MicroED Sample Preparation and Data Collection For Protein Crystals

Bu, Guanhong; Nannenga, Brent L.

doi:10.1007/978-1-0716-0966-8_13

Cited by 7 publications

(3 citation statements)

References 37 publications

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“…To expedite the process of cryo-FIB milling, the large DNA crystals were initially broken into smaller fragments by pipetting them vigorously within the drops (de la Cruz et al, 2017). These fragments were promptly placed on holey carbon EM grids and vitrified using conventional MicroED sample preparation techniques (Bu and Nannenga, 2021).…”

Section: Resultsmentioning

confidence: 99%

Structure determination of a DNA crystal by MicroED

Haymaker

Bardin

Gonen

et al. 2023

Preprint

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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 angstrom 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.

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Section: Resultsmentioning

confidence: 99%

Structure determination of a DNA crystal by MicroED

Haymaker

Bardin

Gonen

et al. 2023

Preprint

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“…The MicroED sample preparation, data collection, and data processing pipeline for proteins as well as small molecules has been described previously ( Shi et al, 2016 ; Bu and Nannenga, 2021 ; de la Cruz, 2021 ; Martynowycz and Gonen, 2021 ; Zee et al, 2021 ). A MicroED structure elucidation workflow for natural products and specialized targeted metabolites has been optimized from previously published methods ( Figure 2 ).…”

Section: Microed Structure Analysis Workflowmentioning

confidence: 99%

Recent Developments Toward Integrated Metabolomics Technologies (UHPLC-MS-SPE-NMR and MicroED) for Higher-Throughput Confident Metabolite Identifications

Ghosh

Nannenga

et al. 2021

Front. Mol. Biosci.

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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.

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“…In this study, we have determined the first experimental structure of Clostridium histolyticum Clostripain in its active state at 3.6 Å resolution by the cryogenic electron microscope (Cryo-EM) method, microcrystal electron diffraction (MicroED). MicroED is a robust method used to determine structures of different samples including small molecules, peptides, natural products and proteins using vanishingly small crystals (Bu and Nannenga, 2021; Mu et al, 2021; Nannenga and Gonen, 2019; Nannenga et al, 2014; Shi et al, 2016). The structure consists of a typical Clan CD α/β/α sandwich architecture and the Cys231/His176 catalytic dyad in the active site.…”

Section: Introductionmentioning

confidence: 99%

MicroED structure of the C11 cysteine protease Clostripain

Ruma,

Bu,

Gonen

2024

Preprint

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Clostripain secreted from Clostridium histolyticum is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of C. histolyticum Clostripain determined at 3.6 Angstrom resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of Clostripain shows a typical Clan CD α/β/α; sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 to 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as Clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.

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MicroED Sample Preparation and Data Collection For Protein Crystals

Cited by 7 publications

References 37 publications

Structure determination of a DNA crystal by MicroED

Structure determination of a DNA crystal by MicroED

Recent Developments Toward Integrated Metabolomics Technologies (UHPLC-MS-SPE-NMR and MicroED) for Higher-Throughput Confident Metabolite Identifications

MicroED structure of the C11 cysteine protease Clostripain

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