- Thursday 11/19/2015
Elden R. Strahm Emeritus Professor of Structural Virology
Ghost protocol: Seeing the invisible with cryoEM variance maps.
All imaging techniques based on multiple copies of the object imaged (e.g. X-ray crystallography and cryoEM reconstructions) depend on closely similar appearances from one observation of the object to the next. Unlike crystallography, CryoEM places no constraint on the object and at the moment of freezing a biological specimen can assume a range of spatial occupancies that depend on the intrinsic dynamic restraints of the specimen. A novel analytical approach to cryoEM reconstructions will be presented that characterizes the continuous 3 dimensional heterogeneity of an ensemble of particles resulting in variance maps that reveal dynamic features that are not obvious in the cryoEM density. Three examples will be discussed where variance maps revealed intrinsic, localized particle heterogeneity associated with chemical mechanisms of virus maturation. (Wang, Q., Matsui, T., Domitrovic, T., Zheng, Y., Doerschuk, P. C., and Johnson, J. E. 2013. Dynamics in cryo EM reconstructions visualized with maximum-likelihood derived variance maps. J Struct Biol 181:195-206).
- Wednesday 9/16/2015
Florida State University
Automated Fiducial-less Tilt-Series Alignment with Protomo.
For more than a decade, tilt-series collection using gold fiducial beads has allowed for structural determination of in situ cellular structures as well as heterogeneous macromolecular structures. However, not all specimen are amenable to the use of fiducials. The emergence of direct electron detectors has enabled the collection of low dose, high contrast images, opening the door for reliable correlation-based tilt-series alignment. I will present a set of automated, correlation-based tilt-series alignment, CTF correction, and reconstruction workflows from within Appion with the goal of making the production of high quality tomograms accessible to all users.
- Friday 8/28/2015
NIH Nat'l Cancer Institute
Localization of small-ligand binding to pharmacological targets using cryo-electron microscopy.
Advances in cryo-electron microscopy are driving the determination of small protein complexes at near-atomic resolution with unprecedented rapidity. For example, computer driven image acquisition together with streamlined image processing pipelines offer the opportunity to localize the binding of ligands with low molecular weight to their target proteins in a relatively automated way. Moreover, improvements in classification algorithms enable the reconstruction of sub-nanometer resolution density maps of distinct conformations from a mixture in single droplet of protein solution.
To test the practicality of this approach, we combined automated procedures and advanced with direct electron detectors to localize ADP (427 Da) and other small ligands on glutamate dehydrogenase (GDH), a clinically significant 365 kDa enzyme that is a relevant pharmaceutical target for cancer, Parkinson's, and diabetes.
Images from single specimens collected in a single session provided enough information to localize nucleotides in a complex at ~3.5 Å resolution. The validity of the results was confirmed by comparison with the crystallographic coordinates of GDH in complex with a variety of ligands. The methods we present provide a streamlined path to rapidly solve the structure of macromolecular complexes and to image the binding target of drug molecules at near atomic resolution.
- Friday 7/10/2015
Max Planck Institute for Brain Research
Dynamo: a complete framework for subtomogram averaging of cryo electron tomography data.
While Cryo Electron Tomography offers an unique potential for direct visualization of the three dimensional structure of macromolecular compounds in their cellular environment, the severely noisy nature of the low dose images delivered by the microscope makes this imaging technique heavily dependent on a suitable computational treatment of the raw data. In particular, structure determination through Subtomogram Averaging requires the localization of large number of noisy copies of the compound of interest and extraction of the common signal through classification and alignment. This presentation focuses on several data processing challenges appearing in different stages of this technique, discussed in the context of the unified processing framework provided by the Dynamo software package. The new Catalogue module covers the logistical tasks attached to the organization of large datasets, providing specific tools for visualization and geometrical modeling of tomogram content by manual or semiautomated identification and annotation of structures as membranes, filaments or lattice-like arrangements of particles.
Further modules address the analysis of the identified subtomograms, guiding the users through the use of standardized analysis steps or rather supporting the design of new, case specific approaches. The software can harness High Performance Computing environments like CPU or GPU clusters, in order to cope with the numerically demanding nature of the technique, an aspect that will be showcased by different case studies.
Following this forum there will be a hands on demonstration of the Dynamo package, during which time attendees can see a preliminary introduction.
- Wednesday 7/8/2015
Max Planck Institute of Biophysics
Structure of mitochondrial ATP synthases and their role in shaping cristae membranes.
Mitochondria are the powerhouses of eukaryotic cells and the main site of ATP synthesis in cells performing aerobic respiration. Located in the cristae membranes, the ancient nano-machine, ATP synthase, uses the energy stored in an electrochemical gradient of protons to catalyses the conversion of ADP and inorganic phosphate to ATP. The mechanism of proton-coupled ATP synthesis has remained poorly understood for decades due to the lack of structural information on the membrane-embedded subunits of this complex. By single-particle cryo-EM analysis, we have revealed a bundle of long horizontal, membrane intrinsic helices adjacent to the rotor ring of a mitochondrial ATP synthase thereby providing a structural basis for understanding how proton movement across a membrane drives ATP synthesis. Moreover, by electron cryo-tomography and subtomogram averaging we have shown that mitochondrial ATP synthases play a major role in shaping cristae, which in turn affects cellular fitness.
- Wednesday 7/1/2015
Salk Institute for Biological Studies
Structural Investigation of Retroviral Fusion and Integration Machineries by High-Resolution CryoEM.
- Tuesday 6/30/2015
Scripps Research Institute
Near Atomic Resolution Using Automated Electron Microscopy.
Recent developments in detector hardware and image-processing software have revolutionized single particle cryo-electron microscopy (cryoEM) and led to a wave of near-atomic resolution (typically ~3.3 Å) reconstructions. Reaching resolutions higher than 3 Å is a prerequisite for structure-based drug design and for cryoEM to become widely interesting to pharmaceutical industries. We report here the structure of the 700 kDa Thermoplasma acidophilum 20S proteasome (T20S), determined at 2.8 Å resolution by single-particle cryoEM. The quality of the reconstruction enables identifying the rotameric conformation adopted by some amino-acid side chains (rotamers) and resolving ordered water molecules, in agreement with the expectations for crystal structures at similar resolutions. The results described in this manuscript demonstrate that single particle cryoEM is capable of competing with X-ray crystallography for determination of protein structures of suitable quality for rational drug design.
- Monday 6/29/2015
Salk Institute for Biological Studies
Analysis of Large-Scale Structural Heterogeneity Using CryoEM.
Single particle cryo-electron microscopy (cryoEM) is an important component of a structural biologist’s toolkit, as improvements in instrumentation, software, automation, and specimen preparation are making this technique increasingly powerful for the analysis of large (>100 kDa) macromolecules and macromolecular complexes. One of the primary advantages of the methodology is the ability to analyze heterogeneous macromolecular assemblies, i.e. those that exhibit either conformational mobility within distinct regions or compositional heterogeneity exhibited by loosely and sub-stoichiometrically associated components. By employing classification techniques, it is possible to place each individual particle image into one of several, potentially many, groups, according to homogeneity. I will describe recent advances in the use of classification techniques to decipher structural heterogeneity within macromolecular complexes by cryoEM. Using a highly heterogeneous experimental dataset of a 60S ribosomal subunit with several associated non-ribosomal cofactors, I will show how global classification tools can recover numerous conformational and compositional states of mobile components, including subnanometer resolution reconstructions of loosely associated and sub-stoichiometric proteins. I will then zoom in on remaining areas of heterogeneity using a focused classification approach to show how one can deconvolute the mobility of small mobile regions which was not possible using a global classification approach alone, including a single flexible RNA helix. Lastly, I will discuss strategies for improving classification methods and progress toward capturing and deciphering large-scale structural heterogeneity within a single cryoEM data set.
- Friday 6/19/2015
Iowa State University
Visualizing Conformational and Compositional Heterogeneity by Single-Particle EM.
Macromolecular interactions that dictate biological processes are often highly dynamic and transient, leading to conformational and compositional heterogeneity of functionally relevant complexes. Single-particle electron microscopy (EM) facilitates the structural determination of distinct species present within complex mixtures, enabling the identification of multiple biologically important conformations from a single sample. In collaboration with NRAMM, we have used automated negative-stain EM data collection and single-particle analysis to characterize the structures of particle sub-populations from highly heterogeneous samples. Automation was key to our previous studies, in which we determined the composition and conformation of in vivo ribosome assembly intermediates from Escherichia coli. Similarly, automation has been crucial for our current work, which focuses on determining the structure and oligomeric state of heterogeneous Tec kinase complexes. Tec kinases play essential roles in immune cell activation, and their activity is controlled by intramolecular interactions that may result in the formation of kinase oligomers. Our initial analysis of oligomeric Tec kinase by single-particle EM provides the first structural data for higher-order complexes formed by these enzymes and will be critical in informing our understanding of kinase autoregulation.
- Friday 6/5/2015
Vlaams Instituut voor Biotechnologie
Structural Insight into Calcium Signalling in Muscular Contraction.
Concentration of calcium ions in the cytoplasm of cells regulates many physiological processes, including transcription, cell cycle, apoptosis, and muscular contraction. Muscles are contracted at high calcium concentrations and relaxed at low calcium concentrations in the cytoplasm, and contraction is initiated by the release of Ca2+ from the sarcoplasmic reticulum into the cytoplasm of myocytes through ryanodine receptors (RyR). RyRs are the 2.2 MDa homotetrameric ion channels that are primarily gated by changes in concentration of calcium ions in the cytoplasm, and are regulated by multiple factors, including ions, small organic molecules, and interactions with other proteins. Hundreds of mutations in RyRs have been associated with human diseases, but the molecular mechanism underlying the complex regulation of RyR is still poorly understood. I will present the architecture of rabbit RyR1 determined at resolution of 6.1 Å by single particle cryo-EM, and show how changes in calcium concentration induce conformational changes in RyR resulting in the channel gating.
- Tuesday 4/28/2015
Baylor College of Medicine
Comparative Refinement as a Tool for Structural Validation.
The need for structural validation increases as electron cryomicroscopy structures go to a higher resolution. Comparative refinement utilizes the same data set that has already been collected to perform reconstructions in multiple software packages, and this can be used as a tool to perform the needed validation. When it is used in conjunction with other validation techniques, such as tilt-validation or class-average comparison, comparative refinement has the ability to show structural variability due to algorithmic bias. The greatest advantage of comparative refinement versus another validation technique, (e.g. tilt-validation), is that it requires no additional data collection. In this forum we will look at reconstructions of different specimens and examine what comparative refinement demonstrates.
- Wednesday 3/18/2015
Building Proteins in a Day: Efficient 3D Reconstruction for Cryo-EM.
Transmission electron microscopy (TEM) is a powerful technique for visualizing structure at nanometer or Angstrom resolution. However, multiple factors reduce the resolution and signal-to-noise ratio (SNR) of TEM images. These include the microscope instrumentation, dynamic specimen processes (e.g. drift, beam-induced motion, charging, radiation damage, etc.), and inefficient electron detectors. Direct detection cameras have overcome many of these obstacles, dramatically boosting the resolution and efficiency of TEMs for biology, and in addition to improved detection efficiency and resolution, the architecture of direct detection cameras allows for fast, continuous streaming with no dead time between consecutive frames (known as "movie-mode"). Several studies have already shown how movies can be exploited to correct stage drift and beam-induced specimen motion, however movies also provide intrinsic dose fractionation, which allows microscopists to choose their image exposure ex post facto by using subsets of frames from each movie. Users may also exploit movie-mode imaging with "damage compensation," which applies a low-pass filter to each movie frame based on the expected radiation damage at the corresponding cumulative specimen dose. Using this method allows microscopists to collect data with exposures beyond the conventional radiation damage limit of their specimen, which yields a very high contrast without sacrificing resolution.
This talk will introduce the DE-20 Camera System (which is now installed on the JEOL 3200FSC at NYSBC), discuss the best practices for data collection and image processing with the DE-20's movie-mode, and share recent results from Direct Electron's cameras.
- Friday 2/20/2015
Brookhaven National Laboratory
Cryo-EM for Structural Biology and DNA Nanotechnology.
I will give two examples to demonstrate the application of single particle cryo-EM in structural biology and DNA nanotechnology. The first example will be a cryo-EM investigation of the prokaryotic proteasomal activation by a novel cofactor in an ATP-independent manner. At an intermediate resolution, I showed that the cofactor enlarges the substrate entrance of prokaryotic proteasome core particles. Additionally, I identified the cofactor-binding site on the proteasomes. The second example will be a visualization of 3D architectures inside inorganic metal nanoparticle clusters that are organized by a robust designer DNA frame. DNA-templated self-assembly is an efficient way to achieve programmable spatial arrangement of nanostructured clusters, such as nanoparticles. However self-assembled 3D architectures are difficult to characterize; I show that cryo-EM is an ideal tool to address this challenge, because it is capable of obtaining high-resolution 3D structures with minimal structural disturbance. By adapting the existing computational image processing methods, I found that cryo-EM should be a very useful tool for characterizing 3D bio-hybrid systems in the future.
- Friday 1/23/2015
Buchmann Institute for Molecular Life Sciences
3D Electron Microscopy on Different Scales and Resolution: Future Directions
Modern 3D electron microscopy methods cover a large variety of applications at different resolutions. They serve to further elucidate the structure of macromolecular complexes or to visualize the cellular structure of larger tissue compartments. These methods will be demonstrated using three examples of biological research.
First, using cryo-electron tomography (cryoET) and sub tomogram averaging we were able to resolve the mouse liver desmosomal ultrastructure. The cryoET data from these experiments yielded insights into the native organization of epithelial cell junctions of mammalian desmosomes.
Second, our developments in sub-nanometer cryo tomography will be presented. By optimizing sample preparation, tomogram acquisition and post processing techniques we were able to visualize secondary structural features of tobacco mosaic virus coat proteins for the first time.
Third, our developments using FIB/SEM for 3D electron microscopy will be discussed. The application of FIB/SEM in conjunction with a versatile correlative light EM (CLEM) approach with high spatial precision will be presented along with the analysis of complete parts of the C.elegans nerve cord structure and its synaptic vesicles.