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Cholesterol induces faster collapse by compressed films of pulmonary surfactant. Because collapse prevents films from reaching the high surface pressures achieved in the alveolus, most therapeutic surfactants remove or omit cholesterol. The studies here determined the structural changes by which cholesterol causes faster collapse by films of dipalmitoyl phosphatidylcholine, used as a simple model for the functional alveolar film. Measurements of isobaric collapse, with surface pressure held constant at 52 mN/m, showed that cholesterol had little effect until the mol fraction of cholesterol, X

Structural biologists are using cryogenic electron microscopy to resolve atomic-scale structures of proteins from nanocrystals.

Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous-rotation data were collected using an ultra-low exposure rate to enable electron counting in diffraction. For the first sample, triclinic lysozyme extending to a resolution of 0.87 Å, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a resolution of 1.5 Å. These results demonstrate that macromolecules can be determined to sub-ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data.

Crystallization of membrane proteins, such as G protein-coupled receptors (GPCRs), is challenging and frequently requires the use of lipidic cubic phase (LCP) crystallization methods. These typically yield crystals that are too small for synchrotron X-ray crystallography, but ideally suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, the viscous nature of LCP makes sample preparation challenging. The LCP layer is often too thick for transmission electron microscopy (TEM), and crystals buried in LCP cannot be identified topologically using a focused ion-beam and scanning electron microscope (FIB/SEM). Therefore, the LCP needs to either be converted to the sponge phase or entirely removed from the path of the ion-beam to allow identification and milling of these crystals. Unfortunately, conversion of the LCP to sponge phase can also deteriorate the sample. Methods that avoid LCP conversion are needed. Here, we employ a novel approach using an integrated fluorescence light microscope (iFLM) inside of a FIB/SEM to identify fluorescently labelled crystals embedded deep in a thick LCP layer. The crystals are then targeted using fluorescence microscopy and unconverted LCP is removed directly using a plasma focused ion beam (pFIB). To assess the optimal ion source to prepare biological lamellae, we first characterized the four available gas sources on standard crystals of the serine protease, proteinase K. However, lamellae prepared using either argon and xenon produced the highest quality data and structures. Fluorescently labelled crystals of the human adenosine receptor embedded in thick LCP were placed directly onto EM grids without conversion to the sponge phase. Buried microcrystals were identified using iFLM, and deep lamellae were created using the xenon beam. Continuous rotation MicroED data were collected from the exposed crystalline lamella and the structure was determined using a single crystal. This study outlines a robust approach to identifying and milling LCP grown membrane protein crystals for MicroED using single microcrystals, and demonstrates plasma ion-beam milling as a powerful tool for preparing biological lamellae.

Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å. Even though a beam stop was not used in these studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED.

Microcrystal electron diffraction (MicroED) is a powerful technique utilizing electron cryo-microscopy (cryo-EM) for protein structure determination of crystalline samples too small for X-ray crystallography. Electrons interact with the electrostatic potential of the sample, which means that scattered electrons carry informing about the charged state of atoms and can provide strong contrast for visualizing hydrogen atoms. Accurately identifying the positions of hydrogen atoms, and by extension the hydrogen bonding networks, is of importance for drug discovery and electron microscopy can enable such visualization. Using subatomic resolution MicroED data obtained from triclinic hen egg-white lysozyme, we identified hundreds of individual hydrogen atom positions and directly visualize hydrogen bonding interactions and the charged states of residues. Over a third of all hydrogen atoms are identified from strong difference peaks, the most complete view of a macromolecular hydrogen network visualized by electron diffraction to date. These results show that MicroED can provide accurate structural information on hydrogen atoms and non-covalent hydrogen bonding interactions in macromolecules. Furthermore, we find that the hydrogen bond lengths are more accurately described by the inter-nuclei distances than the centers of mass of the corresponding electron clouds. We anticipate that MicroED, coupled with ongoing advances in data collection and refinement, can open further avenues for structural biology by uncovering and understanding the hydrogen bonding interactions underlying protein structure and function.

Understanding the relationship between molecular structure and solid-state arrangement informs about the design of new organic semiconductor (OSC) materials with improved optoelectronic properties. However, determining their atomic structure remains challenging. Here, we report the lattice organization of two non-fullerene acceptors (NFAs) determined using microcrystal electron diffraction (MicroED) from crystals not traceable by X-ray crystallography. The MicroED structure of o-IDTBR was determined from a powder without crystallization, and a new polymorph of ITIC-Th is identified with the most distorted backbone of any NFA. Electronic structure calculations elucidate the relationships between molecular structures, lattice arrangements, and charge-transport properties for a number of NFA lattices. The high dimensionality of the connectivity of the 3D wire mesh topology is the best for robust charge transport within NFA crystals. However, some examples suffer from uneven electronic coupling. MicroED combined with advanced electronic structure modeling is a powerful new approach for structure determination, exploring polymorphism and guiding the design of new OSCs and NFAs.

When compressed by the shrinking alveolar surface area during exhalation, films of pulmonary surfactant in situ reduce surface tension to levels at which surfactant monolayers collapse from the surface in vitro. Vesicles of pulmonary surfactant added below these monolayers slow collapse. X-ray scattering here determined the structural changes induced by the added vesicles. Grazing incidence X-ray diffraction on monolayers of extracted calf surfactant detected an ordered phase. Mixtures of dipalmitoyl phosphatidylcholine and cholesterol, but not the phospholipid alone, mimic that structure. At concentrations that stabilize the monolayers, vesicles in the subphase had no effect on the unit cell, and X-ray reflection showed that the film remained monomolecular. The added vesicles, however, produced a concentration-dependent increase in the diffracted intensity. These results suggest that the enhanced resistance to collapse results from enlargement by the additional material of the ordered phase.

Structure determination of novel biological macromolecules by X-ray crystallo­graphy can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective search models for molecular replacement to overcome the phase problem. Independence from the need for a complete pre-existing model with sequence similarity to the crystallized molecule is the primary appeal of ARCIMBOLDO, a suite of programs which employs this ab initio algorithm for phase determination. Here, the use of ARCIMBOLDO is investigated to overcome the phase problem with the electron cryomicroscopy (cryoEM) method known as microcrystal electron diffraction (MicroED). The results support the use of the ARCIMBOLDO_SHREDDER pipeline to provide phasing solutions for a structure of proteinase K from 1.6 Å resolution data using model fragments derived from the structures of proteins sharing a sequence identity of as low as 20%. ARCIMBOLDO_SHREDDER identified the most accurate polyalanine fragments from a set of distantly related sequence homologues. Alternatively, such templates were extracted in spherical volumes and given internal degrees of freedom to refine towards the target structure. Both modes relied on the rotation function in Phaser to identify or refine fragment models and its translation function to place them. Model completion from the placed fragments proceeded through phase combination of partial solutions and/or density modification and main-chain autotracing using SHELXE. The combined set of fragments was sufficient to arrive at a solution that resembled that determined by conventional molecular replacement using the known target structure as a search model. This approach obviates the need for a single, complete and highly accurate search model when phasing MicroED data, and permits the evaluation of large fragment libraries for this purpose., This work was performed as part of STROBE, an NSF Science and Technology Center, through grant DMR-1548924. This work was also supported by DOE grant DE-FC02-02ER63421 and NIH–NIGMS grants R35 GM128867 and P41GM136508. LSR is supported by USPHS National Research Service Award 5T32GM008496. RJB received a fellowship from FAPESP (16/24191-8 and 17/13485-3). CM is grateful to MICINN for her BES-2015-71397 scholarship associated with the Structural Biology Maria de Maeztu Unit of Excellence. This work was supported by grants BIO2015-64216-P, PGC2018-101370-B-100 and MDM2014-0435-01 (the Spanish Ministry of Economy and Competitiveness) and Generalitat de Catalunya (2017SGR-1192). JAR is supported as a Searle Scholar, a Pew Scholar and a Beckman Young Investigator. The Gonen laboratory is supported by the Howard Hughes Medical Institute; this work was also partially supported by the Janelia Research Campus Visitor Exchange Program.

Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that was collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion-beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous rotation data were collected using an ultra-low exposure rate on a Falcon 4 direct electron detector in electron-counting mode. For the first sample, triclinic lysozyme extending to 0.87 Å resolution, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a modest 1.5 Å resolution. These results demonstrate that macromolecules can be determined to sub-Ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data collected on a direct electron detector.

Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous-rotation data were collected using an ultra-low exposure rate to enable electron counting in diffraction. For the first sample, triclinic lysozyme extending to a resolution of 0.87 Å, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a resolution of 1.5 Å. These results demonstrate that macromolecules can be determined to sub-ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data.

Significance A systematic investigation of the effects of sample thickness on electron scattering in electron cryo-microscopy (cryo-EM) was previously not feasible. Here, methods are employed to investigate the effects of increasing sample thickness. Near identical protein crystals are used as samples, and microcrystal electron diffraction data are used to assess the effects of thickness. These experiments were conducted using the three most-common accelerating voltages in cryo-EM, and data were compared using the calculated inelastic mean free path. Structures may be determined using crystals up to twice the inelastic mean free path. No coherent information remains at thicknesses over four times the mean free path. This study provides limits for biological specimen thickness with implications for all cryo-EM methods., 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.

G Protein-Coupled Receptors (GPCRs), or 7-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without signaling partners bound requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here we show that the structure of a human A2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by cryoFIB milling. We determined the structure of the A2A receptor to 2.8 Å resolution and resolved an antagonist in its orthosteric ligand-binding site as well as 4 cholesterol molecules bound to the receptor. This study lays the groundwork for future GPCR structural studies using single microcrystals that would otherwise be impossible by other crystallographic methods.One sentence summaryFIB milled LCP-GPCR structure determined by MicroED

The relationship between sample thickness and quality of data obtained by microcrystal electron diffraction (MicroED) is investigated. Several 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 scanning electron microscope (FIB/SEM) where the crystals were then systematically thinned into lamellae between 95 nm and 1650 nm thick. MicroED data were collected at either 120, 200, or 300 kV accelerating voltages. Lamellae thicknesses were converted to multiples of the calculated inelastic mean free path (MFP) of electrons at each accelerating voltage to allow the results to be compared on a common scale. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined from crystalline lamellae only 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 quality was insufficient to yield structures. No diffraction data were observed from lamellae thicker than four times the calculated inelastic mean free path. The quality of the determined structures and crystallographic statistics were similar for all lamellae up to 2x the inelastic mean free path in thickness, but quickly deteriorated at greater thicknesses. This study provides a benchmark with respect to the ideal limit for biological specimen thickness with implications for all cryo-EM methods.SignificanceA systematic investigation of the effects of thickness on electron scattering from protein crystals was previously not feasible, because there was no accurate method to control sample thickness. Here, the recently developed methods for preparing protein crystals into lamellae of precise thickness by ion-beam milling are used to investigate the effects of increasing sample thickness on MicroED data quality. These experiments were conducted using the three most common accelerating voltages in cryo-EM. Data across these accelerating voltages and thicknesses were compared on a common scale using their calculated inelastic mean free path lengths. It is found that structures may accurately be determined from crystals up to twice the inelastic mean free path length in thickness, regardless of the acceleration voltage.

Gram-negative bacteria are protected from their environment by an outer membrane that is primarily composed of lipopolysaccharides (LPSs). Under stress, pathogenic serotypes of Salmonella enterica remodel their LPSs through the PhoPQ two-component regulatory system that increases resistance to both conventional antibiotics and antimicrobial peptides (AMPs). Acquired resistance to AMPs is contrary to the established narrative that AMPs circumvent bacterial resistance by targeting the general chemical properties of membrane lipids. However, the specific mechanisms underlying AMP resistance remain elusive. Here we report a 2-fold increase in bacteriostatic concentrations of human AMP LL-37 for S. enterica with modified LPSs. LPSs with and without chemical modifications were isolated and investigated by Langmuir films coupled with grazing-incidence X-ray diffraction (GIXD) and specular X-ray reflectivity (XR). The initial interactions between LL-37 and LPS bilayers were probed using all-atom molecular dynamics simulations. These simulations suggest that initial association is nonspecific to the type of LPS and governed by hydrogen bonding to the LPS outer carbohydrates. GIXD experiments indicate that the interactions of the peptide with monolayers reduce the number of crystalline domains but greatly increase the typical domain size in both LPS isoforms. Electron densities derived from XR experiments corroborate the bacteriostatic values found in vitro and indicate that peptide intercalation is reduced by LPS modification. We hypothesize that defects at the liquid-ordered boundary facilitate LL-37 intercalation into the outer membrane, whereas PhoPQ-mediated LPS modification protects against this process by having innately increased crystallinity. Since induced ordering has been observed with other AMPs and drugs, LPS modification may represent a general mechanism by which Gram-negative bacteria protect against host innate immunity.

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.

Microcrystal electron diffraction (MicroED) enables atomic resolution structures to be determined from vanishingly small crystals. Soluble proteins typically grow crystals that are tens to hundreds of microns in size for X-ray crystallography. But membrane protein crystals often grow crystals that are too small for X-ray diffraction and yet too large for MicroED. These crystals are often formed in thick, viscous media that challenge traditional cryoEM grid preparation. Here, we describe two approaches for preparing membrane protein crystals for MicroED data collection: application of a crystal slurry directly to EM grids, and focused ion beam milling in a Scanning Electron Microscope (FIB-SEM). We summarize the case of preparing an ion channel, NaK, and the workflow of focused ion-beam milling. By milling away the excess media and crystalline material, crystals of any size may be prepared for MicroED. Finally, an energy filter may be used to help minimize inelastic scattering leading to lower noise on recorded images.

A near-atomic resolution structure of the mouse voltage dependent anion channel (mVDAC) is determined by combining cryogenic focused ion-beam (FIB) milling and microcrystal electron diffraction (MicroED). The crystals were grown in a viscous modified bicelle suspension which limited their size and made them unsuitable for conventional X-ray crystallography. Individual thin, plate-like crystals were identified using scanning electron microscopy (SEM) and focused ion-beam (FIB) imaging at high magnification. Three crystals were milled into thin lamellae. MicroED data were collected from each lamellae and merged to increase completeness. Unmodelled densities were observed between protein monomers, suggesting the presence of lipids that likely mediate crystal contacts. This work demonstrates the utility of milling membrane protein microcrystals grown in viscous media using a focused ion-beam for subsequent structure determination by MicroED for samples that are not otherwise tractable by other crystallographic methods. To our knowledge, the structure presented here is the first of a membrane protein crystallized in a lipid matrix and solved by MicroED.

Summary A high throughout method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoelectron microscopy vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of approximately 200 nm using a focused ion beam, and MicroED data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Both the number of ligands bound and their occupancy is higher using on-grid soaking of microcrystals compared with much larger crystals treated similarly and investigated by X-ray crystallography. These results indicate that on-grid soaking ligands into microcrystals results in efficient uptake of ligands into protein microcrystals.

When compressed by the shrinking alveolar surface area during exhalation, films of pulmonary surfactantin situreduce surface tension to levels, at which surfactant monolayers collapse from the surfacein vitro. Vesicles of pulmonary surfactant added below these monolayers slow collapse. X-ray scattering here determined the structural changes that improve stability. Grazing incidence X-ray diffraction on monolayers of extracted calf surfactant detected an ordered phase. Mixtures of dipalmitoyl phosphatidylcholine and cholesterol, but not the phospholipid alone, mimic that structure. At concentrations that stabilize the monolayers, vesicles in the subphase had no effect on the unit cell, and the film remained monomolecular. The added vesicles, however, produced a concentration-dependent increase in the diffracted intensity. These results suggest that the enhanced resistance to collapse results from components of an ordered interfacial phase which partition from subphase to the surface, increasing the area of the ordered structure.SIGNIFICANCELow alveolar surface tensions are essential for maintaining the integrity of the pulmonary air-sacks during normal breathing. Films of pulmonary surfactant cause the low tensions. The interfacial structures required for the low surface tensions remain uncertain. These studies used X-ray scattering to determine the initial structure of pulmonary surfactant monolayers, and to establish how vesicles of pulmonary surfactant enhance the ability of those initial monolayers to sustain low tensions. The initial monolayers contained ordered structures that differ from the crystalline forms widely speculated to occur in alveolar films. The added vesicles had no effect on the local structure of the initial monolayer, but substantially increased the area of the ordered regions. This structural change reasonably explains the functional improvement.

We previously engineered the tryptophan synthase beta-subunit (TrpB), which catalyzes the condensation reaction between L-serine and indole to form L-tryptophan, to synthesize a range of modified tryptophans from serine and indole derivatives. In this study, we used directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C–C bonds leading to new quaternary stereocenters. At first, the TrpBs that could use 3-substituted oxindoles preferentially formed N–C bonds by attacking the oxindole N1 atom. We found, however, that protecting the nitrogen encouraged evolution towards C-alkylation, which persisted even when this protection was removed. After seven rounds of evolution leading to a 400-fold improvement in activity, variant Pfquat efficiently alkylates 3-substituted oxindoles to selectively form new stereocenters at the γ-position of the amino acid products. The configuration of the new γ-stereocenter of one of the products was determined from the crystal structure obtained by microcrystal electron diffraction (MicroED). Substrates structurally related to 3-methyloxindole such as lactones and ketones can also be used by the enzyme for quaternary carbon bond formation, where the biocatalyst exhibits excellent regioselectivity for the tertiary carbon atom. Highly thermostable and expressed at > 500 mg/L E. coli culture, TrpB Pfquat provides an efficient and environmentally-friendly platform for the preparation of noncanonical amino acids bearing quaternary carbons.

Synchrotron-based X-ray scattering measurements of phase-separated surfactant monolayers at the air-water interface provide molecular-level structural information about the packing and ordering of film components. In this work, grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity (XR) measurements were used to collect crystallographic structural information for binary mixed monolayers of arachidic acid (AA, C19H39COOH) with perfluorotetradecanoic acid (PA, C13F27COOH), a system that has previously been investigated using a variety of thermodynamic and micron-scale structural characterization methods. GIXD measurements at surface pressures of π = 5, 15, and 30 mN/m indicated that AA in pure and mixed films forms a rectangular lattice at π = 5 and 15 mN/m but a hexagonal lattice at π = 30 mN/m. PA formed hexagonal lattices under all conditions, with films being highly ordered and crystalline (as determined by Bragg peak width) at even the lowest surface pressures investigated. Phase separation occurred for all mixed monolayer film compositions and surface pressures, manifesting as diffraction peaks characteristic of the individual components appearing at different in-plane scattering vector qxy. For both pure and mixed films, the molecular tilt angle of the AA hydrocarbon chain toward the nearest-neighbor was substantial at low pressures but decreased with increasing pressure. The PA fluorocarbon chain showed negligible molecular tilt under all conditions, and was oriented normal to the subphase surface regardless of surface pressure or the presence of AA in the films. In all cases, the two components in the mixed film behaved entirely independently of film composition, which is exactly the expected result for a fully phase-separated, immiscible system. XR measurements of film thickness at the air-water interface supported these results; overall film thickness approached the calculated ideal surfactant tail lengths with increasing surface pressure, indicating nearly normal oriented surfactants. The overall surfactant packing and crystallographic features of the mixed monolayers are discussed in terms of the lipophobic nature of the perfluorinated surfactant as well as in context of thermodynamic miscibility and domain structure formation reported elsewhere in the literature for these mixed monolayer systems.

Hydrophobic interactions govern specificity for natural antimicrobial peptides. No such relationship has been established for synthetic peptoids that mimic antimicrobial peptides. Peptoid macrocycles synthesized with five different aromatic groups are investigated by minimum inhibitory and hemolytic concentration assays, epifluorescence microscopy, atomic force microscopy, and X-ray reflectivity. Peptoid hydrophobicity is determined using high performance liquid chromatography. Disruption of bacterial but not eukaryotic lipid membranes is demonstrated on the solid supported lipid bilayers and Langmuir monolayers. X-ray reflectivity studies demonstrate that intercalation of peptoids with zwitterionic or negatively charged lipid membranes is found to be regulated by hydrophobicity. Critical levels of peptoid selectivity are demonstrated and found to be modulated by their hydrophobic groups. It is suggested that peptoids may follow different optimization schemes as compared to their natural analogues.

Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.

The peptidomimetic approach has emerged as a powerful tool for overcoming the inherent limitations of natural antimicrobial peptides, where the therapeutic potential can be improved by increasing the selectivity and bioavailability. Restraining the conformational flexibility of a molecule may reduce the entropy loss upon its binding to the membrane. Experimental findings demonstrate that the cyclization of linear antimicrobial peptoids increases their bactericidal activity against Staphylococcus aureus while maintaining high hemolytic concentrations. Surface X-ray scattering shows that macrocyclic peptoids intercalate into Langmuir monolayers of anionic lipids with greater efficacy than for their linear analogues. It is suggested that cyclization may increase peptoid activity by allowing the macrocycle to better penetrate the bacterial cell membrane.

The structure of ice Ih is solved from a single nanocrystal to a resolution of 0.53Å using the cryoEM method microcrystal electron diffraction (MicroED). Data were collected at just above liquid nitrogen temperatures (~80K) in ultra-high vacuum (~8 x 10-7 Pa) using a total exposure of less than 1e- Å-2.

Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.

Synthetic polymers mimicking antimicrobial peptides have drawn considerable interest as potential therapeutics. N-substituted glycines, or peptoids, are recognized by their in vivo stability and ease of synthesis. Peptoids are thought to act primarily on the negatively charged lipids that are abundant in bacterial cell membranes. A mechanistic understanding of lipid-peptoid interaction at the molecular level will provide insights for rational design and optimization of peptoids. Here, we highlight recent studies that utilize synchrotron liquid surface X-ray scattering to characterize the underlying peptoid interactions with bacterial and eukaryotic membranes. Cellular membranes are highly complex, and difficult to characterize at the molecular level. Model systems including Langmuir monolayers, are used in these studies to reduce system complexity. The general workflow of these systems and the corresponding data analysis techniques are presented alongside recent findings. These studies investigate the role of peptoid physicochemical characteristics on membrane activity. Specifically, the roles of cationic charge, conformational constraint via macrocyclization, and hydrophobicity are shown to correlate their membrane interactions to biological activities in vitro. These structure-activity relationships have led to new insights into the mechanism of action by peptoid antimicrobials, and suggest optimization strategies for future therapeutics based on peptoids.

In the many scientific endeavors that are driven by organic chemistry, unambiguous identification of small molecules is of paramount importance. Over the past 50 years, NMR and other powerful spectroscopic techniques have been developed to address this challenge. While almost all of these techniques rely on inference of connectivity, the unambiguous determination of a small molecule’s structure requires X-ray and/or neutron diffraction studies. In practice, however, X-ray crystallography is rarely applied in routine organic chemistry due to intrinsic limitations of both the analytes and the technique. Here we report the use of the electron cryo-microscopy (cryoEM) method microcrystal electron diffraction (MicroED) to provide routine and unambiguous structural determination of small organic molecules. From simple powders, with minimal sample preparation, we could collect high-quality MicroED data from nanocrystals (∼100 nm, ∼10–15 g) resulting in atomic resolution (, From powder to structure: The cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) is described as a new technique for routine structural analysis providing high-resolution crystal structures from seemingly amorphous powders.

Superwarfarins were developed following the emergence of warfarin resistance in rodents. Compared to warfarin, superwarfarins have much longer half-lives and stronger affinity to vitamin K epoxide reductase and therefore can cause death in warfarin-resistant rodents. By the mid-1970s, the superwarfarins brodifacoum and difenacoum were the most widely used rodenticides throughout the world. Unfortunately, increased use was accompanied by a rise in accidental poisonings, reaching >16,000 per year in the United States. Risk of exposure has become a concern since large quantities, up to hundreds of kilograms of rodent bait, are applied by aerial dispersion over regions with rodent infestations. Reports of intentional use of superwarfarins in civilian and military scenarios raise the specter of larger incidents or mass casualties. Unlike warfarin overdose, for which 1-2 days of treatment with vitamin K is effective, treatment of superwarfarin poisoning with vitamin K is limited by extremely high cost and can require daily treatment for a year or longer. Furthermore, superwarfarins have actions that are independent of their anticoagulant effects, including both vitamin K-dependent and -independent effects, which are not mitigated by vitamin K therapy. In this review, we summarize superwarfarin development, biology and pathophysiology, their threat as weapons, and possible therapeutic approaches.

Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while thediffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.

The atomic structure of the infectious, protease-resistant, β-sheet-rich and fibrillar mammalian prion remains unknown. Through the cryo-EM method, MicroED, we reveal the sub-1Å resolution structure of a protofibril formed by a wild-type segment from the β2-α2 loop of the bank vole prion protein. The structure of this protofibril reveals a stabilizing network of hydrogen bonds that link polar zippers within a sheet, producing motifs we name ‘polar clasps’., Summary sentence: Ultrahigh-resolution cryo-EM structure reveals a prion protofibril stabilized by a dense three-dimensional network of hydrogen bonds.

Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. Herein we present the structure of the largest aqueous gold cluster, Au146(p-MBA)57 (p-MBA: para-mercaptobenzoic acid), solved by electron diffraction (MicroED) to subatomic resolution (0.85 Å) and by X-ray diffraction at atomic resolution (1.3 Å). The 146 gold atoms may be decomposed into two constituent sets consisting of 119 core and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure whereas the surface gold atoms follow a C2 rotational symmetry about an axis bisecting the twinning plane. The protective layer of 57 p-MBAs fully encloses the cluster and comprises bridging, monomeric, and dimeric staple motifs. Au146(p-MBA)57 is the largest cluster observed exhibiting a bulk-like FCC structure as well as the smallest gold particle exhibiting a stacking fault.

Low dose imaging procedures are key for a successful cryoEM experiment (whether by electron cryotomography, single particle analysis, electron crystallography, or MicroED). We present a method to minimize magnetic hysteresis of the condenser lens system in the JEOL JEM-3200FSC transmission electron microscope (TEM) in order to maintain a stable optical axis for the beam path of low-dose imaging. The simple procedure involves independent voltage ramping of the CL1 and CL2 lenses immediately before switching to the focusing and exposure beam settings for data collection.

Theoretical calculations suggest that crystals exceeding 100 nm thickness are excluded by dynamical scattering from successful structure determination using microcrystal electron diffraction (MicroED). These calculations are at odds with experimental results where MicroED structures have been determined from significantly thicker crystals. Here we systematically evaluate the influence of thickness on the accuracy of MicroED intensities and the ability to determine structures from protein crystals one micrometer thick. To do so, we compare ab initio structures of a human prion protein segment determined from thin crystals to those determined from crystals up to one micrometer thick. We also compare molecular replacement solutions from crystals of varying thickness for a larger globular protein, proteinase K. Our results indicate that structures can be reliably determined from crystals at least an order of magnitude thicker than previously suggested by simulation, opening the possibility for an even broader range of MicroED experiments.SummaryAtomic resolution protein structures can be determined by MicroED from crystals that surpass the theoretical maximum thickness limit by an order of magnitude.

Recently, significant technological innovations have enabled the measurement of both X-ray and electron diffraction from protein microcrystals. These new microcrystallography experiments are useful when large crystals cannot be obtained, but also in other cases, such as when large crystals suffer from long-range disorder, or when uniform perturbations need to be applied rapidly to the entire crystal volume. Optimizing the preparation of protein microcrystals for this new class of experiments presents new challenges for crystallographers, who have traditionally sought to grow large, single crystals. To better understand these new challenges, we optimized the production of microcrystalline samples of cyclophilin A (CypA), starting from conditions that produced millimeter scale crystals. Next, we used these microcrystals to determine CypA structures by serial femtosecond crystallography (SFX) at two XFEL lightsources, and by microcrystal electron diffraction (microED) in an electron cryomicroscope. Here, I will present our optimization strategy for protein microcrystallization, and compare the results of X-ray and electron microcrystallography experiments with CypA. I will focus on the unique caveats of sample delivery for each method, and compare the resulting structures. The goal will be to provide insight into which microcrystallography experiment is most appropriate for which types of samples, and to share our experience with sample preparation and delivery for each type of experiment.

Achieving structurally well-defined catalytic species requires a fundamental understanding of surface chemistry. Detailed structural characterization of the catalyst binding sites in situ, such as single site catalysts on silica supports, is technically challenging or even unattainable. Octadecyltrioxysilane (OTOS) monolayers formed from octadecyltrimethoxysilane (OTMS) at the air-liquid interface after hydrolysis and condensation at low pH were chosen as a model system of surface binding sites in silica-supported Zn(2+) catalysts. We characterize the system by grazing incidence X-ray diffraction, X-ray reflectivity (XR), and X-ray fluorescence spectroscopy (XFS). Previous X-ray and infrared surface studies of OTMS/OTOS films at the air-liquid interface proposed the formation of polymer OTOS structures. According to our analysis, polymer formation is inconsistent with the X-ray observations and structural properties of siloxanes; it is energetically unfavorable and thus highly unlikely. We suggest an alternative mechanism of hydrolysis/condensation in OTMS leading to the formation of structurally allowed cyclic trimers with the six-membered siloxane rings, which explain well both the X-ray and infrared results. XR and XFS consistently demonstrate that tetrahedral [Zn(NH3)4](2+) ions bind to hydroxyl groups of the film at a stoichiometric ratio of OTOS:Zn ∼ 2:1. The high binding affinity of zinc ions to OTOS trimers suggests that the six-membered siloxane rings are binding locations for single site Zn/SiO2 catalysts. Our results show that OTOS monolayers may serve as a platform for studying silica surface chemistry or hydroxyl-mediated reactions.

Superwarfarins were developed following the emergence of warfarin resistance in rodents. Superwarfarins have much longer half-lives and stronger affinity to vitamin K epoxide reductase versus warfarin, and therefore can cause death in warfarin-resistant rodents. By the mid-1970s, the superwarfarins brodifacoum (BDF) and difenacoum (DiF) were the most widely used rodenticides throughout the world. Unfortunately, increased use was accompanied by a rise in accidental poisonings, reaching >16,000 per year in the United States. Risk of exposure has become a concern since large quantities, up to hundreds of kilograms of rodent bait, are applied by aerial dispersion over regions with rodent infestations. Reports of intentional use of superwarfarins in civilian and military scenarios raises the specter of larger incidents or mass casualties. Unlike warfarin overdose, for which 1–2 days of treatment with vitamin K is effective, treatment of superwarfarin poisoning with vitamin K is limited by extremely high cost and can require daily treatment for a year or longer. Furthermore, superwarfarins have actions that are independent of their anticoagulant effects, including both vitamin K–dependent and –independent effects, which are not mitigated by vitamin K therapy. In this review, we will summarize superwarfarin development, biology and pathophysiology, their threat as weapons, and possible therapeutic approaches.

Superwarfarins are modified analogs of warfarin with additional lipophilic aromatic rings, up to 100-fold greater potency, and longer biological half-lives. We hypothesized that increased hydrophobicity allowed interactions with amphiphilic membranes and modulation of biological responses. We find that superwarfarins brodifacoum and difenacoum increase lactate production and cell death in neuroblastoma cells. In contrast, neither causes changes in glioma cells that have higher cholesterol content. After choleterol depletion, lactate production was increased and cell viability was reduced. Drug-membrane interactions were examined by surface X-ray scattering using Langmuir monolayers of dipalmitoylphosphatidylcholine and/or cholesterol. Specular X-ray reflectivity data revealed that superwarfarins, but not warfarin, intercalate between dipalmitoylphosphatidylcholine molecules, whereas grazing incidence X-ray diffraction demonstrated changes in lateral crystalline order of the film. Neither agent showed significant interactions with monolayers containing >20% cholesterol. These findings demonstrate an affinity of superwarfarins to biomembranes and suggest that cellular responses to these agents are regulated by cholesterol content.

Gram-negative bacteria possess a double membrane system, with the external leaflet of the outer membrane composed of lipopolysaccharides (LPS) that act as the first barrier to drugs. The importance of studying the permeability of LPS barrier comes from its correlation with strain susceptibility. In this study, we extracted LPS from two isogenic Salmonella sp strains, that contains alterations in the lipid A portion regulated by the phoPQ-system. Interaction between phoP- (LPS with no modification) or phoPc (palmitoylation of the 3-hydroxyl group in the 3-OH-myristyl residue, addition of 4-aminoarabinose to the 4' phosphate group and addition of a 2-hydroxy group to the myristate residue at the position 3') LPS and novobiocin was evaluated by using Langmuir monolayers. Novobiocin was chosen due to its size (612 Da), large enough to permeate cells throughout the LPS, and not through the porin channels. MIC of novobiocin for phoP- was 14 μg mL-1 and for phoPc, 40 μg mL-1. p-A isotherms indicated that phoP- occupies a limiting area of 150A2, while for phoPc this value is 175A2. However, addition of Mg2+ promotes condensation of 20A2 for phoP-, and 70A2 for phoPc. Epifluorescence microscopy reveals phoP- has fluid-like morphology .In contrast, phoPc displays circular-shaped domains that increase in size and number with the compression. The addition of novobiocin to the subphase promoted slight changes in phoP- morphology. However, for phoPc monolayer, changes in morphology were observed for higher concentrations of novobiocin at which domains started to aggregate generating structures like a "pearl necklace". Structural organization was evaluated by GIXD, and data corroborates the one obtained by epifluorescence, in which novobiocin interaction promotes liquid-crystalline phase formation. Electron density profile probed that novobiocin interacts with phoP- hydrophobic portion, but much higher concentration is needed to permeate phoPc.

A novel supported bilayer system at the air-water interface has been developed for use in synchrotron X-ray scattering experiments in order to drastically increase signal to noise ratio. This system gives a significant resolution improvement comparing to more traditional solid-supported bilayer system and is better suited for specular X-Ray reflectivity and grazing incidence X-ray off-specular scattering. Using this system we have successfully characterized anionic bilayers mimetic of Gram-positive bacterial membranes interacting with engineered biomimetic oligomers (peptoids), zwitterionic bilayers interacting with human islet amyloid polypeptide (IAPP), and insertion differences between zwitterionic membranes with and without cholesterol and the glycoprotein-41 subunit fragment of the viral envelope glycoprotein-160. Results from anionic and net neutral bilayers correspond to previous studies on monolayer insertion studies, where peptoid ML 2-6 acts by associating throughout the lipophilic regions and zwitterionic bilayers without cholesterol were completely penetrated by the GP-41 fusion peptide. Resolution of the dynamic formation of a zwitterionic lipid bilayer system in minute scale time resolution via Grazing Incidence X-Ray Off-Specular Scattering (GIXOS) was also observed, and found to be in agreement with the proposed mechanisms associated with vesicle fusion on a solid support.