Biology subjects

Gonen, T.

Publications and source records attributed to Gonen, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Electron-counting in MicroED

The combination of high sensitivity and rapid readout makes it possible for electron-counting detectors to record cryogenic electron microscopy data faster and more accurately without increasing the exposure. This is especially useful for MicroED of macromolecular crystals where the strength of the diffracted signal at high resolution is comparable to the surrounding background. The ability to decrease the exposure also alleviates concerns about radiation damage which limits the information that can be recovered from a diffraction measurement. However, the dynamic range of electron-counting detectors requires careful data collection to avoid errors from coincidence loss. Nevertheless, these detectors are increasingly deployed in cryo-EM facilities, and several have been successfully used for MicroED. Provided coincidence loss can be minimized, electron-counting detectors bring high potential rewards.

biochemistry↗

bioRxiv2023

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 ({beta}3AR) 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.

biochemistry↗

bioRxiv2023

Improving the solubility of pseudo-hydrophobic Alzheimer's Disease medicinal chemicals through co-crystal formulation

Natural products are ligands and potential inhibitors of Alzheimers disease (AD) tau. Dihydromyricetin (DHM) is a CNS active natural product. Despite having signature polyphenolic character, DHM is ostensibly hydrophobic owing to intermolecular hydrogen bonds that shield hydrophilic phenols. Our research shows DHM becomes ionized at near-neutral pH allowing formulation of salts with transformed solubility. The MicroED co-crystal structure with trolamine reveals DHM salts as metastable solids with unlocked hydrogen bonding and a thermodynamic bent to solubilize in water. All salt formulations show better inhibitory activity against AD tau than the non-salt form, with efficacies correlating to enhanced solubilities. These results underscore the role of structural chemistry in guiding selection of solubilizing agents for chemical formulation. We propose DHM salts are appropriate formulations for research as dietary supplements to promote healthy aging by combating protein misfolding. Additionally, DHM is a suitable lead for medicinal chemistry and possible development of CNS pharmaceuticals.

pharmacology and toxicology↗

bioRxiv2023

Structure determination of a DNA crystal by MicroED

Microcrystal electron diffraction (MicroED) is a powerful tool for determining high-resolution structures of microcrystals from a diverse array of biomolecular, chemical, and material samples. In this study, we apply MicroED to DNA crystals, which have not been previously analyzed using this technique. We utilized the d(CGCGCG)2 DNA duplex as a model sample and employed cryo-FIB milling to create thin lamella for diffraction data collection. The MicroED data collection and subsequent processing resulted in a 1.10 [A] resolution structure of the d(CGCGCG)2 DNA, demonstrating the successful application of cryo-FIB milling and MicroED to the investigation of nucleic acid crystals.

biochemistry↗

bioRxiv2023

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.

biochemistry↗

bioRxiv2023

Design and implementation of suspended drop crystallization

We have developed a novel crystal growth method known as suspended drop crystallization. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber which we designed, allowing for vapor diffusion to occur from both sides of the drop. A UV transparent window above and below the grid enables the monitoring of crystal growth via light, UV, or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for x-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, we grew crystals of the enzyme proteinase K and determined its structure by MicroED following FIB/SEM milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress, and/or suffering from preferred orientation on EM grids.

biochemistry↗

bioRxiv2022

MicroED structure of a protoglobin reactive carbene intermediate

Microcrystal electron diffraction (MicroED) is an emerging technique which has shown great potential for describing new chemical and biological molecular structures. [1] Several important structures of small molecules, natural products and peptides have been determined using ab initio methods. [2] However, only a couple of novel protein structures have thus far been derived by MicroED. [3, 4] 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 alphahelix of ApePgb into a dynamic loop making the catalytic active site more readily accessible. After exposing the tiny crystals to substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineered ApePgbs 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 formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enables investigation of structures that were previously beyond reach.

biochemistry↗

bioRxiv2022

A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals

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.

biochemistry↗

bioRxiv2022

Electron-counting MicroED data with the K2 and K3 direct electron detectors

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 [A] and 2.8 [A]. 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.

biochemistry↗

bioRxiv2022

Lipid flipping in the omega-3 fatty-acid transporter

Mfsd2a is the primary transporter for the docosahexaenoic acid (DHA), an omega-3 fatty acid, across the blood brain barrier (BBB). Defects in Mfsd2a are linked to ailments from behavioral, learning, and motor dysfunctions to severe microcephaly. Mfsd2a typically transports long-chain unsaturated fatty-acids, including DHA and -Linolenic acid (ALA), that are attached to the zwitterionic lysophosphatidylcholine (LPC) headgroup. Even with two recently determined structures of Mfsd2a the molecular details of how this transporter performs the energetically unfavorable task of translocating and flipping lysolipids across the lipid bilayer remained unclear. Here, we report five single-particle cryo-EM structures of the Danio rerio Mfsd2a (drMfsd2a): in the inward-open conformation in the ligand-free state and bound to ALA-LPC at four unique positions along the substrate translocation pathway. These Mfsd2a snapshots detail the Na+-dependent flipping mechanism of the lipid-LPC from outer to inner membrane leaflet during ligand translocation through the Mfsd2a substrate tunnel and release for membrane integration on the cytoplasmic side. These results also map Mfsd2a mutants that disrupt lipid-LPC transport and are associated with known disease. Together these results provide a model for omega-3 fatty-acid transport and has the potential for the design of the delivery strategies for amphipathic drugs across the BBB.

biophysics↗

bioRxiv2022

Hydrogens and hydrogen-bond networks in macromolecular MicroED data

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.

biochemistry↗

bioRxiv2021

Ab initio phasing macromolecular structures using electron-counted MicroED data

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 [A] 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 [A] resolution. These results demonstrate that macromolecules can be determined to sub-[A]ngstrom resolution by MicroED and that ab initio phasing can be successfully applied to counting data collected on a direct electron detector.

biochemistry↗

bioRxiv2021

Benchmarking ideal sample thickness in cryo-EM using 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.

biochemistry↗

bioRxiv2020

MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP

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 [A] 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

biophysics↗

bioRxiv2020

MicroED structure of lipid-embedded mammalian mitochondrial voltage dependent anion channel

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.

biophysics↗

bioRxiv2020

Efficient, high-throughput ligand incorporation into protein microcrystals by on-grid soaking

A method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoEM 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 200nm using a focused ion-beam, and microcrystal electron diffraction (MicroED) data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Compared to much larger crystals investigated by X-ray crystallography, both the number of ligands bound and their occupancy was higher in MicroED. These results indicate that soaking ligands into microcrystals in this way results in a more efficient uptake than in larger crystals that are typically used in drug discovery pipelines by X-ray crystallography.

biochemistry↗

bioRxiv2019

Experimental phasing of MicroED data using radiation damage

We present an example of an experimentally phased structure using only MicroED. The structure of a seven-residue peptide is determined starting from differences to the diffraction intensities induced by structural changes due to radiation damage. The same wedge of reciprocal space was recorded twice by continuous rotation MicroED from a set of 11 individual crystals. The data from the first pass were merged to make a \"low-dose dataset\". The data from the second pass were similarly merged to form a \"damaged dataset.\" Differences between these two datasets were used to calculate a Patterson difference map and to identify a single heavy atom site, from which initial phases were generated. The structure was then completed by iterative cycles of modeling and refinement.

biophysics↗

bioRxiv2019

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometer to micron scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges not encountered in traditional macromolecular crystallography experiments. Here, we describe XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A (CypA). Our results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample preparation and delivery methods required for each type of experiment effect the crystal structure of the enzyme.

biophysics↗