Search

Metabolomics has emerged as a powerful discipline to study complex biological systems from a small molecule perspective. The success of metabolomics hinges upon reliable annotations of spectral features obtained from MS and/or NMR. In spite of tremendous progress with regards to analytical instrumentation and computational tools, < 20% of spectral features are confidently identified in most untargeted metabolomics experiments. This article explores the integration of multiple analytical instruments such as UHPLC-MS/MS-SPE-NMR and the cryo-EM method MicroED to achieve large-scale and confident metabolite identifications in a higher-throughput manner. UHPLC-MS/MS-SPE allows for the simultaneous automated purification of metabolites followed by offline structure elucidation and structure validation by NMR and MicroED. Large-scale study of complex metabolomes such as that of the model plant legume Medicago truncatula can be achieved using an integrated UHPLC-MS/MS-SPE-NMR metabolomics platform. Additionally, recent developments in MicroED to study structures of small organic molecules have enabled faster, easier and precise structure determinations of metabolites. A MicroED small molecule structure elucidation workflow (e.g., crystal screening, sample preparation, data collection and data processing/structure determination) has been described. Ongoing MicroED methods development and its future scope related to structure elucidation of specialized metabolites and metabolomics are highlighted. The incorporation of MicroED with a UHPLC-MS/MS-SPE-NMR instrumental ensemble offers the potential to accelerate and achieve higher rates of metabolite identification.

Structural characterization of small molecules is a crucial component of organic synthesis. In this work, we applied microcrystal electron diffraction (MicroED) to analyze the structure of the product of an enzymatic reaction that was intended to produce the unnatural amino acid 2,4-dihydroxyphenylalanine (24DHF). Characterization of our isolated product with nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) suggested that an isomer of 24DHF had been formed. Microcrystals present in the isolated product were then used to determine its structure to 0.62 Å resolution, which confirmed its identity as 2-amino-2-(2,4-dihydroxyphenyl)propanoic acid (24DHPA). Moreover, the MicroED structural model indicated that both enantiomeric forms of 24DHPA were present in the asymmetric unit. Notably, the entire structure determination process including setup, data collection, and refinement was completed in ~1 h. The MicroED data not only bolstered previous results obtained using NMR and MS but also immediately provided information about the stereoisomers present in the product, which is difficult to achieve using NMR and MS alone. Our results therefore demonstrate that MicroED methods can provide useful structural information on timescales that are similar to many commonly used analytical methods and can be added to the existing suite of small molecule structure determination tools in future studies.

The PII-like protein SbtB has been identified as a regulator of SbtA, which is one of the key bicarbonate transporters in cyanobacteria. While SbtB from Synechocystis sp. PCC 6803 has previously been shown to be a trimer, a new crystal form is reported here which crystallizes in what is thought to be a non-native tetramer in the crystal, with the C-terminus in an extended conformation. The crystal structure shows the formation of an intermolecular disulfide bond at Cys94 between SbtB monomers, which may stabilize this conformation in the crystal. This motivates the need for future studies to investigate the potential role that the oxidation and reduction of these cysteines may play in the activation and/or function of SbtB.

The high-flux/low-affinity cyanobacterial bicarbonate transporter BicA is a member of sulfate permease/solute carrier 26 (SulP/SLC26) family and plays a major role in cyanobacterial inorganic carbon uptake. In order to study this important membrane protein, robust platforms for over-expression and protocols for purification are required. In this work we have optimized the expression and purification of BicA from strain Synechocystis sp. PCC 6803 (BicA6803) in Escherichia coli. It was determined that expression with C43 (DE3) Rosetta2 at 37 °C produced the highest levels of over-expressed BicA6803 relative to other strains screened, and membrane solubilization with n-dodecyl-β- d -maltopyranoside facilitated the purification of high levels of stable and homogenous BicA6803. Using these expression and purification strategies, the final yields of purified BicA were 6.5 ± 1.0 mg per liter of culture.

Microcrystal electron diffraction, or MicroED, is a method that is capable of determining structure from very small and thin 3D crystals using a transmission electron microscope. MicroED has been successfully used on microcrystalline samples, including proteins, peptides, and small organic molecules, in many cases to very high resolutions. In this work, the MicroED workflow will be briefly described and areas of future method development will be highlighted. These areas include improvements in sample preparation, data collection, and structure determination.

In 2013 we established a cryo-electron microscopy (cryo-EM) technique called microcrystal electron diffraction (MicroED). Since that time, data collection and analysis schemes have been fine-tuned, and structures for more than 40 different proteins, oligopeptides and organic molecules have been determined. Here we review the MicroED technique and place it in context with other structure-determination methods. We showcase example structures solved by MicroED and provide practical advice to prospective users.

MicroED is a method which combines cryo-EM sample preparation and instrumentation, with electron and X-ray crystallography data analysis, and it has been employed to solve many protein crystal structures at high resolution. Initially, the main doubts of this method for structure determination were the dynamic scattering of electrons, which would cause severe inaccuracies in the measured intensities. In this paper, we will review the evolution of MicroED data collection and processing, the major differences of multiple scattering effects in protein crystals and inorganic material, and the advantages of continuous rotation data collection. Additionally, because of the periodic nature of the crystalline sample, radiation doses can be kept significantly lower than those used in single particle data collection. We review the work where this was used to assess the radiation damage of a high-energy electron beam on the protein molecules at much lower dose ranges compared to imaging.

The collection and processing of MicroED data are presented., MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals. A major limiting factor in X-ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges.

hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19–29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15–25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19–29 S20G may serve as a model for the toxic spine of hIAPP. DOI: http://dx.doi.org/10.7554/eLife.19273.001, eLife digest In Type-II Diabetes, an individual’s cells fail to respond correctly to the hormone insulin, leaving them unable to counteract high levels of sugar in the blood. Another hormone, human islet amyloid polypeptide (hIAPP), works with insulin to regulate blood sugar levels. hIAPP is an amyloid protein, which means that it can lose its normal structure and form fibrils. Fibrils are difficult for cells to break down and are often associated with disease. Indeed, fibrils of hIAPP often form in the pancreas as part of Type-II Diabetes. Some studies have shown that hIAPP fibrils are toxic to pancreatic cells and worsen the symptoms of Type-II Diabetes. Others suggest that it is the process of fibril formation that is toxic, not the fibrils themselves. Although the structures of the fibrils have been described, whether these structures cause cell toxicity has not been investigated. Krotee et al. have now explored the structures of two overlapping segments of hIAPP using a new cryo electron microscopy method called MicroED that is ideal for studying such segments. One segment, called 19-29 S20G, forms a standard amyloid fibril structure that is similar to the structure of full-length hIAPP fibrils. Adding these segments to human cells causes similar levels of toxicity as the full-length hIAPP fibrils. The second segment, called 15-25 WT, forms a non-toxic structure that is less stable than standard amyloid fibrils. The results presented by Krotee et al. support the view that standard amyloid fibril structures are toxic to cells and suggest that 19-29 S20G may be a good model to use when studying how full-length hIAPP fibrils behave. The structure of 19-29 S20G may also be useful as a template for designing molecules that block amyloid fibril growth. If amyloid fibrils cause cell toxicity in the pancreas, then these molecules could be used to treat Type-II Diabetes. DOI: http://dx.doi.org/10.7554/eLife.19273.002

Because membrane proteins are difficult to express, our understanding of their structure and function is lagging. In Escherichia coli, α-helical membrane protein biogenesis usually involves binding of a nascent transmembrane segment (TMS) by the signal recognition particle (SRP), delivery of the SRP-ribosome nascent chain complexes (RNC) to FtsY, a protein that serves as SRP receptor and docks to the SecYEG translocon, cotranslational insertion of the growing chain into the translocon, and lateral transfer, packing and folding of TMS in the lipid bilayer in a process that may involve chaperone YidC. Here, we explored the feasibility of reprogramming this pathway to improve the production of recombinant membrane proteins in exponentially growing E. coli with a focus on: (i) eliminating competition between SRP and chaperone trigger factor (TF) at the ribosome through gene deletion; (ii) improving RNC delivery to the inner membrane via SRP overexpression; and (iii) promoting substrate insertion and folding in the lipid bilayer by increasing YidC levels. Using a bitopic histidine kinase and two heptahelical rhodopsins as model systems, we show that the use of TF-deficient cells improves the yields of membrane-integrated material threefold to sevenfold relative to the wild type, and that whereas YidC coexpression is beneficial to the production of polytopic proteins, higher levels of SRP have the opposite effect. The implications of our results on the interplay of TF, SRP, YidC, and SecYEG in membrane protein biogenesis are discussed.

The signal recognition particle (SRP) dependent secretion pathway is as an attractive alternative to Sec-dependent export for the production of disulfide-bonded and/or fast-folding recombinant proteins in the Escherichia coli periplasm. SRP, which shares a ribosomal attachment site with the molecular chaperone trigger factor (TF), recognizes highly hydrophobic signal sequence as they emerge from the ribosome and delivers ribosome nascent chain complexes to FtsY for subsequent cotranslational translocation of target proteins across the SecYEG pore. However, like in the case of Sec-dependent export, secretory yields can be limited by the accumulation of precursor proteins in the cytoplasm. Using leech carboxypeptidase inhibitor (LCI) fused to the SRP-dependent DsbA signal sequence as a model system, we show that a null mutation in the gene encoding TF (Deltatig) or SRP co-expression reduce pre-LCI accumulation by half, and that quantitative export can be achieved by combining the two strategies. Interestingly, enhanced precursor processing did not alter periplasmic LCI levels but increased the amount of protein excreted in the growth medium. All mature LCI was nearly fully active and an 80% increase in productivity was achieved in Deltatig cells alone due to their faster growth. Our results show that competition between SRP and TF can interfere with efficient export of recombinant proteins targeted to the SRP pathway and establish TF-deficient strains and SRP co-expression as a simple solution to improve yields.

We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1–1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name ‘MicroED’, that may have wide applicability in structural biology. DOI: http://dx.doi.org/10.7554/eLife.01345.001, eLife digest X-ray crystallography has been used to work out the atomic structure of a large number of proteins. In a typical X-ray crystallography experiment, a beam of X-rays is directed at a protein crystal, which scatters some of the X-ray photons to produce a diffraction pattern. The crystal is then rotated through a small angle and another diffraction pattern is recorded. Finally, after this process has been repeated enough times, it is possible to work backwards from the diffraction patterns to figure out the structure of the protein. The crystals used for X-ray crystallography must be large to withstand the damage caused by repeated exposure to the X-ray beam. However, some proteins do not form crystals at all, and others only form small crystals. It is possible to overcome this problem by using extremely short pulses of X-rays, but this requires a very large number of small crystals and ultrashort X-ray pulses are only available at a handful of research centers around the world. There is, therefore, a need for other approaches that can determine the structure of proteins that only form small crystals. Electron crystallography is similar to X-ray crystallography in that a protein crystal scatters a beam to produce a diffraction pattern. However, the interactions between the electrons in the beam and the crystal are much stronger than those between the X-ray photons and the crystal. This means that meaningful amounts of data can be collected from much smaller crystals. However, it is normally only possible to collect one diffraction pattern from each crystal because of beam induced damage. Researchers have developed methods to merge the diffraction patterns produced by hundreds of small crystals, but to date these techniques have only worked with very thin two-dimensional crystals that contain only one layer of the protein of interest. Now Shi et al. report a new approach to electron crystallography that works with very small three-dimensional crystals. Called MicroED, this technique involves placing the crystal in a transmission electron cryo-microscope, which is a fairly standard piece of equipment in many laboratories. The normal ‘low-dose’ electron beam in one of these microscopes would normally damage the crystal after a single diffraction pattern had been collected. However, Shi et al. realized that it was possible to obtain diffraction patterns without severely damaging the crystal if they dramatically reduced the normal low-dose electron beam. By reducing the electron dose by a factor of 200, it was possible to collect up to 90 diffraction patterns from the same, very small, three-dimensional crystal, and then—similar to what happens in X-ray crystallography—work backwards to figure out the structure of the protein. Shi et al. demonstrated the feasibility of the MicroED approach by using it to determine the structure of lysozyme, which is widely used as a test protein in crystallography, with a resolution of 2.9 Å. This proof-of principle study paves the way for crystallographers to study protein that cannot be studied with existing techniques. DOI: http://dx.doi.org/10.7554/eLife.01345.002

Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three-dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X-ray crystallography. Electron crystallography employs two-dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well-ordered two-dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well-ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution.

The synthesis of crystalline one-dimensional polymers provides a fundamental understanding about the structure??? property relationship in polymeric materials and allows the preparation of materials with enhanced thermal, mechanical, and conducting properties. However, the synthesis of crystalline one-dimensional polymers remains a challenge because polymers tend to adopt amorphous or semicrystalline phases. Herein, we report the synthesis of a crystalline one-dimensional polymer in solution by dynamic covalent chemistry. The structure of the polymer has been unambiguously confirmed by microcrystal electron diffraction that together with charge transport studies and theoretical calculations show how the ir-stacked chains of the polymer generate optimal channels for charge transport. This work was carried out with support from the Basque Science Foundation for Science (Ikerbasque) , POLYMAT, the University of the Basque Country, Diputaci?n de Guipu ? zcoa, Gobierno Vasco (PIBA_2022_1_0031 and BERC program) , Generalitat Valenciana (PROMETEU/2021/054) and Go- bierno de Espan ? a (Projects CEX2020-001067-M, PID2021- 124484OB-I00,PID2020-118117RB-I00 and CEX2019- 0 0 0 9 1 9-M financed by MCIN/AEI/10.13039/501100011033) . Project (PCI2022-132921) funded by the Agencia Estatal de Investigaci?n through the PCI 2022 and M- ERA.NET 2021 calls. Technical and human support provided by SGIker of UPV/EHU and European funding (ERDF and ESF) is acknowledged. This project has received funding from the European Research Council (ERC) under the European Union?s Horizon 2020 research and innovation program (Grant Agreement No. 722951 and 714122) . This project has received funding from the European Union?s Horizon 2020 research and innovation program under grant agreement No. 899895. This work was funded by the ? uropean Union under the Horizon Europe grant 101046231. This project has received support from the National Institutes of Health (R21GM135784) and the use of the Titan Krios within the Eyring Materials Center at Arizona State University (NSF DBI 1531991) . Support through project IF/00894/2015 and within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020, financed by national funds through the FCT/MEC (PID- DAC) , is gratefully acknowledged. We thank the Nanoscale and Microscale Research Centre (nmRC) , University of Nottingham, for access to EM instrumentation. N.M.P. thanks La Caixa Foundation for a Postdoctoral Junior Leader - Retaining Fellowship (ID 100010434 and fellowship code LCF/BQ/PR20/11770014) .