture). By performing QR for three protonation states of the bridging solvent molecule, we could show that it is undoubtedly a water molecule in the reduced XFEL and MicroED structures (it is not present in the SCX structure) and that it is not water in the oxidized structures. The XFEL data indicate that it is O²⁻ in the oxidized XFEL structure, in agreement with the spectroscopic results. However, for the SCX structure, O²⁻ and OH⁻ give comparable results, whereas OH⁻ is slightly preferred in the MicroED structure. This indicates that the SCX and MicroED structures may be partly photoreduced during data collection.

Serial femtosecond crystallography (SFX) and continuous serial electron diffraction (c-SerialED) both enable high-resolution structure determination from protein microcrystals with minimal radiation damage, making it ideal for studying redox-active metalloenzymes. Here, c-SerialED and SFX were used to solve structures of the class Ia ribonucleotide reductase R2 subunit in oxidized (Fe^III-Fe^III), reduced (Fe^II-Fe^II), and re-oxidized states at ∼1.8 Å resolution, capturing three points in a redox reaction. These results demonstrate that c-SerialED can track reversible changes at the redox-site, enabling future time-resolved studies. Comparison between c-SerialED structures and SFX diffraction and emission data confirmed minimal radiation damage. Furthermore, previously reported structures use mercury in the crystallization condition and show mercury-induced conformational changes. Here, we use mercury-free crystallization conditions and reveal a water molecule in the redox center of the reduced state, absent in the previous structures, making these structures more representative of the physiological state.

Microcrystal electron diffraction (MicroED), also known as three-dimensional electron diffraction (3D ED), allows the collection of diffraction data from submicrometre-sized crystals under low electron-dose conditions. Despite having several advantages over conventional X-ray crystallographic techniques, susceptibility to radiation damage is a great challenge that remains to be solved in MicroED. Similar to X-ray crystallography, radiation damage to the macromolecular crystal structures in MicroED manifests in two forms: global damage that affects the overall order of the crystal lattice and site-specific damage that affects highly sensitive residues and moieties in macromolecules. Traditionally, the unit e⁻ Å⁻² has been used for electron-dose estimations, which does not consider the interaction between the incident electron beam and the sample. In this study, we clarify the terminology for describing `dose' in electron crystallography, including the procedure for converting values from e⁻ Å⁻² to grays (Gy). Furthermore, we investigated data-processing strategies that could be used to limit the effects of radiation damage to the crystal. During MicroED data collection, radiation damage increases with the number of acquired ED frames because the accumulated electron dose increases. Data collected from several crystals and processed in this way can be merged to increase the completeness and subsequently be used for structure refinement. According to our results, this approach improves the resolution of the data, the data statistics, the structure determination and the quality of the final structure.

3D electron diffraction (3D ED) has shown great potential in crystal structure determination in materials, small organic molecules, and macromolecules. In this work, an automated, low-dose and low-bias 3D ED protocol has been implemented to identify six phases from a multiple-phase melt-crystallisation product of an active pharmaceutical ingredient, griseofulvin (GSF). Batch data collection under low-dose conditions using a widely available commercial software was combined with automated data analysis to collect and process over 230 datasets in three days. Accurate unit cell parameters obtained from 3D ED data allowed direct phase identification of GSF Forms III, I and the known GSF inclusion complex (IC) with polyethylene glycol (PEG) (GSF-PEG IC-I), as well as three minor phases, namely GSF Forms II, V and an elusive new phase, GSF-PEG IC-II. Their structures were then directly determined by 3D ED. Furthermore, we reveal how the stabilities of the two GSF-PEG IC polymorphs are closely related to their crystal structures. These results demonstrate the power of automated 3D ED for accurate phase identification and direct structure determination of complex, beam-sensitive crystallisation products, which is significant for drug development where solid form screening is crucial for the overall efficacy of the drug product. 

Background: IgE cross-sensitization to major birch pollen allergen Bet v 1 and pathogenesis-related (PR10) plant food allergens is responsible for the pollen-food allergy syndrome.

Methods: We designed a recombinant protein, AB-PreS, consisting of non-allergenic peptides derived from the IgE-binding sites of Bet v 1 and the cross-reactive apple allergen, Mal d 1, fused to the PreS domain of HBV surface protein as immunological carrier. AB-PreS was expressed in E. coli and purified by chromatography. The allergenic and inflammatory activity of AB-PreS was tested using basophils and PBMCs from birch pollen allergic patients. The ability of antibodies induced by immunization of rabbits with AB-PreS and birch pollen extract-based vaccines to inhibit allergic patients IgE binding to Bet v 1 and Mal d 1 was assessed by ELISA.

Results: IgE-binding experiments and basophil activation test revealed the hypoallergenic nature of AB-PreS. AB-PreS induced lower T-cell activation and inflammatory cytokine production in cultured PBMCs from allergic patients. IgG antibodies induced by five injections with AB-PreS inhibited allergic patients' IgE binding to Bet v 1 and Mal d 1 better than did IgG induced by up to 30 injections of six licensed birch pollen allergen extract-based vaccines. Additionally, immunization with AB-PreS induced HBV-specific antibodies potentially protecting from infection with HBV.

Conclusion: The recombinant AB-PreS-based vaccine is hypoallergenic and superior over currently registered allergen extract-based vaccines regarding the induction of blocking antibodies to Bet v 1 and Mal d 1 in animals.

Millions of people have been vaccinated with Gam-COVID-Vac but fine specificities of induced antibodies have not been fully studied. Plasma from 12 naïve and 10 coronavirus disease 2019 (COVID-19) convalescent subjects was obtained before and after two immunizations with Gam-COVID-Vac. Antibody reactivity in the plasma samples (n = 44) was studied on a panel of micro-arrayed recombinant folded and unfolded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and 46 peptides spanning the spike protein (S) and by immunoglobulin G (IgG) subclass enzyme-linked immunosorbent assay (ELISA). The ability of Gam-COVID-Vac-induced antibodies to inhibit binding of the receptor-binding domain (RBD) to its receptor angiotensin converting enzyme 2 (ACE2) was investigated in a molecular interaction assay (MIA). The virus-neutralizing capacity of antibodies was studied by the pseudo-typed virus neutralization test (pVNT) for Wuhan-Hu-1 and Omicron. We found that Gam-COVID-Vac vaccination induced significant increases of IgG₁ but not of other IgG subclasses against folded S, spike protein subunit 1 (S1), spike protein subunit 2 (S2), and RBD in a comparable manner in naïve and convalescent subjects. Virus neutralization was highly correlated with vaccination-induced antibodies specific for folded RBD and a novel peptide (i.e., peptide 12). Peptide 12 was located close to RBD in the N-terminal part of S1 and may potentially be involved in the transition of the pre- to post-fusion conformation of the spike protein. In summary, Gam-COVID-Vac vaccination induced S-specific IgG₁ antibodies in naive and convalescent subjects in a comparable manner. Besides the antibodies specific for RBD, the antibodies induced against a peptide close to the N-terminus of RBD were also associated with virus-neutralization.

Background: The determinants of successful humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of critical importance for the design of effective vaccines and the evaluation of the degree of protective immunity conferred by exposure to the virus. As novel variants emerge, understanding their likelihood of suppression by population antibody repertoires has become increasingly important.

Methods: In this study, we analyzed the SARS-CoV-2 polyclonal antibody response in a large population of clinically well-characterized patients after mild and severe COVID-19 using a panel of microarrayed structurally folded and unfolded SARS-CoV-2 proteins, as well as sequential peptides, spanning the surface spike protein (S) and the receptor-binding domain (RBD) of the virus.

Results: S- and RBD-specific antibody responses were dominated by immunoglobulin G (IgG), mainly IgG₁, and directed against structurally folded S and RBD and three distinct peptide epitopes in S2. The virus neutralization activity of patients´ sera was highly correlated with IgG antibodies specific for conformational but not sequential RBD epitopes and their ability to prevent RBD binding to its human receptor angiotensin-converting enzyme 2 (ACE2). Twenty percent of patients selectively lacked RBD-specific IgG. Only immunization with folded, but not with unfolded RBD, induced antibodies against conformational epitopes with high virus-neutralizing activity. Conformational RBD epitopes required for protection do not seem to be altered in the currently emerging virus variants.

Conclusion: These results are fundamental for estimating the protective activity of antibody responses after natural infection or vaccination and for the design of vaccines, which can induce high levels of SARS-CoV-2–neutralizing antibodies conferring sterilizing immunity.

Three-dimensional electron diffraction (3D ED)/microcrystal electron diffraction (MicroED) techniques are gaining in popularity. However, the data processing often does not fit existing graphical user interface software, instead requiring the use of the terminal or scripting. Scipion-ED, described in this article, provides a graphical user interface and extendable framework for processing of 3D ED/MicroED data. An illustrative project is described, in which multiple 3D ED/MicroED data sets collected on tetragonal lysozyme were processed with DIALS through the Scipion-ED interface. The ability to resolve unmodelled features in the electrostatic potential map was compared between three strategies for merging data sets.