scholarly journals Ab initio phasing macromolecular structures using electron-counted MicroED data

2021 ◽  
Author(s):  
Michael Williams Martynowycz ◽  
Max T.B. Clabbers ◽  
Johan Hattne ◽  
Tamir Gonen
Keyword(s):  
Ab Initio ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Proteinase K ◽  
Exposure Rate ◽  
Electron Detector ◽  
Counting Data ◽  
Continuous Rotation ◽  
Counting Mode ◽  
Low Exposure

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-Angstrom resolution by MicroED and that ab initio phasing can be successfully applied to counting data collected on a direct electron detector.

Novel Approach of Improving Secondary Electron Detector in FIB System

2018 ◽  
Author(s):  
Steve Wang ◽  
Jim McGinn ◽  
Peter Tvarozek ◽  
Amir Weiss
Keyword(s):  
Integrated Circuit ◽  
Secondary Electron ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Vital Role ◽  
Electron Detector ◽  
System A ◽  
Novel Approach

Abstract Secondary electron detector (SED) plays a vital role in a focused ion beam (FIB) system. A successful circuit edit requires a good effective detector. Novel approach is presented in this paper to improve the performance of such a detector, making circuit altering for the most advanced integrated circuit (IC) possible.

Method for Cross-sectional Thin Specimen Preparation from a Specific Site Using a Combination of a Focused Ion Beam System and Intermediate Voltage Electron Microscope and Its Application to the Characterization of a Precipitate in a Steel

2001 ◽  
Vol 7 (3) ◽  
pp. 287-291
Author(s):  
Toshie Yaguchi ◽  
Hiroaki Matsumoto ◽  
Takeo Kamino ◽  
Tohru Ishitani ◽  
Ryoichi Urao
Keyword(s):  
Electron Microscope ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Specific Site ◽  
Specimen Preparation ◽  
Thin Specimen ◽  
Cross Sectional ◽  
Electron Detector ◽  
Specimen Holder

AbstractIn this study, we discuss a method for cross-sectional thin specimen preparation from a specific site using a combination of a focused ion beam (FIB) system and an intermediate voltage transmission electron microscope (TEM). A FIB-TEM compatible specimen holder was newly developed for the method. The thinning of the specimen using the FIB system and the observation of inside structure of the ion milled area in a TEM to localize a specific site were alternately carried out. The TEM fitted with both scanning transmitted electron detector and secondary electron detector enabled us to localize the specific site in a halfway milled specimen with the positional accuracy of better than 0.1 µm. The method was applied to the characterization of a precipitate in a steel. A submicron large precipitate was thinned exactly at its center for the characterization by a high-resolution electron microscopy and an elemental mapping.

scholarly journals Collection of continuous rotation MicroED Data from Ion Beam Milled Crystals of Any Size

2018 ◽  
Author(s):  
Michael W. Martynowycz ◽  
Wei Zhao ◽  
Johan Hattne ◽  
Grant J. Jensen ◽  
Tamir Gonen
Keyword(s):  
Data Collection ◽  
Sample Preparation ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Air Interface ◽  
Structure Solution ◽  
Continuous Rotation ◽  
Macromolecular Protein ◽  
Subsequent Sample

AbstractMicrocrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoEM as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to ideal dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.

scholarly journals A Workflow for Protein Structure Determination from Thin Crystal Lamella by Micro-Electron Diffraction

2020 ◽  
Author(s):  
Emma V. Beale ◽  
David G. Waterman ◽  
Corey Hecksel ◽  
Jason van Rooyen ◽  
James B. Gilchrist ◽  
...  
Keyword(s):  
Electron Diffraction ◽  
Data Processing ◽  
Structure Determination ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Protein Structure Determination ◽  
Proteinase K ◽  
Protein Crystal ◽  
Electron Diffraction Data ◽  
X Ray Crystallography

AbstractMicro-Electron Diffraction (MicroED) has recently emerged as a powerful method for the analysis of biological structures at atomic resolution. This technique has been largely limited to protein nanocrystals which grow either as needles or plates measuring only a few hundred nanometres in thickness. Furthermore, traditional microED data processing uses established X-ray crystallography software that is not optimised for handling compound effects that are unique to electron diffraction data. Here, we present an integrated workflow for microED, from sample preparation by cryo-focused ion beam milling, through data collection with a standard Ceta-D detector, to data processing using the DIALS software suite, thus enabling routine atomic structure determination of protein crystals of any size and shape using microED. We demonstrate the effectiveness of the workflow by determining the structure of proteinase K to 2.0 Å resolution and show the advantage of using protein crystal lamellae over nanocrystals.

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

2020 ◽  
Author(s):  
Michael W. Martynowycz ◽  
Tamir Gonen
Keyword(s):  
High Resolution ◽  
Electron Diffraction ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Proteinase K ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Resolution Structure ◽  
Efficient Uptake ◽  
Resolution Structure

AbstractA 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.

scholarly journals Benchmarking ideal sample thickness in cryo-EM using MicroED

2021 ◽  
Author(s):  
Michael W. Martynowycz ◽  
Max T. B. Clabbers ◽  
Johan Unge ◽  
Johan Hattne ◽  
Tamir Gonen
Keyword(s):  
Free Path ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Mean Free Path ◽  
Sample Thickness ◽  
Proteinase K ◽  
Specimen Thickness ◽  
Quality Of Data ◽  
Inelastic Mean Free Path

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.

Nanocomposite Polyurethane Foams Via POSS Blends

2002 ◽  
Vol 733 ◽  
Author(s):  
Brock McCabe ◽  
Steven Nutt ◽  
Brent Viers ◽  
Tim Haddad
Keyword(s):  
High Temperature ◽  
Sample Preparation ◽  
Room Temperature ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Polyurethane Foams ◽  
Development Projects ◽  
Polymer Systems ◽  
Polyurethane Resin ◽  
Military Development

AbstractPolyhedral Oligomeric Silsequioxane molecules have been incorporated into a commercial polyurethane formulation to produce nanocomposite polyurethane foam. This tiny POSS silica molecule has been used successfully to enhance the performance of polymer systems using co-polymerization and blend strategies. In our investigation, we chose a high-temperature MDI Polyurethane resin foam currently used in military development projects. For the nanofiller, or “blend”, Cp7T7(OH)3 POSS was chosen. Structural characterization was accomplished by TEM and SEM to determine POSS dispersion and cell morphology, respectively. Thermal behavior was investigated by TGA. Two methods of TEM sample preparation were employed, Focused Ion Beam and Ultramicrotomy (room temperature).

An Study of Influence of Imperfections on the Delamination of Diamond-Like Carbon Films

2002 ◽  
Vol 719 ◽  
Author(s):  
Myoung-Woon Moon ◽  
Kyang-Ryel Lee ◽  
Jin-Won Chung ◽  
Kyu Hwan Oh
Keyword(s):  
Cross Sections ◽  
Focused Ion Beam ◽  
Imaging System ◽  
Ion Beam ◽  
Carbon Films ◽  
Diamond Like Carbon ◽  
Glass Substrates ◽  
Atomic Force ◽  
Initiation And Propagation

AbstractThe role of imperfections on the initiation and propagation of interface delaminations in compressed thin films has been analyzed using experiments with diamond-like carbon (DLC) films deposited onto glass substrates. The surface topologies and interface separations have been characterized by using the Atomic Force Microscope (AFM) and the Focused Ion Beam (FIB) imaging system. The lengths and amplitudes of numerous imperfections have been measured by AFM and the interface separations characterized on cross sections made with the FIB. Chemical analysis of several sites, performed using Auger Electron Spectroscopy (AES), has revealed the origin of the imperfections. The incidence of buckles has been correlated with the imperfection length.

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