scholarly journals Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids

2016 ◽  
Author(s):  
Joost Snijder ◽  
Andrew J. Borst ◽  
Annie Dosey ◽  
Alexandra C. Walls ◽  
Anika Burrell ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Particle Density ◽  
Protein Complexes ◽  
Cost Effective ◽  
Carbon Support ◽  
Cryo Electron Microscopy ◽  
Sample Application ◽  
Single Field

Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain a dense distribution of particles within a single field of view remains a major bottleneck for the success of such experiments. Here, we describe a simple and cost-effective method to increase the density of frozen-hydrated particles on grids with holey carbon support films. It relies on performing multiple rounds of sample application and blotting prior to plunge freezing in liquid ethane. We show that this approach is generally applicable and significantly increases particle density for a range of samples, such as small protein complexes, viruses and filamentous assemblies. The method is versatile, easy to implement, minimizes sample requirements and can enable characterization of samples that would otherwise resist structural studies using single particle cryoEM.

scholarly journals Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids

2017 ◽  
Vol 198 (1) ◽  
pp. 38-42 ◽  
Author(s):  
Joost Snijder ◽  
Andrew J. Borst ◽  
Annie Dosey ◽  
Alexandra C. Walls ◽  
Anika Burrell ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Particle Density ◽  
Cryo Electron Microscopy ◽  
Sample Application

Uncovering the structures of modular polyketide synthases

2015 ◽  
Vol 32 (3) ◽  
pp. 436-453 ◽  
Author(s):  
Kira J. Weissman
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Structural Characterization ◽  
Small Angle ◽  
Single Particle ◽  
Polyketide Synthases ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Microscopy

This review covers a breakthrough in the structural biology of the gigantic modular polyketide synthases (PKS): the structural characterization of intact modules by single-particle cryo-electron microscopy and small-angle X-ray scattering.

scholarly journals Structure of the Ebola virus nucleoprotein – RNA complex

2019 ◽  
Author(s):  
Robert N. Kirchdoerfer ◽  
Erica Ollmann Saphire ◽  
Andrew B. Ward
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Ebola Virus ◽  
Viral Proteins ◽  
Cryo Electron Microscopy ◽  
Emerging Virus ◽  
Deadly Disease ◽  
Rna Complex ◽  
Viral Nucleocapsid

AbstractEbola virus is an emerging virus capable of causing a deadly disease in humans. Replication, transcription and packaging of the viral genome is carried out by the viral nucleocapsid. The nucleocapsid is a complex of the viral nucleoprotein, RNA and several other viral proteins. The nucleoprotein NP forms large, RNA-bound, helical filaments and acts as a scaffold for additional viral proteins. The 3.1 Å single-particle cryo-electron microscopy structure of the nucleoprotein-RNA helical filament presented here resembles previous structures determined at lower resolution while providing improved molecular details of protein-protein and protein-RNA interactions. The higher resolution of the structure presented here will facilitate the design and characterization of novel and specific Ebola virus therapeutics targeting the nucleocapsid.SynopsisThe 3.1 Å single-particle cryo-electron microscopy structure of the RNA-bound, Ebola virus nucleoprotein helical filament provides molecular details of protein-protein and protein-RNA interactions.

scholarly journals Low cost, high performance processing of single particle cryo-electron microscopy data in the cloud

2015 ◽  
Author(s):  
Michael A. Cianfrocco ◽  
Andres E. Leschziner
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
High Performance ◽  
Low Cost ◽  
Cost Effective ◽  
Cryo Electron Microscopy ◽  
Software Packages ◽  
Electron Microscopes ◽  
Microscopy Data ◽  
New Generation

The advent of a new generation of electron microscopes and direct electron detectors has realized the potential of single particle cryo-electron microscopy (cryo-EM) as a technique to generate high-resolution structures. However, calculating these structures requires high performance computing clusters, a resource that may be limiting to many likely cryo-EM users. To address this limitation and facilitate the spread of cryo-EM, we developed a publicly available ‘off-the-shelf’ computing environment on Amazon’s elastic cloud computing infrastructure. This environment provides users with single particle cryo-EM software packages and the ability to create computing clusters that can range in size from 16 to 480+ CPUs. Importantly, these computing clusters are also cost-effective, as we illustrate here by determining a near-atomic resolution structure of the 80S yeast ribosome for $28.89 USD in ~10 hours.

scholarly journals A Bayesian approach to extracting free-energy profiles from cryo-electron microscopy experiments

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Julian Giraldo-Barreto ◽  
Sebastian Ortiz ◽  
Erik H. Thiede ◽  
Karen Palacio-Rodriguez ◽  
Bob Carpenter ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Free Energy ◽  
Single Molecule ◽  
Single Particle ◽  
Particle Density ◽  
Bound State ◽  
Complete Characterization ◽  
Energy Profiles ◽  
Cryo Electron Microscopy ◽  
Free Energy Profiles

AbstractCryo-electron microscopy (cryo-EM) extracts single-particle density projections of individual biomolecules. Although cryo-EM is widely used for 3D reconstruction, due to its single-particle nature it has the potential to provide information about a biomolecule’s conformational variability and underlying free-energy landscape. However, treating cryo-EM as a single-molecule technique is challenging because of the low signal-to-noise ratio (SNR) in individual particles. In this work, we propose the cryo-BIFE method (cryo-EM Bayesian Inference of Free-Energy profiles), which uses a path collective variable to extract free-energy profiles and their uncertainties from cryo-EM images. We test the framework on several synthetic systems where the imaging parameters and conditions were controlled. We found that for realistic cryo-EM environments and relevant biomolecular systems, it is possible to recover the underlying free energy, with the pose accuracy and SNR as crucial determinants. We then use the method to study the conformational transitions of a calcium-activated channel with real cryo-EM particles. Interestingly, we recover not only the most probable conformation (used to generate a high-resolution reconstruction of the calcium-bound state) but also a metastable state that corresponds to the calcium-unbound conformation. As expected for turnover transitions within the same sample, the activation barriers are on the order of $$k_BT$$ k B T . We expect our tool for extracting free-energy profiles from cryo-EM images to enable more complete characterization of the thermodynamic ensemble of biomolecules.

scholarly journals CASSPER: A Semantic Segmentation based Particle Picking Algorithm for Single Particle Cryo-Electron Microscopy

2020 ◽  
Author(s):  
Blesson George ◽  
Anshul Assaiya ◽  
Robin Jacob Roy ◽  
Ajit Kembhavi ◽  
Radha Chauhan ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Deep Learning ◽  
Structure Determination ◽  
Single Particle ◽  
High Efficiency ◽  
Protein Complexes ◽  
Semantic Segmentation ◽  
Particle Identification ◽  
Learning Tool ◽  
Cryo Electron Microscopy

AbstractSingle-particle cryo-electron microscopy has emerged as the method of choice for structure determination of proteins and protein complexes. However, particle identification and selection which is a prerequisite for achieving high-resolution still poses a major bottleneck for automating the steps of structure determination. Here, we present a generalised deep learning tool, CASSPER, for the automated detection and isolation of protein particles in transmission microscope images. This deep learning tool uses Semantic Segmentation and a collection of visually prepared training samples to capture the differences in the transmission intensities of protein, ice, carbon and other impurities found in the micrograph. CASSPER is the first method to do pixel level classification and completely eliminates the need of manual particle picking. Integration of Contrast Limited Adaptive Histogram Equalization (CLAHE) in CASSPER enables high-fidelity particle detection even in micrographs with variable ice thickness and contrast. In addition, our generalized model for cross molecule picking works with high efficiency on unseen datasets and can potentially pick particles on-the-fly, thereby, enabling automation of data processing.

scholarly journals Application of Cryo-Electron Microscopy on Drug Discovery

2021 ◽  
Vol 27 (S1) ◽  
pp. 3250-3250
Author(s):  
Viswanath Vittaladevaram ◽  
Kranthi Kuruti
Keyword(s):  
Electron Microscopy ◽  
Protein Complexes ◽  
Lead Discovery ◽  
Biologically Relevant ◽  
Biological Targets ◽  
3 Dimensional ◽  
Drug Molecules ◽  
Cryo Electron Microscopy ◽  
Therapeutic Molecules ◽  
Novel Drug

AbstractThe key aspect for development of novel drug molecules is to perform structural determination of target molecule associated with its ligand. One such tool that provides insights towards structure of molecule is Cryo-electron microscopy which covers biological targets that are intractable. Examination of proteins can be carried out in native state, as the samples are frozen at -175 degree Celsius i.e. cryogenic temperatures. In addition to this, there were no limits for molecular and functional structures of proteins that can be imagined in 3-dimensional form. This includes ligands which unravel mechanisms that are biologically relevant. This will enable to better understand the mechanisms that are used for development of new therapeutics. Application of Cryo-electron microscopy is not limited to protein complexes and is considered as non-specific. Intervention of Cryo-EM would allow to analyse the structures and also able to dissect the interaction with therapeutic molecules. The study determines the usage of cryo-EM for providing resolutions that are acceptable for lead discovery. It also provides support for lead optimization and also for discovery of vaccines and therapeutics.

scholarly journals Electron microscopy snapshots of single particles from single cells

2018 ◽  
Vol 294 (5) ◽  
pp. 1602-1608 ◽  
Author(s):  
Xiunan Yi ◽  
Eric J. Verbeke ◽  
Yiran Chang ◽  
Daniel J. Dickinson ◽  
David W. Taylor
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Protein Complexes ◽  
Signal To Noise Ratio ◽  
Single Cells ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particle Analysis ◽  
Whole Cells ◽  
Potential Applications

Cryo-electron microscopy (cryo-EM) has become an indispensable tool for structural studies of biological macromolecules. Two additional predominant methods are available for studying the architectures of multiprotein complexes: 1) single-particle analysis of purified samples and 2) tomography of whole cells or cell sections. The former can produce high-resolution structures but is limited to highly purified samples, whereas the latter can capture proteins in their native state but has a low signal-to-noise ratio and yields lower-resolution structures. Here, we present a simple, adaptable method combining microfluidic single-cell extraction with single-particle analysis by EM to characterize protein complexes from individual Caenorhabditis elegans embryos. Using this approach, we uncover 3D structures of ribosomes directly from single embryo extracts. Moreover, we investigated structural dynamics during development by counting the number of ribosomes per polysome in early and late embryos. This approach has significant potential applications for counting protein complexes and studying protein architectures from single cells in developmental, evolutionary, and disease contexts.

scholarly journals Single-particle cryo-EM—How did it get here and where will it go

Science ◽  
2018 ◽  
Vol 361 (6405) ◽  
pp. 876-880 ◽  
Author(s):  
Yifan Cheng
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Single Particle ◽  
Electron Crystallography ◽  
The Past ◽  
Cryo Electron Microscopy ◽  
Technological Advances ◽  
The Future ◽  
Electron Cryotomography

Cryo–electron microscopy, or simply cryo-EM, refers mainly to three very different yet closely related techniques: electron crystallography, single-particle cryo-EM, and electron cryotomography. In the past few years, single-particle cryo-EM in particular has triggered a revolution in structural biology and has become a newly dominant discipline. This Review examines the fascinating story of its start and evolution over the past 40-plus years, delves into how and why the recent technological advances have been so groundbreaking, and briefly considers where the technique may be headed in the future.

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