Eva Nogales, University of California, Berkeley

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

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

Titan Krios

The Titan Krios is the most powerful and flexible high resolution electron microscope for 2D and 3D characterization of protein structures and protein complexes. With automated sample loading and reliable unattended operation, Titan Krios achieves a level of productivity that is unmatched.

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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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 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
  • Structure of a Pancreatic ATP-sensitive Potassium Channel
    Author: Li, N. et al.
    Taken on a FEI TITAN KRIOS at 5.6 Å resolution
  • Products for Single Particle Analysis

    Titan Krios TEM for Life Sciences
    The FEI Titan Krios TEM is the most powerful and flexible high resolution electron microscope for 2D and 3D characterization of protein structures and protein complexes. With automated sample loading and reliable unattended operation, the Titan Krios TEM achieves a level of productivity that is unmatched.
    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 FEI 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 FEI 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 FEI TalosTM 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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