Ab Initio Determination of Peptide Structures by MicroED

Zee, Chih-Te; Saha, Ambarneil; Sawaya, M.R.; Rodríguez, José A.

doi:10.1007/978-1-0716-0966-8_17

Cited by 3 publications

(3 citation statements)

References 37 publications

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“…Cryogenic temperatures also delay the onset and progression of radiation damage, which is frequently quite severe for macromolecular crystals at RT. For proteins and oligopeptides, several step-by-step protocols detailing cryo-preservation procedures have been published. , A glow-discharged EM grid is first loaded with 2–3 μL of an aqueous suspension of protein crystals (usually immersed in mother liquor from a successful crystallization trial, such as the hanging drop in Figure C). Subsequently, the grid is blotted and rapidly plunged into a small reservoir (∼4 mL) of liquid ethane.…”

Section: Methodsmentioning

confidence: 99%

“…With minimal modification, well-established programs such as iMosflm, 108 DIALS, 109 and XDS 110 have all been successfully applied to continuousrotation 3D ED data reduction. Detailed tutorials (such as for DIALS 111 and XDS 65 ) easily comprehensible to any practicing X-ray crystallographer have subsequently appeared in the literature. Likewise, current processing pipelines for serial diffraction (such as crystFEL 112 ) have also been ported to ED data.…”

Section: Data Reductionmentioning

confidence: 99%

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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%

Section: Data Reductionmentioning

confidence: 99%

Electron Diffraction of 3D Molecular Crystals

2022

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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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Scaling up cryo-EM for biology and chemistry: The journey from niche technology to mainstream method

de la Cruz,

Eng

2023

Structure

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Ab Initio Determination of Peptide Structures by MicroED

Cited by 3 publications

References 37 publications

Electron Diffraction of 3D Molecular Crystals

Electron Diffraction of 3D Molecular Crystals

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

Scaling up cryo-EM for biology and chemistry: The journey from niche technology to mainstream method

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