scholarly journals High-yield monolayer graphene grids for near-atomic resolution cryoelectron microscopy

2019 ◽  
Vol 117 (2) ◽  
pp. 1009-1014 ◽  
Author(s):  
Yimo Han ◽  
Xiao Fan ◽  
Haozhe Wang ◽  
Fang Zhao ◽  
Christopher G. Tully ◽  
...  
Keyword(s):  
Structural Biology ◽  
Structural Investigation ◽  
High Yield ◽  
Monolayer Graphene ◽  
Minimal Amount ◽  
Biological Macromolecules ◽  
Atomic Structures ◽  
Specific Proteins ◽  
Protein Density ◽  
Working Mechanisms

Cryogenic electron microscopy (cryo-EM) has become one of the most powerful techniques to reveal the atomic structures and working mechanisms of biological macromolecules. New designs of the cryo-EM grids—aimed at preserving thin, uniform vitrified ice and improving protein adsorption—have been considered a promising approach to achieving higher resolution with the minimal amount of materials and data. Here, we describe a method for preparing graphene cryo-EM grids with up to 99% monolayer graphene coverage that allows for more than 70% grid squares for effective data acquisition with improved image quality and protein density. Using our graphene grids, we have achieved 2.6-Å resolution for streptavidin, with a molecular weight of 52 kDa, from 11,000 particles. Our graphene grids increase the density of examined soluble, membrane, and lipoproteins by at least 5-fold, affording the opportunity for structural investigation of challenging proteins which cannot be produced in large quantity. In addition, our method employs only simple tools that most structural biology laboratories can access. Moreover, this approach supports customized grid designs targeting specific proteins, owing to its broad compatibility with a variety of nanomaterials.

scholarly journals High Yield Monolayer Graphene Grids for Near-Atomic Resolution Cryo-Electron Microscopy

2019 ◽  
Author(s):  
Yimo Han ◽  
Xiao Fan ◽  
Haozhe Wang ◽  
Fang Zhao ◽  
Christopher G. Tully ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Sample Preparation ◽  
Three Dimensional ◽  
Atomic Resolution ◽  
High Yield ◽  
Monolayer Graphene ◽  
Minimal Amount ◽  
Biological Macromolecules ◽  
Atomic Structures ◽  
Protein Density

AbstractCryogenic electron microscopy (cryo-EM) has become one of the most powerful techniques to reveal the atomic structures and working mechanisms of biological macromolecules. New designs of the cryo-EM grids—aimed at preserving thin, uniform vitrified ice and improving protein adsorption—have been considered a promising approach to achieving higher resolution with the minimal amount of materials and data. Here, we describe a method for preparing graphene cryo-EM grids with 99% monolayer graphene coverage that allows for more than 70% grid squares for effective data acquisition with improved image quality and protein density. Using our graphene grids, we have achieved 2.6 Å resolution for streptavidin, with a molecular weight of 52 kDa, from 11,000 particles. Our graphene grids increase the density of examined soluble, membrane, and lipo-proteins by at least five times, affording the opportunity for structural investigation of challenging proteins which cannot be produced in large quantity. In addition, our method employs only simple tools that most structural biology laboratories can access. Moreover, our approach allows for customized grid designs targeting specific proteins, due to its broad compatibility with a variety of nanomaterials.Significance statementSingle particle cryogenic electron microscopy (cryo-EM) represents the cutting-edge technology to determine three-dimensional atomic structures of bio-macromolecules. However, issues of cryo-sample preparation limit the cryo-EM to achieve higher resolution. Here, we demonstrated a high yield, monolayer graphene supporting film to improve the cryo-sample quality. Using our approach, we have achieved so far, the highest resolution structure of the smallest protein by cryo-EM with the minimal number of datasets. Our technique paves the way for universal cryo-sample preparation for near-atomic resolution cryo-EM.

Cryo-EM Is a Powerful Tool, But Helical Applications Can Have Pitfalls

Soft Matter ◽  
2021 ◽  
Author(s):  
Edward Egelman ◽  
Fengbin Wang
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Macromolecular Complexes ◽  
Atomic Structures ◽  
Cryo Electron Microscopy ◽  
Main Technique

In structural biology, cryo-electron microscopy (cryo-EM) has emerged as the main technique for determining the atomic structures of macromolecular complexes. This has largely been due to the introduction of direct...

scholarly journals Protein dynamics revealed by NMR relaxation methods

2018 ◽  
Vol 2 (1) ◽  
pp. 93-105 ◽  
Author(s):  
Fa-An Chao ◽  
R. Andrew Byrd
Keyword(s):  
Protein Dynamics ◽  
Structural Biology ◽  
Dynamic Models ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Data Bank ◽  
Biological Macromolecules ◽  
Relaxation Methods ◽  
Genomic Databases ◽  
Wide Range

Structural biology often focuses primarily on three-dimensional structures of biological macromolecules, deposited in the Protein Data Bank (PDB). This resource is a remarkable entity for the worldwide scientific and medical communities, as well as the general public, as it is a growing translation into three-dimensional space of the vast information in genomic databases, e.g. GENBANK. There is, however, significantly more to understanding biological function than the three-dimensional co-ordinate space for ground-state structures of biomolecules. The vast array of biomolecules experiences natural dynamics, interconversion between multiple conformational states, and molecular recognition and allosteric events that play out on timescales ranging from picoseconds to seconds. This wide range of timescales demands ingenious and sophisticated experimental tools to sample and interpret these motions, thus enabling clearer insights into functional annotation of the PDB. NMR spectroscopy is unique in its ability to sample this range of timescales at atomic resolution and in physiologically relevant conditions using spin relaxation methods. The field is constantly expanding to provide new creative experiments, to yield more detailed coverage of timescales, and to broaden the power of interpretation and analysis methods. This review highlights the current state of the methodology and examines the extension of analysis tools for more complex experiments and dynamic models. The future for understanding protein dynamics is bright, and these extended tools bring greater compatibility with developments in computational molecular dynamics, all of which will further our understanding of biological molecular functions. These facets place NMR as a key component in integrated structural biology.

scholarly journals VIRTUAL LABORATORY MOLDYNGRID AS A PART OF SCIENTIFIC INFRASTRUCTURE FOR BIOMOLECULAR SIMULATIONS

2010 ◽  
pp. 295-301
Author(s):  
Andrii Salnikov ◽  
Ievgen Sliusar ◽  
Oleksandr Sudakov ◽  
Oleksandr Savytskyi ◽  
Alexander Kornelyuk
Keyword(s):  
Structural Biology ◽  
Md Simulations ◽  
Virtual Laboratory ◽  
Biological Macromolecules ◽  
Grid Infrastructure ◽  
Processing Power ◽  
Biomolecular Simulations ◽  
High Processing ◽  
Scientific Infrastructure ◽  
Very High

Virtual laboratory MolDynGrid has been established for computational researches in structural biology and bioinformatics, especially for molecular dynamics (MD) simulations of biological macromolecules and their complexes. Biomolecular simulation usually requires a very high processing power and huge storage space for MD trajectories. In this study we describe the development of virtual laboratory infrastructure to run massive MD simulations on a Grid infrastructure in a distributed manner. The aim of MolDynGrid development was to provide an efficient infrastructure for automation of MD job processing in Grid. It will assist researchers to use grid technology without having them to be familiar with highly complicated internals of the Grid. Web-portal of MolDynGrid virtual laboratory has been developed (http://moldyngrid.org), which consists of computational, analytical and educational blocks аnd data base of MD simulations.

scholarly journals Structural investigation of cyclic nucleotide binding proteins from Trypanosoma cruzi

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Gabriel Ferri ◽  
Martin Edreira ◽  
Ivan Campeotto
Keyword(s):  
Life Cycle ◽  
Structural Biology ◽  
Structural Investigation ◽  
Buenos Aires ◽  
Therapeutic Agents ◽  
Parasite Life Cycle ◽  
Structure Based Drug Design ◽  
Antiparasitic Drugs ◽  
Nucleotide Binding Proteins ◽  
Biology Group

Fora targeted therapy of Trypanosomiasis, new antiparasitic drugs should be specifically directed against essential pathways in the parasite life cycle. Among these potential targets are signal transduction pathways, which have remained largely unexplored in Trypanosoma species. Of special interest is cAMP-mediated signaling, since cAMP has been shown to play critical roles in the life cycle of T. cruzi and in host cell during invasion. The presented research focuses on the identification and characterisation of novel cAMP response proteins (CARPs) in T. cruzi by using a multi disciplinary approach involving the parasitology group of Dr Martin Edreira (University of Buenos Aires, Argentina) and the structural biology group of Dr Ivan Campeotto (University of Leicester, UK). The aim of the project is not only to increase our knowledge about T. cruzi biology but also to target CARPs for the design and development of novel therapeutic agents against Chagas disease. To date, protein crystals of one of the members of the CARP family have been obtained, paving the way for structure determination and for a structure-based drug design approach.

scholarly journals Illuminating the Secrets of Crystals - Microcrystal Electron Diffraction in Structural Biology

2018 ◽  
Author(s):  
Rob Barringer ◽  
Thomas Meier
Keyword(s):  
Electron Diffraction ◽  
Structural Biology ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Novel Method ◽  
Practical Level ◽  
Crystallographic Theory

An exploration of the crystallographic theory of the relatively novel method of Microcrystal Electron Diffraction (MicroED), via comparison to X-ray crystallography at the theoretical and practical level as it applies to biological macromolecules. We then attempt to outline the limitations and challenges that the technique currently faces in structural biology, and suggest future areas of study that may improve and optimize the technique.

scholarly journals Exfoliation Behavior of Large Anionic Graphite Flakes in Liquid Produced by Salt-Assisted Ball Milling

Processes ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 28 ◽  
Author(s):  
Yoshihiko Arao ◽  
Jonathon D. Tanks ◽  
Kojiro Aida ◽  
Masatoshi Kubouchi
Keyword(s):  
Ball Milling ◽  
Polar Solvent ◽  
High Yield ◽  
Monolayer Graphene ◽  
Methyl Ethyl ◽  
Natural Graphite ◽  
Solvent Type ◽  
Liquid Phase Exfoliation ◽  
Average Size

Functionalization of graphite is crucial for efficient and effective exfoliation to graphene. When negative charges are fixed to the edges of natural graphite, the resulting anionic graphite shows negative charging in a polar solvent. This enhanced negative charging is assumed to contribute the exfoliation of graphite during liquid-phase exfoliation (LPE). In this study, we prepared large anionic graphite flakes (~10 μm) by salt-assisted ball milling, as well as natural graphite flakes of the same size for comparison. During the LPE process, centrifugation speed and solvent type have dominant effects on graphene concentration and quality (e.g., size and thickness), so we investigated these factors for anionic graphite flakes in detail. The anionic graphite showed higher exfoliation efficiency in every type of solvent (isopropanol, methyl ethyl ketone, acetone, and water-based cosolvent) compared with the natural graphite. Monolayer graphene, with an average size of 80–200 nm, was obtained with relatively high yield (>10%) at only 3 min of sonication. The small size of graphene was due to edge fragmentation during the LPE process. The recyclability of the sediment and the characterization of the exfoliated powders for anionic graphene were also investigated.

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