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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.

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:

  • Resolves protein structures from samples that are difficult to crystallize
  • Analyze challenging, heterogeneous samples and antibodies

How cryo-TEM complements X-ray crystallography and NMR

Integrative Structural Biology

In collaboration with the editors of Science Magazine, we invite you to download this comprehensive booklet, containing articles from the Science family of journals, perspectives on integrative structural biology, and interviews with leading researchers.

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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. FEI 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

FEI 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

FEI EPU Automated SPA Software

As part of single particle analysis workflow, FEI offers powerful EPU automated acquisition software for data collection. FEI 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.

FEI Phase Plate

Vitrified samples typically exhibit low intrinsic contrast and require low-dose imaging techniques. FEI 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.

FEI 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.
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.

2.
H. Zhao, K. Li, A. Lynn, K. Aron, G. Yu, W. Jiang, L. Tang   (2017)   Structure of a headful DNA-packaging bacterial virus at 2.9 Å resolution by electron cryo-microscopy.   Proceedings of the National Academy of Sciences of the United States of America   114

DOI:  10.1073/pnas.1615025114

References PDB protein(s):  5L35

Read abstract

Structure of a headful DNA-packaging bacterial virus at 2.9 Å resolution by electron cryo-microscopy.

H. Zhao, K. Li, A. Lynn, K. Aron, G. Yu, W. Jiang, L. Tang

The enormous prevalence of tailed DNA bacteriophages on this planet is enabled by highly efficient self-assembly of hundreds of protein subunits into highly stable capsids. These capsids can stand with an internal pressure as high as ∼50 atmospheres as a result of the phage DNA-packaging process. Here we report the complete atomic model of the headful DNA-packaging bacteriophage Sf6 at 2.9 Å resolution determined by electron cryo-microscopy. The structure reveals the DNA-inflated, tensed state of a robust protein shell assembled via noncovalent interactions. Remarkable global conformational polymorphism of capsid proteins, a network formed by extended N arms, mortise-and-tenon-like intercapsomer joints, and abundant β-sheet-like mainchain:mainchain intermolecular interactions, confers significant strength yet also flexibility required for capsid assembly and DNA packaging. Differential formations of the hexon and penton are mediated by a drastic α-helix-to-β-strand structural transition. The assembly scheme revealed here may be common among tailed DNA phages and herpesviruses.

3.
X. Wang, P. Cimermancic, C. Yu, A. Schweitzer, N. Chopra, J. Engel, C. Greenberg, A. Huszagh, F. Beck, E. Sakata, Y. Yang, E. Novitsky, A. Leitner, P. Nanni, A. Kahraman, X. Guo, J. Dixon, S. Rychnovsky, R. Aebersold, W. Baumeister, A. Sali, L. Huang   (2017)   Molecular Details Underlying Dynamic Structures and Regulation of the Human 26S Proteasome.   Molecular & cellular proteomics : MCP   

DOI:  10.1074/mcp.M116.065326

References PDB protein(s):  5LN3

Read abstract

Molecular Details Underlying Dynamic Structures and Regulation of the Human 26S Proteasome.

X. Wang, P. Cimermancic, C. Yu, A. Schweitzer, N. Chopra, J. Engel, C. Greenberg, A. Huszagh, F. Beck, E. Sakata, Y. Yang, E. Novitsky, A. Leitner, P. Nanni, A. Kahraman, X. Guo, J. Dixon, S. Rychnovsky, R. Aebersold, W. Baumeister, A. Sali, L. Huang

The 26S proteasome is the macromolecular machine responsible for ATP/ubiquitin dependent degradation. As aberration in proteasomal degradation has been implicated in many human diseases, structural analysis of the human 26S proteasome complex is essential to advance our understanding of its action and regulation mechanisms. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for elucidating structural topologies of large protein assemblies, with its unique capability of studying protein complexes in cells. To facilitate the identification of cross-linked peptides, we have previously developed a robust amine reactive sulfoxide-containing MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). To better understand the structure and regulation of the human 26S proteasome, we have established new DSSO-based in vivo and in vitro XL-MS workflows by coupling with HB-tag based affinity purification to comprehensively examine protein-protein interactions within the 26S proteasome. In total, we have identified 447 unique lysine-to-lysine linkages delineating 67 inter-protein and 26 intra-protein interactions, representing the largest cross-link dataset for proteasome complexes. In combination with EM maps and computational modeling, the architecture of the 26S proteasome was determined to infer its structural dynamics. In particular, three proteasome subunits Rpn1, Rpn6 and Rpt6 displayed multiple conformations that have not been previously reported. Additionally, cross-links between proteasome subunits and 15 proteasome interacting proteins including 9 known and 6 novel ones have been determined to demonstrate their physical interactions at the amino-acid level. Our results have provided new insights on the dynamics of the 26S human proteasome and the methodologies presented here can be applied to study other protein complexes. .

4.
G. Demo, E. Svidritskiy, R. Madireddy, R. Diaz-Avalos, T. Grant, N. Grigorieff, D. Sousa, A. Korostelev   (2017)   Mechanism of ribosome rescue by ArfA and RF2.   eLife   6

DOI:  10.7554/eLife.23687

References PDB protein(s):  5U9F, 5U9G

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Mechanism of ribosome rescue by ArfA and RF2.

G. Demo, E. Svidritskiy, R. Madireddy, R. Diaz-Avalos, T. Grant, N. Grigorieff, D. Sousa, A. Korostelev

ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2 Å resolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA•RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA 'senses' the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

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)
  • CryoEM Helical Reconstruction of TMV
    Author: Ge, P. et al.
    Taken on a FEI TITAN KRIOS at 3.3 Å resolution
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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 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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