Compositional Analysis of Complex Mixtures using Automatic MicroED Data Collection.

Quantitative analysis of complex mixtures, including compounds having similar chemical properties, is demonstrated using an automatic and high throughput approach to microcrystal electron diffraction (MicroED). Compositional analysis of organic and inorganic compounds can be accurately executed without the need of diffraction standards. Additionally, with sufficient statistics, small amounts of compounds in mixtures can be reliably detected. These compounds can be distinguished by their crystal structure properties prior to structure solution. In addition, if the crystals are of good quality, the crystal structures can be generated on the fly, providing a complete analysis of the sample. MicroED is an effective method for analyzing the structural properties of sub-micron crystals, which are frequently found in small-molecule powders. By developing and using an automatic and high throughput approach to MicroED, and with the use of SerialEM for data collection, data from thousands of crystals allow sufficient statistics to detect even small amounts of compounds reliably.

Unraveling the Structure of Meclizine Dihydrochloride with MicroED.

Meclizine (Antivert, Bonine) is a first-generation H1 antihistamine used in the treatment of motion sickness and vertigo. Despite its wide medical use for over 70 years, its crystal structure and the details of protein-drug interactions remained unknown. Single-crystal X-ray diffraction (SC-XRD) is previously unsuccessful for meclizine. Today, microcrystal electron diffraction (MicroED) enables the analysis of nano- or micro-sized crystals that are merely a billionth the size needed for SC-XRD directly from seemingly amorphous powder. In this study, MicroED to determine the 3D crystal structure of meclizine dihydrochloride is used. Two racemic enantiomers (R/S) are found in the unit cell, which is packed as repetitive double layers in the crystal lattice. The packing is made of multiple strong N-H-Cl- hydrogen bonding interactions and weak interactions like C-H-Cl- and pi-stacking. Molecular docking reveals the binding mechanism of meclizine to the histamine H1 receptor. A comparison of the docking complexes between histamine H1 receptor and meclizine or levocetirizine (a second-generation antihistamine) shows the conserved binding sites. This research illustrates the combined use of MicroED and molecular docking in unraveling elusive drug structures and protein-drug interactions for precision drug design and optimization.

Distinct Conformations of Mirabegron Determined by MicroED.

Mirabegron, commonly known as "Myrbetriq", has been widely prescribed as a medicine for overactive bladder syndrome for over a decade. However, the structure of the drug and what conformational changes it may undergo upon binding its receptor remain unknown. In this study, the authors employed microcrystal electron diffraction (MicroED) to reveal its elusive three-dimensional (3D) structure. They find that the drug adopts two distinct conformational states (conformers) within the asymmetric unit. Analysis of hydrogen bonding and packing demonstrated that the hydrophilic groups are embedded within the crystal lattice, resulting in a hydrophobic surface and low water solubility. Structural comparison revealed the presence of trans- and cis- forms in conformers 1 and 2, respectively. Comparison of the structures of Mirabegron alone with that of the drug bound to its receptor, the beta 3 adrenergic receptor (ß3AR) suggests that the drug undergoes major conformational change to fit in the receptor agonist binding site. This research highlights the efficacy of MicroED in determining the unknown and polymorphic structures of active pharmaceutical ingredients (APIs) directly from powders.

Unraveling the Structure of Meclizine Dihydrochloride with MicroED.

Meclizine (Antivert, Bonine) is a first-generation H1 antihistamine used in the treatment of motion sickness and vertigo. Despite its wide medical use for over 70 years, its crystal structure and the details of protein-drug interactions remained unknown. In this study, we used microcrystal electron diffraction (MicroED) to determine the three-dimensional (3D) crystal structure of meclizine dihydrochloride directly from a seemingly amorphous powder. Two racemic enantiomers (R/S) were found in the unit cell, which packed as repetitive double layers in the crystal lattice. The packing was made of multiple strong N-H⋯Cl- hydrogen bonding interactions and weak interactions like C-H⋯Cl- and pi-stacking. Molecular docking revealed the binding mechanism of meclizine to the histamine H1 receptor. A comparison of the docking complexes between histamine H1 receptor and meclizine or levocetirizine (a second-generation antihistamine) showed the conserved binding sites. This research illustrates the combined use of MicroED and molecular docking in unraveling protein-drug interactions for precision drug design and optimization.

Distinct Conformations of Mirabegron Determined by MicroED.

Mirabegron, commonly known as "Myrbetriq", has been widely prescribed as a medicine for overactive bladder syndrome for over a decade. However, the structure of the drug and what conformational changes it may undergo upon binding its receptor remain unknown. In this study, we employed microcrystal electron diffraction (MicroED) to reveal its elusive three-dimensional (3D) structure. We find that the drug adopts two distinct conformational states (conformers) within the asymmetric unit. Analysis of hydrogen bonding and packing demonstrated that the hydrophilic groups were embedded within the crystal lattice, resulting in a hydrophobic surface and low water solubility. Structural comparison revealed the presence of trans- and cis- forms in conformers 1 and 2, respectively. Comparison of the structures of Mirabegron alone with that of the drug bound to its receptor,1 the beta 3 adrenergic receptor (ß3AR) suggests that the drug undergoes major conformational change to fit in the receptor agonist binding site. This research highlights the efficacy of MicroED in determining the unknown and polymorphic structures of active pharmaceutical ingredients (APIs) directly from powders.

The structure of the neurotoxin palytoxin determined by MicroED.

Palytoxin (PTX) is a potent neurotoxin found in marine animals that can cause serious symptoms such as muscle contractions, haemolysis of red blood cells and potassium leakage. Despite years of research, very little is known about the mechanism of PTX. However, recent advances in the field of cryoEM, specifically the use of microcrystal electron diffraction (MicroED), have allowed us to determine the structure of PTX. It was discovered that PTX folds into a hairpin motif and is able to bind to the extracellular gate of Na,K-ATPase, which is responsible for maintaining the electrochemical gradient across the plasma membrane. These findings, along with molecular docking simulations, have provided important insights into the mechanism of PTX and can potentially aid in the development of molecular agents for treating cases of PTX exposure.

MicroED Structure of a Protoglobin Reactive Carbene Intermediate.

Microcrystal electron diffraction (MicroED) is an emerging technique that has shown great potential for describing new chemical and biological molecular structures. Several important structures of small molecules, natural products, and peptides have been determined using ab initio methods. However, only a couple of novel protein structures have thus far been derived by MicroED. Taking advantage of recent technological advances, including higher acceleration voltage and using a low-noise detector in counting mode, we have determined the first structure of an Aeropyrum pernix protoglobin (ApePgb) variant by MicroED using an AlphaFold2 model for phasing. The structure revealed that mutations introduced during directed evolution enhance carbene transfer activity by reorienting an α helix of ApePgb into a dynamic loop, making the catalytic active site more readily accessible. After exposing the tiny crystals to the substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineered ApePgb's new-to-nature activity, a challenging carbene transfer from a diazirine via a putative metallo-carbene. The bound structure discloses how an enlarged active site pocket stabilizes the carbene bound to the heme iron and, presumably, the transition state for the formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enable investigation of structures that was previously beyond reach.

Benchmarking the ideal sample thickness in cryo-EM.

The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.

The structure of human dermatan sulfate epimerase 1 emphasizes the importance of C5-epimerization of glucuronic acid in higher organisms.

Dermatan sulfate epimerase 1 (DS-epi1, EC 5.1.3.19) catalyzes the conversion of d-glucuronic acid to l-iduronic acid on the polymer level, a key step in the biosynthesis of the glycosaminoglycan dermatan sulfate. Here, we present the first crystal structure of the catalytic domains of DS-epi1, solved at 2.4 Å resolution, as well as a model of the full-length luminal protein obtained by a combination of macromolecular crystallography and targeted cross-linking mass spectrometry. Based on docking studies and molecular dynamics simulations of the protein structure and a chondroitin substrate, we suggest a novel mechanism of DS-epi1, involving a His/double-Tyr motif. Our work uncovers detailed information about the domain architecture, active site, metal-coordinating center and pattern of N-glycosylation of the protein. Additionally, the structure of DS-epi1 reveals a high structural similarity to proteins from several families of bacterial polysaccharide lyases. DS-epi1 is of great importance in a range of diseases, and the structure provides a necessary starting point for design of active site inhibitors.

BioMAX - the first macromolecular crystallography beamline at MAX IV Laboratory.

BioMAX is the first macromolecular crystallography beamline at the MAX IV Laboratory 3 GeV storage ring, which is the first operational multi-bend achromat storage ring. Due to the low-emittance storage ring, BioMAX has a parallel, high-intensity X-ray beam, even when focused down to 20 µm × 5 µm using the bendable focusing mirrors. The beam is tunable in the energy range 5-25 keV using the in-vacuum undulator and the horizontally deflecting double-crystal monochromator. BioMAX is equipped with an MD3 diffractometer, an ISARA high-capacity sample changer and an EIGER 16M hybrid pixel detector. Data collection at BioMAX is controlled using the newly developed MXCuBE3 graphical user interface, and sample tracking is handled by ISPyB. The computing infrastructure includes data storage and processing both at MAX IV and the Lund University supercomputing center LUNARC. With state-of-the-art instrumentation, a high degree of automation, a user-friendly control system interface and remote operation, BioMAX provides an excellent facility for most macromolecular crystallography experiments. Serial crystallography using either a high-viscosity extruder injector or the MD3 as a fixed-target scanner is already implemented. The serial crystallography activities at MAX IV Laboratory will be further developed at the microfocus beamline MicroMAX, when it comes into operation in 2022. MicroMAX will have a 1 µm × 1 µm beam focus and a flux up to 1015 photons s-1 with main applications in serial crystallography, room-temperature structure determinations and time-resolved experiments.

Disubstituted Bis-THF Moieties as New P2 Ligands in Nonpeptidal HIV-1 Protease Inhibitors (II).

A series of darunavir analogues featuring a substituted bis-THF ring as P2 ligand have been synthesized and evaluated. Very high affinity protease inhibitors (PIs) with an interesting activity on wild-type HIV and a panel of multi-PI resistant HIV-1 mutants containing clinically observed, primary mutations were identified using a cell-based assay. Crystal structure analysis was conducted on a number of PI analogues in complex with HIV-1 protease.

Synthesis of P1'-functionalized macrocyclic transition-state mimicking HIV-1 protease inhibitors encompassing a tertiary alcohol.

Seven novel tertiary alcohol containing linear HIV-1 protease inhibitors (PIs), decorated at the para position of the benzyl group in the P1' side with (hetero)aromatic moieties, were synthesized and biologically evaluated. To study the inhibition and antiviral activity effect of P1-P3 macrocyclization, 14- and 15-membered macrocyclic PIs were prepared by ring-closing metathesis of the corresponding linear PIs. The macrocycles were more active than the linear precursors and compound 10f, with a 2-thiazolyl group in the P1' position, was the most potent PI of this new series (Ki 2.2 nM, EC50 0.2 µM). Co-crystallized complexes of both linear and macrocyclic PIs with the HIV-1 protease enzyme were prepared and analyzed.

Design and synthesis of P1-P3 macrocyclic tertiary-alcohol-comprising HIV-1 protease inhibitors.

To study P1-P3 macrocyclizations of previously reported tertiary-alcohol-comprising HIV-1 protease inhibitors (PIs), three new 14- and 15-member macrocyclic PIs were designed, synthesized by ring-closing metathesis, and evaluated alongside with 10 novel linear PIs. Cocrystallized complexes of the macrocyclic PIs and the HIV-1 protease are presented, analyzed, and discussed. The macrocyclic structures exhibited higher activities than the linear precursors with Ki and EC50 values down to 3.1 nM and 0.37 µM, respectively.

The macromolecular crystallography beamline I911-3 at the MAX IV laboratory.

The macromolecular crystallography beamline I911-3, part of the Cassiopeia/I911 suite of beamlines, is based on a superconducting wiggler at the MAX II ring of the MAX IV Laboratory in Lund, Sweden. The beamline is energy-tunable within a range between 6 and 18 keV. I911-3 opened for users in 2005. In 2010-2011 the experimental station was completely rebuilt and refurbished such that it has become a state-of-the-art experimental station with better possibilities for rapid throughput, crystal screening and work with smaller samples. This paper describes the complete I911-3 beamline and how it is embedded in the Cassiopeia suite of beamlines.

Synthesis, X-ray analysis, and biological evaluation of a new class of stereopure lactam-based HIV-1 protease inhibitors.

In an effort to identify a new class of druglike HIV-1 protease inhibitors, four different stereopure ß-hydroxy γ-lactam-containing inhibitors have been synthesized, biologically evaluated, and cocrystallized. The impact of the tether length of the central spacer (two or three carbons) was also investigated. A compound with a shorter tether and (3R,4S) absolute configuration exhibited high activity with a K(i) of 2.1 nM and an EC(50) of 0.64 µM. Further optimization by decoration of the P1' side chain furnished an even more potent HIV-1 protease inhibitor (K(i) = 0.8 nM, EC(50) = 0.04 µM). According to X-ray analysis, the new class of inhibitors did not fully succeed in forming two symmetric hydrogen bonds to the catalytic aspartates. The crystal structures of the complexes further explain the difference in potency between the shorter inhibitors (two-carbon spacer) and the longer inhibitors (three-carbon spacer).

Difluoromethylbenzoxazole pyrimidine thioether derivatives: a novel class of potent non-nucleoside HIV-1 reverse transcriptase inhibitors.

This paper reports the synthesis and antiviral properties of new difluoromethylbenzoxazole (DFMB) pyrimidine thioether derivatives as non-nucleoside HIV-1 reverse transcriptase inhibitors. By use of a combination of structural biology study and traditional medicinal chemistry, several members of this novel class were synthesized using a single electron transfer chain process (radical nucleophilic substitution, S(RN)1) and were found to be potent against wild-type HIV-1 reverse transcriptase, with low cytotoxicity but with moderate activity against drug-resistant strains. The most promising compound 24 showed a significant EC(50) value close to 6.4 nM against HIV-1 IIIB, a moderate EC(50) value close to 54 µM against an NNRTI resistant double mutant (K103N + Y181C), but an excellent selectivity index >15477 (CC(50) > 100 µM).

On the routine use of soft X-rays in macromolecular crystallography. Part V. Molecular replacement and anomalous scattering.

Currently, about two thirds of all new macromolecular structures are determined by molecular replacement. In general the method works reliably, but it reaches its limits when the search model differs too much from the target structure in terms of coordinate deviations or completeness. Since anomalously scattering substructures are better conserved than the overall structure, these substructures and the corresponding anomalous intensity differences can be utilized to enhance the performance of molecular-replacement approaches. It is demonstrated that the combined and concomitant use of structure-factor amplitudes and anomalous differences constitutes a promising approach to push the limits of molecular replacement and to make more structures amenable to structure solution by this technique.

Two-carbon-elongated HIV-1 protease inhibitors with a tertiary-alcohol-containing transition-state mimic.

A new generation of HIV-1 protease inhibitors encompassing a tertiary-alcohol-based transition-state mimic has been developed. By elongation of the core structure of recently reported inhibitors with two carbon atoms and by varying the P1' group of the compounds, efficient inhibitors were obtained with Ki down to 2.3 nM and EC50 down to 0.17 microM. Two inhibitor-enzyme X-ray structures are reported.

Potential for dramatic improvement in sequence alignment against structures of remote homologous proteins by extracting structural information from multiple structure alignment.

A novel method has been developed for acquiring the correct alignment of a query sequence against remotely homologous proteins by extracting structural information from profiles of multiple structure alignment. A systematic search algorithm combined with a group of score functions based on sequence information and structural information has been introduced in this procedure. A limited number of top solutions (15,000) with high scores were selected as candidates for further examination. On a test-set comprising 301 proteins from 75 protein families with sequence identity less than 30%, the proportion of proteins with completely correct alignment as first candidate was improved to 39.8% by our method, whereas the typical performance of existing sequence-based alignment methods was only between 16.1% and 22.7%. Furthermore, multiple candidates for possible alignment were provided in our approach, which dramatically increased the possibility of finding correct alignment, such that completely correct alignments were found amongst the top-ranked 1000 candidates in 88.3% of the proteins. With the assistance of a sequence database, completely correct alignment solutions were achieved amongst the top 1000 candidates in 94.3% of the proteins. From such a limited number of candidates, it would become possible to identify more correct alignment using a more time-consuming but more powerful method with more detailed structural information, such as side-chain packing and energy minimization, etc. The results indicate that the novel alignment strategy could be helpful for extending the application of highly reliable methods for fold identification and homology modeling to a huge number of homologous proteins of low sequence similarity. Details of the methods, together with the results and implications for future development are presented.