Protocol for the use of focused ion-beam milling to prepare crystalline lamellae for microcrystal electron diffraction (MicroED)

Martynowycz, Michael W.; Gonen, Tamir

doi:10.1016/j.xpro.2021.100686

Cited by 17 publications

(14 citation statements)

References 31 publications

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“…In cases where a suitably inert drop-casting solvent is unavailable, simply grinding dry powder between two glass coverslips can achieve an analogous effect via shear force. Alternatively, focused ion-beam (FIB) milling can shave excessively thick crystals down to thin electron-transparent lamellae with precision. − Although quite powerful, FIB milling requires usage of specialized ancillary equipment (a scanning electron microscope), as well as multiple cumbersome cryo-transfer steps if dealing with vitrified samples.…”

Section: Methodsmentioning

confidence: 99%

Electron Diffraction of 3D Molecular Crystals

2022

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Electron crystallography has a storied history which rivals that of its more established X-ray-enabled counterpart. Recent advances in data collection and analysis have sparked a renaissance in the field, opening a new chapter for this venerable technique. Burgeoning interest in electron crystallography has spawned innovative methods described by various interchangeable labels (3D ED, MicroED, cRED, etc.). This Review covers concepts and findings relevant to the practicing crystallographer, with an emphasis on experiments aimed at using electron diffraction to elucidate the atomic structure of threedimensional molecular crystals. CONTENTS 1. Introduction and Historical Background 13883 2. Theoretical Foundations 13884 2.1. Differences Between X-ray and Electron Scattering 13884 2.2. Differences Between X-ray and Electron Wavelengths 13886 2.3.

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Section: Methodsmentioning

confidence: 99%

Electron Diffraction of 3D Molecular Crystals

2022

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“…Even at low flux, the ion-beam can damage the sample during imaging (Zhou et al ., 2019). Milling is typically conducted at much higher beam currents than imaging (Schaffer et al ., 2017; Beale et al ., 2020; Martynowycz & Gonen, 2021). For this purpose, it has become a routine to protect the samples by coating the specimens in a thick layer of platinum using a GIS.…”

Section: Methodsmentioning

confidence: 99%

“…Grids were made from this sponge phase mixture and blotted using standard protocols. The microcrystals in the blotted sponge phase grids were visible by FIB/SEM and could be thinned by the gallium beam and subsequently determined by MicroED (Martynowycz & Gonen, 2021). In this work, the looped crystals could be kept hydrated using a humidifier, but resulted in thick layers of ice on the grids, and no crystals could be identified in the FIB/SEM.…”

Section: Mainmentioning

confidence: 99%

“…Even at low flux, the ion-beam can damage the sample during imaging and milling (Zhou et al ., 2019). Milling is typically conducted at much higher beam currents than imaging (Schaffer et al ., 2017; Beale et al .,2020; Martynowycz & Gonen, 2021). Although milling is contained to a defined region, the beam is usually much larger than the defined area milled.…”

Section: Mainmentioning

confidence: 99%

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

Martynowycz

Shiriaeva

Clabbers

et al. 2022

Preprint

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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.

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“…Analyzing the response of biologically active compounds at the single cell level can also accelerate the discovery of NP drugs. In order to accurately determine the structure of small molecules, a microcrystal electron diffraction (MicroED) based on cryo-electron microscopy has recently been developed [80]. Biology phenotype chip, microplate high-throughput screening, microfluidics, fluorescence activated droplet sorting system (FADS), Raman light spectrum, Fourier transform infrared spectroscopy or near-infrared spectroscopy and advanced spectral sensor have also been used for strain screening and phenotyping profiling [81,82].…”

Section: The Test Stage For Natural Product Discoverymentioning

confidence: 99%

Synthetic Biology Advanced Natural Product Discovery

2021

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A wide variety of bacteria, fungi and plants can produce bioactive secondary metabolites, which are often referred to as natural products. With the rapid development of DNA sequencing technology and bioinformatics, a large number of putative biosynthetic gene clusters have been reported. However, only a limited number of natural products have been discovered, as most biosynthetic gene clusters are not expressed or are expressed at extremely low levels under conventional laboratory conditions. With the rapid development of synthetic biology, advanced genome mining and engineering strategies have been reported and they provide new opportunities for discovery of natural products. This review discusses advances in recent years that can accelerate the design, build, test, and learn (DBTL) cycle of natural product discovery, and prospects trends and key challenges for future research directions.

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Protocol for the use of focused ion-beam milling to prepare crystalline lamellae for microcrystal electron diffraction (MicroED)

Cited by 17 publications

References 31 publications

Electron Diffraction of 3D Molecular Crystals

Electron Diffraction of 3D Molecular Crystals

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

Synthetic Biology Advanced Natural Product Discovery

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