Courtesy: Robert L. Duda (a), Matthijn Vos (b) and James F. Conway (a); a. University of Pittsburgh; b. FEI Company

Structural Biology

Revealing 3D macromolecular complexes

Structural biology explores the functional mechanistics of molecular structures such as proteins, protein complexes and protein-ribonucleic acid associations - the fundamental building blocks of life. 

Scientists seek to understand how these structures are designed and how structural variations affect functionality. Structural biology research is essential to understanding the origins of events such as pathogen-induced and age related diseases, allergic hypersensitivities, and cell growth and differentiation.

Today, many structural biologists are interested in finding innovative ways to intervene in disease processes and find new preventive measures, treatments, and pharmaceutical agents. This requires the in vivo exploration of the molecular mechanisms of diseases, to unambiguously determine the conformation of flexible protein structures in their natural biological context. Understanding these activities will unravel the fundamentals of life and may consequently lead towards wide scale pharmaceutical solutions for treating fundamental diseases and disorders.

 

Structural Biology Workflow

This FEI Cryo-TEM workflow provides a setp-by-step solution for examining 3D protein complexes. Please select any of the steps in the workflow above to see the detailed description.

Sample Preparation:

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

Learn About :
Sample Preparation

Sample Vitrification

Prepare the sample for Cryo-TEM imaging

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

View Product 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.

Visit the Vitrobot product page

Request More Information »

Back

Back

Imaging:

Vitrification prepares the sample for imaging. This requires the use of a properly configured transmission electron microscope to capture many high quality 2D images for the final 3D reconstruction of the structure of proteins and protein complexes.

Learn About :
Imaging

Imaging platforms

Image macromolecular structures at extremely high resolution

Electron Detection

Capture image information with high sensitivity and efficiency

Single Particle Analysis

Produces the structure of an individual particle or protein from thousands of individual images.

Tomography

Examine the cellular structures in all three dimensions to generate an accurate three-dimensional map of the interior of the cell.

Phase Plates

Improved contrast at low spatial frequencies

Imaging platforms

A properly configured Transmission Electron Microscope (TEM) suited for cryo-TEM experiments needs to be able to perform image acquisition over prolonged periods of times.  Producing 3D macromolecular protein complexes requires capturing thousands of  2D images which are used to create 3D reconstructions of protein complexes as the final result. Effective cryo-TEM imaging platforms support automated sample loader options, 24/7 unattended operation and operating software that enables automated acquisition of data for either Single Particle Analysis, cryo-electron Tomography or both.

View Product Solutions »

Imaging Platforms - FEI Titan Krios & Tecnai Arctica

FEI Produces two TEM platforms for high resolution image acquisition within the Cryo-TEM workflow.Titan Krios and Tecnai Arctica. Both are exclusively designed for high throughput, high resolution results. The Titan Krios offers some distinct advantages over the Tecnai Arctica, take a moment to learn more about them.

Visit the Titan Krios product page

Visit the Tecnai Arctica product page

Visit the Talos product page

Visit the Titan Halo product page

Request More Information »

Back

Back

Electron Detection

Most biological samples are highly beam sensitive. The electron dose is therefore one of the main limiting factors when conducting primary structural biology research applications such as single particle analysis (SPA). Conducting SPA, large numbers of vitrified low contrast molecule/protein complexes are imaged at low electron dose conditions to preserve the sample, so superior detection of the low contrast signal is of significant importance. This also applies for cryo-tomography where the resolution in the tomogram will increase with the number of tomography images that is recorded  at reduced increment angles. For both applications an improved signal to noise ratio is therefore of extreme importance. FEI has developed a complete range of detection cameras and software to support efficient data recording as part of the cryo-TEM workflow.

View Product Solutions »

Falcon (II) Direct Electron Detector

FEI developed the Falcon II taking into account all factors that determine the final result of an experiment: sensitivity, dynamic range, SNR, ease of use, speed, FOV, automation, ease of processing. This resulted in a camera with a very high sensitivity, enabling experiments that result in 3D reconstructions with a minimal amount of Falcon generated images and incredible time to structure and publications.

The Falcon II has been built with a forward thinking design where all features available in subsequent generation Falcon cameras can be fully retrofitted to the Falcon II.

Visit the Falcon II product page

Request More Information »

Back

Back

Single Particle Analysis

Single Particle Analysis produces the structure of an individual particle, protein or macromolecular complex from thousands of individual images. This requires specialized software which takes into account the different orientation of particles and matches similar classes of orientation to deduce the one original structure. Combining many digitized datasets results in high resolution data and results in resolutions of 10 nanometer down to approximately 3.5 nanometer currently.

View Product Solutions »

EPU Automation Software

Exclusively designed for demanding structural biology applications that require high and intermediate resolutions, EPU brings powerful automation and reliability that delivers impressive results when conducting single particle analysis with the FEI TEM microscope platforms. EPU software operates the instrument and collects data, removing the need for full-time operator control and presence. This software automatically focuses the instrument and ensures the most stable conditions before collecting superior images. Unique algorithms allow EPU to select thin specimen patches autonomously, using preset experimental variables like drift limits. EPU will allow you to significantly increase your sample throughput, data collection and time-to-data.

Visit the EPU product page

Request More Information »

Back

Back

Tomography

Cryo-tomography involves examining the cellular structures under varying tilt angles to generate an accurate three-dimensional map of the interior of the cell. It enables researchers to analyze molecular structures in relation to the cellular architecture, the cytoskeleton and the cell organelles. Most proteins do not function as individual entities, but in coordination or dependence with other proteins. The knowledge of the three-dimensional organization is essential to understand protein function at the cellular level. Cellular tomography using transmission electron microscopy (TEM) is the only available technology to chart the inside of a cell and is therefore an essential technology in the cell biologists' tool box.

View Product Solutions »

Tomography 4.0

Tomography 4.0 software provides a fast, easy and complete solution for dual- and single-axis electron tomography using FEI's transmission electron microscopes and FEI's latest detector modules. Similar in concept to techniques used to examine organs and other large structures in clinical medicine like X-ray CAT scans, electron tomography creates accurate, detailed, three dimensional models of intricate biological structures at the sub cellular and molecular scale - from the 3D organization of membranes and organelles down to the molecular configuration of individual proteins and protein.

Visit the Tomography 4.0 product page

Request More Information »

Back

Back

Phase Plates

One of the main characteristics of imaging biological materials within their vitrified context is their low intrinsic contrast. In addition, their radiation sensitivity imposes a limit on the total dose that can be applied for imaging, which results in a poor overall signal to noise ratio in the data. Hence, for the main structural biology applications - tomography and single particle analysis - there is a strong need to image amorphous and/or organic (biological) materials at an improved contrast while maintaining maximum resolution.

FEI offers a solution that achieves a significantly improved contrast at low spatial frequencies, revealing a greater level of detail and thus improving the detection of weak features. With an increase in contrast, more structural and morphological information can be retrieved from e.g. tomographic reconstructions.

View Product Solutions »

A total, complete solution

The FEI Phase Plate solution is a total solution which consists of an aperture holder infrastructure in which a hole free thin film phase plate is mounted, in addition to the presence of conventional apertures. The FEI solution - operating via a Volta induced phase shift - is optimized in enhancing the life time and quality of the phase plate and is embedded into the main TEM platform and applications software for the leading applications in structural biology. The FEI phase plate is a self-generating phase plate, based on the beam-induced Volta effect. The effect provides a close to ideal phase plate principle and also enables a long lifetime of the phase plate. The strong linkage with the latest direct electron detector technology and latest TEM platform developments ensures FEI's phase plate contribution to an increased information output at the highest possible quality and data output. 

FEI Phase Plate

Request More Information »

Back

Back

Data Processing:

Data Processing is the step in the workflow which produces the final result. It combines the many images retrieved from the experiment, and turns them into tomographic reconstructions or single particle structures. This step also allows for multi-dimensional data to be combined into correlative data to add yet another layer of insight.

Learn About :
Data Processing

Cryo-Tomography

Examine the cellular structures in all three dimensions to generate an accurate three-dimensional map of the interior of the cell.

Template Matching

Finding proteins and structures if interest in a tomogram can be very challenging as Single Particle Analysis and Tomography result in structures and data which are very different from one another yet they share common basic information. Template matching is used to reveal that information as it helps to locate the proteins as resulted from the SPA experiment, and positions them into a 3D tomographic dataset.

Cryo-Tomography

Cryo-tomography involves examining the cellular structures under varying tilt angles to generate an accurate three-dimensional map of the interior of the cell. It enables researchers to analyze molecular structures in relation to the cellular architecture, the cytoskeleton and the cell organelles. Most proteins do not function as individual entities, but in coordination or dependence with other proteins. The knowledge of the three-dimensional organization is essential to understand protein function at the cellular level. Cellular tomography using transmission electron microscopy (TEM) is the only available technology to chart the inside of a cell and is therefore an essential technology in the cell biologists' tool box.

View Product Solutions »

Tomography 4.0 software

Tomography 4.0 software provides a fast, easy and complete solution for dual- and single-axis electron tomography using FEI's transmission electron microscopes and FEI's latest detector modules. Similar in concept to techniques used to examine organs and other large structures in clinical medicine like X-ray CAT scans, electron tomography creates accurate, detailed, three dimensional models of intricate biological structures at the sub cellular and molecular scale - from the 3D organization of membranes and organelles down to the molecular configuration of individual proteins and protein.

Visit the Tomography 4.0 product page

Request More Information »

Back

Back

Template Matching

FEI's ARGOS, Automated Recognition of Geometries, Objects and Segmentations, software automatically finds and visualizes the corresponding SPA protein structures in the bigger context of a tomogram and is the ideal solution for template matching because of its high level of automation and ease-of-use.

View Product Solutions »

FEI’s ARGOS

FEI's ARGOS - Automated Recognition of Geometries, Objects and Segmentations - is a software solution that combines high-resolution information from SPA with the cellular context derived from tomography experiments, and increases the amount of knowledge that can be extracted from these two methods. ARGOS is also capable of taking high-resolution structures from SPA analysis and combines these with tomography data. It is not only possible to localize the object of interest in its 3D context but also to define the real orientation of the high resolution object. By doing so, an additional dimension can be obtained which provides structural biologists with a greater understanding of the functioning of molecular complexes within the cell.

Visit the ARGOS product page

Request More Information »

Back

Back

Results:

Learn About :
Results

Structure of the TRPV1 ion channel determined by electron cryo-microscopy

Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

Subnanometre-resolution structure of the intact Thermus thermophilus H1-driven ATP synthase

Structure of the TRPV1 ion channel determined by electron cryo-microscopy

Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4 Å resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane segments 5-6 (S5-S6) and the intervening pore loop, which is flanked by S1-S4 voltage-sensor-like domains. TRPV1 has a wide extracellular 'mouth' with a short selectivity filter. The conserved 'TRP domain' interacts with the S4-S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including amino-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function.

Read full article

View Product Solutions »

No additional information available

Please return to previous page

Request More Information »

Back

Back

Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

The 26S proteasome is at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The "lid" of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAA-ATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates. 

Read full article

View Product Solutions »

No additional information available

Please return to previous page

Request More Information »

Back

Back

Subnanometre-resolution structure of the intact Thermus thermophilus H1-driven ATP synthase

Ion-translocating rotary ATPases serve either as ATP synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. During ATP synthesis, ion translocation through the membrane-bound region of the complex causes rotation of a central rotor that drives conformational changes and ATP synthesis in the catalytic region of the complex. There are no structural models available for the intact membrane region of any ion-translocating rotary ATPase. Here we present a 9.7 Å resolution map of the H(+)-driven ATP synthase from Thermus thermophilus obtained by electron cryomicroscopy of single particles in ice. The 600-kilodalton complex has an overall subunit composition of A(3)B(3)CDE(2)FG(2)IL(12). The membrane-bound motor consists of a ring of L subunits and the carboxy-terminal region of subunit I, which are equivalent to the c and a subunits of most other rotary ATPases, respectively. The map shows that the ring contains 12 L subunits and that the I subunit has eight transmembrane helices. The L(12) ring and I subunit have a surprisingly small contact area in the middle of the membrane, with helices from the I subunit making contacts with two different L subunits. The transmembrane helices of subunit I form bundles that could serve as half-channels across the membrane, with the first half-channel conducting protons from the periplasm to the L(12) ring and the second half-channel conducting protons from the L(12) ring to the cytoplasm. This structure therefore suggests the mechanism by which a transmembrane proton motive force is converted to rotation in rotary ATPases.

Read full article

View Product Solutions »

No additional information available

Please return to previous page

Request More Information »

Back

Back

Want to learn more?

Fill out the form below and an FEI representative will contact you. All fields are required.

* 
* 
*
* 
* 

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

Courtesy: Sriram  Subramaniam, Lab of Cell Biology, National Cancer Institute, National Institutes of Health

Documents

Article - 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.

Download document

Achieving breakthrough results using cryo electron microscopy

Prof. Holger Stark, a leading scientist within the field of structural biology has adopted the FEI workflow to resolve macromolecular structures. Watch this video to explore the capabilities of cryo electron microscopy today and how the study proteins and macromolecular complexes in their native state has led to breakthrough results.

Products for Structural Biology

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.
Falcon II Direct Electron Detector
The Falcon is based upon direct electron detection that enables the acquisition of low-noise images of delicate biological samples and other beam-sensitive materials that require low electron dose interactions to prevent radiation damage of the material. Serving applications like cryo-tomography and single particle analysis, Falcon is designed for optimized image detection of those beam-sensitive specimens that require extreme low-dose conditions. Improved spatial resolution and superior image quality are now available at almost no electron dose.
Vitrobot
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 - Automated Single Particles Acquisition Software
Effective cryo-TEM workflows require automation in order to achieve optimal results and high thoughput. EPU enables effective workflows as it facilitates the process of optimal area selection – the first crucial step of the total single particle analysis workflow.

Need more information?

* fields are required

* 
* 
*
* 
*