| Electron Microscopy Solutions Materials Science Semiconductors Life Sciences Oil and Gas Home / Life Sciences / Single Particle Analysis 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 Featured Video Webcast: Understanding molecular structures – faster and more reliably with cryo-EM In this webcast, we demonstrate the advantages and applications of the new Thermo Scientific™ Glacios™ and Krios™ G3i Cryo-TEMs for acquiring the 3D structure of molecules. This webcast is specifically tailored for those researchers who are interested in the growing field of cryo-EM imaging in the field of single particle analysis (SPA). 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? LEARN MORE Single Particle Analysis Workflow A step-by-step solution for resolving 3D protein complexes 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. LEARN MORE 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. LEARN MORE 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. LEARN MORE Step four: Single Particle Reconstruction For single particle reconstructions, the data produced can immediately be imported into various reconstruction packages. LEARN MORE 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. Download document Quick Links Talos L120C for Life Sciences Datasheet Krios G3i Cryo-TEM datasheet Glacios Cryo-TEM datasheet Talos Arctica Datasheet Falcon 3EC datasheet EPU - Applications Brochure Phase Plate Datasheet 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 Read abstract 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 Read abstract 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 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 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 VIEW ALL PUBLICATIONS Protein Data Bank Structures (Click on images for more details) Structure of F-ATPase from Pichia angusta, state1 Author: Vinothkumar, K.R. et al. DOI: 10.1073/pnas.1615902113 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. DOI: 10.1073/pnas.1615939113 Taken on a FEI TITAN KRIOS at 8.9 Å resolution Ca2+ bound aplysia Slo1 Author: MacKinnon, R. et al. DOI: 10.1038/nature20775 Taken on a FEI TITAN KRIOS at 3.8 Å resolution 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Prev Next SEE MORE STRUCTURES 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. Need more information? 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