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Electron Microscopy Solutions
Courtesy: Robert L. Duda (a), Matthijn Vos (b) and James F. Conway (a); a. University of Pittsburgh; b. Thermo Fisher Scientific

Single Particle Analysis

The Challenges of Protein Complex Analysis

Drug development at the pace requested by today's society requires the study of molecular mechanisms as close as possible to in vivo at high resolution. Until recently, the main technique available to achieve high resolution structures of biological molecules was crystallography.

Cryo-EM, the Nature 2015 Method of the Year, is now able to solve near-atomic-resolution structures with volume and clarity. Using Cryo-EM to understand molecular structures such as proteins, protein complexes, and protein-ribonucleic acid associations-the fundamental building blocks of life-will lead to wide scale pharmaceutical solutions for treating diseases and disorders.

How to Engage in cryo-EM Single Particle Analysis

Challenges in revealing 3D macromolecular protein complexes

Until recently, scientists have had to engineer and crystalize proteins in order to reconstruct and visualize them via X-ray crystallography. Besides the fact that crystallization is a time-consuming process - which may not always be successful as some proteins do not crystallize - the technology is also mainly applicable to crystallization of monomeric and dimeric structures only. Although large protein complexes provide more insight to native function, it has been difficult to stabilize and crystallize these structures.

 Cryo-TEM advantages:

  • Elucidate dynamic biological processes in their native states at near atomic resolution.
  • Resolve protein structures (such as protein membranes) that are difficult to solve with other techniques.
  • Determine three-dimensional structure of protein complexes, aggregates and large virus assemblies.

How cryo-TEM complements X-ray crystallography and NMR

Integrative Structural Biology

Mass spectrometry offers the possibilities of a complete lab and an abundance of information for structural biology. The only thing missing is an actual structure to match the information against. Several techniques are capable of providing this complementing structure; however, especially for the focus on large protein complexes, one technique is simply superior to the others: cryo-electron microscopy (cryo-EM). Are you aware of how the integration of mass spectrometry and cryo-EM can benefit your structural biology research? 

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Single Particle Analysis Workflow

A step-by-step solution for resolving 3D protein complexes

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Step one: Sample Preparation

The first step in the cryo-TEM workflow is key in order to produce the highest quality 3D protein complex structures.

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Step two: Single Particle Imaging

Maintaining sample integrity while transferring to an imaging platform for screening and analysis is the next step towards obtaining structural data.

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Step three: Data Acquistion

We take a series of 2D projection images using very low electron dose and computationally extract them. Software then orients them relative to one another to generate a three dimensional structure.

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Step four: Single Particle Reconstruction

For single particle reconstructions, the data produced can immediately be imported into various reconstruction packages.

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Step one: Sample Preparation

Sample Vitrification

This is the step which prepares the sample for cryo-TEM imaging. Vitrification cools the sample so rapidly that water molecules do not have time to crystallize, forming instead an amorphous solid that does little or no damage to the sample structure. Vitrification can be applied to protein solutions, cell suspensions or thin tissue slices.

To enable optimal results, the vitrification step needs to be standardized and reproducible. Thermo Fisher Scientific achieves exactly this by offering an automated, programmable approach to vitrification

Products & Solutions

Vitrification with Vitrobot

Create high quality vitrified samples for single particle analysis or cryo-tomography research applications with Vitrobot. Vitrobot offers fully automated vitrification, fast and easy. It performs the cryo-fixation process at constant physical and mechanical conditions like temperature, relative humidity, blotting conditions and freezing velocity. This ensures high quality cryo-fixation results and a high sample preparation throughput prior to cryo-TEM observation.

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Step two: Single Particle Imaging

Fixed in an ultra-thin vitrified ice layer, most biological materials are highly beam sensitive. As a result, only low-dose beams can be used in imaging. In the past, this has presented a challenge to acquiring sufficient image resolution and contrast. This is no longer the case.

Products & Solutions

Imaging Platforms

Glacios Cryo-TEM

The new Thermo Scientific™ Glacios™ Cryo Transmission Electron Microscope (Cryo-TEM) delivers a complete and affordable Cryo-EM solution to a broad range of scientists. It features 200 kV XFEG optics, the industry-leading Autoloader (cryogenic sample manipulation robot) and the same innovative automation for ease of use as on the Krios G3i Cryo-TEM. The Glacios Cryo-TEM bundles all this into a small footprint that simplifies installation.

Krios G3i Cryo-TEM

The new Thermo Scientific™ Krios™ G3i Cryo Transmission Electron Microscope (Cryo-TEM) enables life science researchers to unravel life at the molecular level—easier, faster, and more reliably than ever before. Its highly stable 300 kV TEM platform and industry-leading Autoloader (cryogenic sample manipulation robot) are designed for automated applications, such as single particle analysis (SPA) and cryo-tomography. Designed-in connectivity ensures a robust and risk-free pathway throughout the entire workflow, from sample preparation and optimization to image acquisition and data processing.

Talos™ Arctica

The Talos Arctica is a 200kV FEG Transmission and Scanning Electron Microscope (STEM). It is a powerful, stable, and versatile system for delivering high-resolution 3D characterization of biological and biomaterials samples in cell biology, structural biology, and nanotechnology research. Talos enables scientists to quickly obtain better insight and understanding of macromolecular structures, cellular components, cells, and tissues in three dimensions.

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Step three: Data Acquistion

Thermo Fisher Scientific offers a complete range of detection cameras and software to support efficient data recording as part of the single particle analysis workflow. With the ability to efficiently detect low-contrast signals, scientists can now capture image information with high sensitivity for higher resolution images.

Products & Solutions

EPU Automated SPA Software

As part of single particle analysis workflow, Thermo Fisher Scientific offers powerful EPU automated acquisition software for data collection. Our EPU software streamlines the acquisition of large data sets, using thousands or tens of thousands of nominally identical particles. After conformational classification and particle averaging, the result is a high-resolution 3D representation.

Phase Plate

Vitrified samples typically exhibit low intrinsic contrast and require low-dose imaging techniques. Thermo Scientific™ Phase Plate achieves a significantly improved contrast at low spatial frequencies, revealing greater levels of detail, as shown in the example to the right. With higher image contrast, each tilt image can be recorded at a lower electron dose with less damage to the specimen.

Falcon Direct Electron Detector

The Falcon 3EC is the first direct electron detector to benefit from our next-generation image processing pipeline. It is seamlessly connected to a dedicated fast storage server, which makes waiting for frames to be stored a thing of the past. Images, including individual frames or dose fractions, are processed on-the-fly and stored on the storage server. The increased frame rate, further reduced noise levels, and powerful imaging pipeline enable electron counting and even on-the-fly drift correction, providing the highest quality data in the shortest amount of time.

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Step four: Single Particle Reconstruction

Our goal is to improve the rate of data collection and through ease of use, get better structures leading to better publications-ultimately leading to breakthrough discoveries. Publications are key here. They are essentially the scientific currency. Accurate, detailed, three-dimensional models of intricate biological structures at the sub cellular and molecular scale are key to developing these publications.

Documents

Structural Mechanism of Trimeric HIV-1 Envelope Glycoprotein Activation

HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env) to CD4 and a co-receptor on target Tcells. Understanding how these ligands influence the structure of Env is of fundamental interest for HIV vaccine development. Using cryo-electron microscopy, we describe the contrasting structural outcomes of trimeric Env binding to soluble CD4, to the broadly neutralizing, CD4-binding site antibodies VRC01, VRC03 and b12, or to the monoclonal antibody 17b, a co-receptor mimic.

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Publication list

1.
A. Heuer, M. Gerovac, C. Schmidt, S. Trowitzsch, A. Preis, P. Kötter, O. Berninghausen, T. Becker, R. Beckmann, R. Tampé   (2017)   Structure of the 40S-ABCE1 post-splitting complex in ribosome recycling and translation initiation.   Nature structural & molecular biology   

DOI:  10.1038/nsmb.3396

References PDB protein(s):  5LL6

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Structure of the 40S-ABCE1 post-splitting complex in ribosome recycling and translation initiation.

A. Heuer, M. Gerovac, C. Schmidt, S. Trowitzsch, A. Preis, P. Kötter, O. Berninghausen, T. Becker, R. Beckmann, R. Tampé

The essential ATP-binding cassette protein ABCE1 splits 80S ribosomes into 60S and 40S subunits after canonical termination or quality-control-based mRNA surveillance processes. However, the underlying splitting mechanism remains enigmatic. Here, we present a cryo-EM structure of the yeast 40S-ABCE1 post-splitting complex at 3.9-Å resolution. Compared to the pre-splitting state, we observe repositioning of ABCE1's iron-sulfur cluster domain, which rotates 150° into a binding pocket on the 40S subunit. This repositioning explains a newly observed anti-association activity of ABCE1. Notably, the movement implies a collision with A-site factors, thus explaining the splitting mechanism. Disruption of key interactions in the post-splitting complex impairs cellular homeostasis. Additionally, the structure of a native post-splitting complex reveals ABCE1 to be part of the 43S initiation complex, suggesting a coordination of termination, recycling, and initiation.

2.
K. van Pee, A. Neuhaus, E. D'Imprima, D. Mills, W. Kühlbrandt, Ö. Yildiz   (2017)   CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin.   eLife   6

DOI:  10.7554/eLife.23644

References PDB protein(s):  5LY6

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CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin.

K. van Pee, A. Neuhaus, E. D'Imprima, D. Mills, W. Kühlbrandt, Ö. Yildiz

Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain D3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane.

3.
K. Škubník, J. Nováček, T. Füzik, A. Přidal, R. Paxton, P. Plevka   (2017)   Structure of deformed wing virus, a major honey bee pathogen.   Proceedings of the National Academy of Sciences of the United States of America   114

DOI:  10.1073/pnas.1615695114

References PDB protein(s):  5L7Q, 5L8Q, 5MUP, 5MV5, 5MV6

Read abstract

Structure of deformed wing virus, a major honey bee pathogen.

K. Škubník, J. Nováček, T. Füzik, A. Přidal, R. Paxton, P. Plevka

The worldwide population of western honey bees (Apis mellifera) is under pressure from habitat loss, environmental stress, and pathogens, particularly viruses that cause lethal epidemics. Deformed wing virus (DWV) from the family Iflaviridae, together with its vector, the mite Varroa destructor, is likely the major threat to the world's honey bees. However, lack of knowledge of the atomic structures of iflaviruses has hindered the development of effective treatments against them. Here, we present the virion structures of DWV determined to a resolution of 3.1 Å using cryo-electron microscopy and 3.8 Å by X-ray crystallography. The C-terminal extension of capsid protein VP3 folds into a globular protruding (P) domain, exposed on the virion surface. The P domain contains an Asp-His-Ser catalytic triad that is, together with five residues that are spatially close, conserved among iflaviruses. These residues may participate in receptor binding or provide the protease, lipase, or esterase activity required for entry of the virus into a host cell. Furthermore, nucleotides of the DWV RNA genome interact with VP3 subunits. The capsid protein residues involved in the RNA binding are conserved among honey bee iflaviruses, suggesting a putative role of the genome in stabilizing the virion or facilitating capsid assembly. Identifying the RNA-binding and putative catalytic sites within the DWV virion structure enables future analyses of how DWV and other iflaviruses infect insect cells and also opens up possibilities for the development of antiviral treatments.

4.
C. Engel, T. Gubbey, S. Neyer, S. Sainsbury, C. Oberthuer, C. Baejen, C. Bernecky, P. Cramer   (2017)   Structural Basis of RNA Polymerase I Transcription Initiation.   Cell   169

DOI:  10.1016/j.cell.2017.03.003

References PDB protein(s):  5N5Y, 5N5Z, 5N60, 5N61

Read abstract

Structural Basis of RNA Polymerase I Transcription Initiation.

C. Engel, T. Gubbey, S. Neyer, S. Sainsbury, C. Oberthuer, C. Baejen, C. Bernecky, P. Cramer

Transcription initiation at the ribosomal RNA promoter requires RNA polymerase (Pol) I and the initiation factors Rrn3 and core factor (CF). Here, we combine X-ray crystallography and cryo-electron microscopy (cryo-EM) to obtain a molecular model for basal Pol I initiation. The three-subunit CF binds upstream promoter DNA, docks to the Pol I-Rrn3 complex, and loads DNA into the expanded active center cleft of the polymerase. DNA unwinding between the Pol I protrusion and clamp domains enables cleft contraction, resulting in an active Pol I conformation and RNA synthesis. Comparison with the Pol II system suggests that promoter specificity relies on a distinct "bendability" and "meltability" of the promoter sequence that enables contacts between initiation factors, DNA, and polymerase.

5.
J. Gu, M. Wu, R. Guo, K. Yan, J. Lei, N. Gao, M. Yang   (2016)   The architecture of the mammalian respirasome.   Nature   537

DOI:  10.1038/nature19359

References PDB protein(s):  5GPN

Read abstract

The architecture of the mammalian respirasome.

J. Gu, M. Wu, R. Guo, K. Yan, J. Lei, N. Gao, M. Yang

The respiratory chain complexes I, III and IV (CI, CIII and CIV) are present in the bacterial membrane or the inner mitochondrial membrane and have a role of transferring electrons and establishing the proton gradient for ATP synthesis by complex V. The respiratory chain complexes can assemble into supercomplexes (SCs), but their precise arrangement is unknown. Here we report a 5.4 Å cryo-electron microscopy structure of the major 1.7 megadalton SCI1III2IV1 respirasome purified from porcine heart. The CIII dimer and CIV bind at the same side of the L-shaped CI, with their transmembrane domains essentially aligned to form a transmembrane disk. Compared to free CI, the CI in the respirasome is more compact because of interactions with CIII and CIV. The NDUFA11 and NDUFB9 supernumerary subunits of CI contribute to the oligomerization of CI and CIII. The structure of the respirasome provides information on the precise arrangements of the respiratory chain complexes in mitochondria.

Protein Data Bank Structures

(Click on images for more details)
  • Life in the extremes: atomic structure of Sulfolobus Turreted Icosahedral Virus
    Author: Veesler, D. et al.
    Taken on a FEI TITAN KRIOS at 4.5 Å resolution
  • Cryo-EM structure of Dengue virus serotype 3 at 28 degrees C
    Author: Fibriansah, G. et al.
    Taken on a FEI TITAN KRIOS at 6.0 Å resolution
  • Structure of beta-galactosidase at 3.2-A resolution obtained by cryo-electron microscopy
    Author: Bartesaghi, A. et al.
    Taken on a FEI TITAN KRIOS at 3.2 Å resolution
  • CryoEM single particle reconstruction of anthrax toxin protective antigen pore at 2.9 Angstrom resolution
    Author: Jiang, J. et al.
    Taken on a FEI TITAN KRIOS at 2.9 Å resolution
  • Atomic structure of a non-enveloped virus reveals pH sensors for a coordinated process of cell entry
    Author: Zhang, X. et al.
    Taken on a FEI TITAN KRIOS at 3.3 Å resolution
  • Structure of alpha-1 glycine receptor by single particle electron cryo-microscopy, strychnine-bound state
    Author: Du, J. et al.
    Taken on a FEI TITAN KRIOS at 3.9 Å resolution
  • Electron cryo-microscopy of the IST1-CHMP1B ESCRT-III copolymer
    Author: McCullough, J. et al.
    Taken on a FEI TITAN KRIOS at 4.0 Å resolution
  • Structure of Escherichia coli EF4 in posttranslocational ribosomes (Post EF4)
    Author: Zhang, D. et al.
    Taken on a FEI TITAN KRIOS at 3.700 Å resolution
  • Cryo-EM structure of the magnesium channel CorA in the closed symmetric magnesium-bound state
    Author: Matthies, D. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Structure of the yeast 60S ribosomal subunit in complex with Arx1, Alb1 and C-terminally tagged Rei1
    Author: Greber, B.J. et al.
    Taken on a FEI TITAN KRIOS at 3.410 Å resolution
  • Structure of a Chaperone-Usher pilus reveals the molecular basis of rod uncoilin
    Author: Hospenthal, M.K. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Cryo-EM structure of the flagellar hook of Campylobacter jejuni
    Author: Matsunami, H. et al.
    Taken on a FEI TITAN KRIOS at 3.5 Å resolution
  • Structure of F-ATPase from Pichia angusta, state1
    Author: Vinothkumar, K.R. et al.
    Taken on a FEI TITAN KRIOS at 7 Å resolution
  • Cryo-EM structure of a BG505 Env-sCD4-17b-8ANC195 complex
    Author: Wang, H. et al.
    Taken on a FEI TITAN KRIOS at 8.9 Å resolution
  • Ca2+ bound aplysia Slo1
    Author: MacKinnon, R. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Dephosphorylated, ATP-free cystic fibrosis transmembrane conductance regulator (CFTR) from zebrafish
    Author: Zhang, Z. et al.
    Taken on a FEI TITAN KRIOS at 3.73 Å resolution
  • The 2.8 A Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparanoid Pentasaccharide
    Author: Xie, Q. et al.
    Taken on a FEI TITAN KRIOS at 2.8 Å resolution
  • Volta phase plate cryo-electron microscopy structure of a calcitonin receptor-heterotrimeric Gs protein complex
    Author: Liang, Y.L. et al.
    Taken on a FEI TITAN KRIOS at 4.1 Å resolution
  • Cryo-EM structure of the human ether-a-go-go related K+ channel
    Author: Wang, W.W. et al.
    Taken on a FEI TITAN KRIOS at 3.7 Å resolution
  • CryoEM structure of type II secretion system secretin GspD in E.coli K12
    Author: Yan, Z. et al.
    Taken on a FEI TITAN KRIOS at 3.04 Å resolution
  • Structure of a Pancreatic ATP-sensitive Potassium Channel
    Author: Li, N. et al.
    Taken on a FEI TITAN KRIOS at 5.6 Å resolution
  • The resting state of yeast proteasome
    Author: Ding, Z. et al.
    Taken on a FEI TITAN KRIOS at 6.3 Å resolution
  • Structure of a eukaryotic voltage-gated sodium channel at near atomic resolution
    Author: Shen, H. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Prefusion structure of MERS-CoV spike glycoprotein, three-fold symmetry
    Author: Yuan, Y. et al.
    Taken on a FEI TITAN KRIOS at 3.2 Å resolution
  • Prefusion structure of MERS-CoV spike glycoprotein, conformation 2
    Author: Yuan, Y. et al.
    Taken on a FEI TITAN KRIOS at 3.2 Å resolution
  • Structure of SARS-CoV spike glycoprotein
    Author: Gui, M. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Nucleosome : Class 1
    Author: Bilokapic, S. et al.
    Taken on a FEI TITAN at 3.7 Å resolution
  • Products for Single Particle Analysis

    Krios G3i Cryo-TEM for Life Sciences
    The new Thermo Scientific™ Krios™ G3i Cryo Transmission Electron Microscope (Cryo-TEM) enables life science researchers to unravel life at the molecular level—easier, faster, and more reliably than ever before. Its highly stable 300 kV TEM platform and industry-leading Autoloader (cryogenic sample manipulation robot) are designed for automated applications, such as single particle analysis (SPA) and cryo-tomography. Designed-in connectivity ensures a robust and risk-free pathway throughout the entire workflow, from sample preparation and optimization to image acquisition and data processing.
    Glacios Cryo-TEM for Life Sciences

    The new Thermo Scientific™ Glacios™ Cryo Transmission Electron Microscope (Cryo-TEM) delivers a complete and affordable Cryo-EM solution to a broad range of scientists. It features 200 kV XFEG optics, the industry-leading Autoloader (cryogenic sample manipulation robot) and the same innovative automation for ease of use as on the Krios G3i Cryo-TEM. The Glacios Cryo-TEM bundles all this into a small footprint that simplifies installation.

    Talos L120C TEM for Life Sciences
    An ideal entry-level solution for imaging and tomography that can be configured as a basic cryo-TEM imaging platform. Fully upgradeable, the Talos L120C TEM will meet your needs, whether those needs are in Cryo or Room Temperature or 2D imaging or 3D imaging and multi-modality imaging experiments.
    Talos F200C TEM for Life Sciences
    The Thermo Scientific™ Talos™ is a 200kV S/TEM designed for fast, precise and quantitative characterization of nanomaterials in multiple dimensions. It accelerates materials nanoanalysis based on higher data quality, faster acquisition, simplified, easy and automated operation.
    Talos Arctica TEM for Life Sciences
    The Thermo Scientific™ Talos™ Arctica is a 200kV FEG Transmission and Scanning Electron Microscope (S/TEM). It is a powerful, stable, and versatile system for delivering high-resolution 3D characterization of biological and biomaterials samples in cell biology, structural biology, and nanotechnology research. The Talos S/TEM enables scientists to quickly obtain better insight and understanding of macromolecular structures, cellular components, cells, and tissues in three dimensions.
    Talos F200C TEM for Life Sciences
    The Thermo Scientific™ Talos™ is a 200kV S/TEM designed for fast, precise and quantitative characterization of nanomaterials in multiple dimensions. It accelerates materials nanoanalysis based on higher data quality, faster acquisition, simplified, easy and automated operation.
    Vitrobot for Life Sciences
    Vitrobot completely automates the vitrification process to provide fast, easy, reproducible sample preparation - the first step in obtaining high quality images and repeatable experimental results.
    EPU for Life Sciences
    Bringing powerful automation and reliability to single particle analysis for effective cryo-TEM workflows.

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