Frontiers and Careers in cryoEM Symposium | April 27-28, 2018
“How CryoEM is helping to understand the molecular basis of neurodegeneration”
Some 40 devastating diseases are all associated with protein fibrils termed amyloid. Some of these diseases are among the most prevalent and the most burdensome to individuals, families, and society, such as Alzheimer’s, Parkinson’s and other neurodegernative diseases, and diabetes type 2. Each disease is associated with one or a few particular proteins that form amyloid fibrils. Despite thousands of scientific papers published every year about amyloid, not a single drug is available that halts progression of these conditions. One reason may be that pharma has ignored the power of molecular structures to speed drugs. Recently both x-ray diffraction and several forms of cryoEM have contributed structures relevant to understanding amyloid conditions. These methods include micro-electron diffraction, single-particle averaging, and cryo-tomagraphy. Once amyloid structures are known, scientists can design inhibitors of amyloid formation which may halt disease progression.
“In Situ Structures of the Bacterial Type IVB Secretion System by Electron Cryotomography”
Bacteria harbor at least nine different types of secretion systems to transfer macromolecules across cellular envelopes. The type IV secretion system (T4SS) is arguably the most elaborate and diverse, and central to the pathogenesis of multiple human diseases. Based on their composition and genetic organization, the T4SS family has been classified into two major groups: type IVA (T4ASS) and type IVB (T4BSS). The T4BSS is prevalent in some human pathogens such as Legionella pneumophila and Coxiella burnetii, and is more complex than most T4ASS with three times as many components.
In this study, using electron cryotomography (ECT) and subtomogram averaging, we report the in situ structure of the Dot/Icm T4BSS utilized by the human pathogen L. pneumophila. This is the first structure of a T4BSS and also the first structure of any T4SS in situ. The L. pneumophila Dot/Icm T4BSS is comprised of two distinct curved layers – the larger just below the outer membrane (OM) and the smaller in the middle of the periplasm with an overall characteristic shape of a “Wi-Fi” symbol. We also resolved some of the inner membrane (IM) associated cytoplasmic densities that represent locations of the ATPases. By systematically imaging mutants lacking defined T4BSS components, we have mapped the locations of many of the outer membrane and periplasmic components, thereby providing insights into the assembly process and structural organization of this complex.
“MicroED opens a new era for biological structure determination”
My laboratory studies the structures of membrane proteins that are important in maintaining homeostasis in the brain. Understanding structure (and hence function) requires scientists to build an atomic resolution map of every atom in the protein of interest, that is, an atomic structural model of the protein of interest captured in various functional states. In 2013 we unveiled the method MicroED, electron diffraction of microscopic crystals, and demonstrated that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). The CryoEM is used in diffraction mode for structural analysis of proteins of interest using vanishingly small crystals. The crystals are often a billion times smaller in volume than what is normally used for other structural biology methods like x-ray crystallography. In this seminar I will describe the basics of this method, from concept to data collection, analysis and structure determination, and illustrate how samples that were previously unattainable can now be studied by MicroED. I will conclude by highlighting how this new method is helping us understand major brain diseases like Parkinson’s disease.
“Unraveling the molecular details of mammalian cell nanoarchitecture: tying cryo-light microscopy and cellular tomography”
Dorit Hanein is a Professor at the Sanford Burnham Prebys Medical Discovery Institute (SBP) in La Jolla, California with an adjunct appointment in the Pathology Department at University of California, San Diego. Dr. Hanein benefitted from a unique training that combines physical chemistry, biochemistry, and structural biology. Dr. Hanein received her Ph.D. in Chemistry from The Weizmann Institute, Israel, and pursued a Fulbright postdoctoral fellowship with Dr. David DeRosier, one of the founders of 3D image TEM analysis, at Brandeis University and Dr. Paul Mastudaira at MIT, Whitehead Institute.
Central to Dr. Hanein’s research is a critical question in cell biology: how do cells employ large, macromolecular machines in cellular processes? To pursue these studies, Hanein’s laboratory pushes the envelope in developing and employing a unique set of powerful tools, merging state-of-the-art light and electron microscopy imaging with micromechanical engineering, computational methods, cell biology, and protein biochemistry. Currently, Dr. Hanein is employing a new generation of electron cryo-microscopy equipment at SBP, including a Titan Krios electron cryo-microscope, to broaden and extend the development of this hybrid technology platform into a robust workflow. The quantitative analysis of molecular machines in unperturbed, intact mammalian cells, at nanometer resolution, would provide the means to resolve single cytoskeletal filaments and macromolecular complexes.
Here, Dr. Hanein will describe advances in direct visualization and quantification of single molecular components of the girders and cables that control the shapes and movements of cells. These mechanosensitive assemblies transmit force across the cell membrane and regulate biochemical signals in response to changes in the mechanical environment. These combined functions in force transmission, signaling and mechanosensing are crucial for cell behaviors in development, homeostasis and disease. Despite advances in our understanding of the protein composition, interactions and regulation if these large multiprotein assemblies, our understanding of how forces affect their dynamic organization and how they induce specific signaling events remains limited. Insights across multiple structural levels are acutely needed to establish the molecular basis of mechanotransduction.
Dr. Hanein will describe how the dedicated effort in the development of scale integration techniques to tie the meso with the nano scale, harnessing correlative light microscopy guided by designated fluorescence in conjunction with electron cryo-tomography, can be used to identify macromolecular assemblies and their three-dimensional architecture at the nano scale in intact mammalian cells. The new generation cryo-TEM hardware, combined with these correlative approaches, and once tied to image analysis and data mining, allow to directly view the nano-architecture of single molecular components of these fascinating mechano-sensing nanomachines while in their native cellular environment. The resulting quantitative integration of scales between macroscopic cellular behavior and high-resolution structural changes will position these efforts at the front end of science with high impact in both medicine and basic biological research.
“CryoEM study of anion exchanger 1 reveals multiple conformations”
Anion exchanger 1 (AE1; also known as Band 3) is the major membrane protein in red blood cell membrane that plays a key role in Cl?/HCO3? exchange and anchoring of cytoskeletal proteins. Full-length AE1 exists as obligated dimers under physiological conditions. Each protomer of AE1 consists of an N-terminal cytoplasmic domain and a C-terminal membrane domain whose crystal structures are known individually; however, how the cytoplasmic and membrane domains coordinate with one another and how the two protomers work together as a transporter are unknown. Here, we have determined the structure of full-length AE1 at near atomic resolution by cryo electron microscopy (cryoEM), revealing multiple conformations at different states of anion exchange.
“No Phase Plate? No Problem! Smaller and Smaller using conventional TEM approaches”
Determining the high-resolution structures of sub-100kDa complexes that have been recalcitrant to crystallization has been a long-term goal of the cryo-EM community. Recently, the Volta Phase Plate has used to solve the structures of numerous small biological specimens, and it is now widely accepted that resolving small-sized samples is only possible with a phase plate. We show that it is possible to solve high-resolution structures of asymmetric, conformationally flexible specimens that are smaller than 100 kDa using conventional cryo-EM methodologies, without the need for a phase plate or energy filter.
“In Situ structural biology – Advances in Cryo-Electron Microscopy“
Cryo-transmission electron microscopy (Cryo-TEM), and particularly single particle analysis, is rapidly becoming the premier method for determining the three dimensional structure of protein complexes, and viruses. In the last several years there have been dramatic technological improvements in Cryo-TEM, such as advancements in automation and use of improved direct electron detectors, as well as improved image processing techniques. Furthermore it is now becoming possible to determine the structure of proteins in situ with the help of focused ion beam technology. Many cells are too thick to study intact in cryo electron tomography. Before the interior of a frozen hydrated cell can be imaged, it must be thinned to electron transparency while maintaining cryogenic conditions. In order to do so, a Aquilos Cryo DualBeam(tm) cryo focused-ion-beam (cryo-FIB) microscope is used. Utilizing the ion beam of the cryo-FIB microscope, frozen specimens are thinned down to the appropriate thickness of 200-300 nm. Cryo-FIB milling [1-4] opens relatively large and distortion-free windows into the cell’s interior, enabling targeting of structural features within the cellular context. Using a so called in-situ lamella milling or on-the-grid thinning strategy allows directly targeting vitrified cells on EM grids with the ion beam. Thereby, multiple regions of interest, containing high quality electron cryo-lamellas, can be created straight on EM grids. These grids, harboring cryolamellas, are then transferred to a Titan Krios(tm) transmission electron microscope for high-resolution imaging by cryo electron tomography.
“Single-Particle Cryo-EM Studies of lipopolysaccharide transport”
The transbilayer movement of most lipids is energetically unfavorable and requires the facilitation by lipid flippases. The mechanisms by which flippases recognize and translocate specific lipid molecules have remained elusive. Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for their cell envelope assembly. LPS synthesized in the cytoplasmic leaflet of the inner membrane is flipped to the periplasmic leaflet by MsbA, an ATP-binding cassette transporter.
We have used single-particle cryo-EM to elucidate the structures of lipid nanodisc-embedded MsbA in three functional states. Our study uncovers the long-sought-after structural basis for LPS recognition by MsbA, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases.
“High-throughput cryo-electron tomography: Visualizing transient channel during viral infection”
While recent breakthrough has revolutionized single particle cryo-electron microscopy as a major tool for structural biology, cryo-electron tomography (cryo-ET) is the method of choice for visualizing native cellular landscapes with the potential to achieve near-atomic resolution in conjunction with advanced image analysis. However, the technique is far from mature and its full potential still needs to be realized. My laboratory has been dedicated in developing a high-throughput cryo-ET pipeline in which both data collection and image analysis have been streamlined and automated in every possible way. Our high-throughput cryo-ET pipeline has become increasingly powerful, not only enabling us to visualize over 100,000 cells in last 10 years, but also providing unlimited opportunities to visualize in situ structures of molecular machines in their native cellular context at high resolution not possible by any other technique. Here, I will discuss some of our recent progresses in using high-throughput cryo-ET to visualize transient channel during bacteriophage infection.
“New methods and developments in cryoSPARC”
As single-particle cryo-EM makes rapid progress, several computational challenges remain in the quest for higher resolutions on challenging targets, as well as in the widespread and routine use of cryo-EM. This talk will introduce new methods in the cryo-EM data processing pipeline, along with implementations within the cryoSPARC software system, to address some of these challenges. Specifically, new algorithms for motion correction, particle picking, and refinement of membrane proteins will be discussed. The talk will also cover new tools for workflow management and automation of the processing pipeline.
“Atomic Resolution Electron Diffraction of Amyloid Nano-Structures”
Despite modern advances in structural biology, structures of many biomedically relevant macromolecular assemblies remain out of reach or lack atomic resolution detail. Large crystals required for conventional crystallography experiments are a challenge to grow, and determination of structures from small or imperfect crystals by x-ray crystallography remains limited. Cryo-electron microscopy (cryo-EM) methods promise to bring new life to high-throughput approaches in macromolecular structure determination through methods like micro electron diffraction (MicroED). MicroED exploits the strong interaction between electrons and nano-scale three-dimensional crystals by leveraging emerging cryo-EM instrumentation against established crystallographic knowledge. These technological advances, coupled with the greater availability of advanced cryoEM instruments, present an opportunity for further improvement of high-throughput structure determination. The development of new and more efficient approaches to structure determination by MicroED allow us to determine structures from small, fragile, and imperfect crystals. We focus on infectious and/or toxic filamentous nanoassemblies associated with disease including prion structures. Through our efforts in determining these challenging structures, we find inspiration to guide the improvement and development of cryoEM techniques, particularly MicroED.
“Supporting drug discovery with cryo-EM and image processing”
In 2017 Genentech’s Structural Biology department established an internal cryo-EM effort to better support drug discovery and biological research, particularly for projects where protein targets are difficult to study with X-ray crystallography. I will give an overview of the role of cryo-EM within Genentech, including requirements on information management, number of supported projects, target turnaround times and resolution, etc. Recent advances in cryo-EM software development supported by Genentech will also be described.
“Structural studies of coronavirus fusion proteins”
Coronaviruses are enveloped viruses responsible for 30% of mild respiratory infections and atypical pneumonia in humans worldwide. The emergence of the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and of the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 demonstrated these zoonotic viruses can transmit to humans from various animal species and suggested that additional emergence events are likely to occur. The fatality rate of SARS-CoV and MERS-CoV infections are about 10-37% and there are no approved antiviral treatments or vaccines against human coronaviruses.
Entry of coronaviruses into cells is mediated by the trimeric transmembrane spike glycoprotein, which carries receptor-binding and membrane fusion functions. It also contains the principal antigenic determinants and is the target of neutralizing antibodies during infection. Coronavirus spike glycoproteins had proven reluctant to structural characterization using traditional approaches since these proteins are metastable and heavily glycosylated. We determined the first high-resolution structure of a coronavirus spike glycoprotein trimer using cryoEM and showed that coronavirus spike and paramyxovirus F proteins share a common core, implicating mechanistic similarities and an evolutionary connection between these viral fusion proteins. We subsequently determined 3D reconstructions of the spike glycoprotein trimer of HCoV-NL63, which is an ?-coronavirus that can cause severe lower-respiratory-tract infections requiring hospitalization, and of a porcine deltacoronavirus (PDCoV) with a major impact for the food industry. The significant resolution improvement as compared with earlier studies revealed the presence of an extended glycan shield obstructing the protein surface and provided a structural framework to understand accessibility to neutralizing antibodies. Our results also suggested that HCoV-NL63, PDCoV and other coronaviruses use molecular trickery, based on epitope masking with glycans and activating conformational changes, to evade the immune system of infected hosts, in a manner similar to that described for HIV-1.
Most recently, we obtained the first snapshot of a coronavirus spike glycoprotein in the post fusion conformation using high-resolution cryoEM and biochemical techniques. The outcomes of this study defined the conformational trajectory undergone by this molecular machine to drive fusion of the viral and host membranes (i.e. cell entry) and elucidated the mode of action of several neutralizing antibodies against these viruses.
Rebecca M. Voorhees
“Mechanism of protein targeting to the endoplasmic reticulum”
Secreted and integral membrane proteins compose ~30% of the eukaryotic proteome and are essential for a range of cellular functions including intracellular trafficking, cell signaling, and the transport of molecules across the lipid bilayer. Defects in membrane protein maturation underlie numerous protein misfolding diseases, and more than half of all therapeutic drugs bind a membrane protein target. The essential roles of these proteins, as well as the consequences of their failed maturation, underscore the importance of understanding the molecular details of membrane protein biogenesis.
The majority of secreted and integral membrane proteins are co-translationally targeted to the ER by the signal recognition particle (SRP), and are then inserted into the lipid bilayer by the universally conserved translocation channel, Sec61. Accurate sorting of proteins to the ER is essential to maintaining cellular homeostasis and preventing protein mislocalization, which can lead to disease. Specificity during this process is achieved via two selection steps: the first carried out by SRP in the cytosol, and the second by Sec61 at the membrane. In order to examine the molecular basis for substrate recognition, we have used cryo-electron microscopy (cryo-EM) to visualize a series of structures that trace the path of a hydrophobic sequence from its recognition by SRP to its insertion and translocation by Sec61. Together these structures suggest a common strategy employed by both SRP and Sec61 for selective recognition of hydrophobic sequences that ensures fidelity during membrane protein targeting.
“Structural Investigation of Excitation-Contraction Coupling”
The voltage-gated calcium (Cav) channels convert membrane electrical signals to intracellular Ca2+-mediated events. Malfunction or dysregulation of Cav channels are associated with various neurological, cardiovascular, and muscular disorders, making Cav channels major targets for drug development. Among the ten subtypes of Cav channels in mammals, Cav1.1 is specified for the excitation–contraction coupling of skeletal muscles, acting as a voltage sensor for the downstream calcium release channel RyR1 in the sarcoplasmic reticulum membrane. Different from their prokaryotic homologs, the eukaryotic Cav channels are always constituted by multiple subunits, forming a large complex. The ion-conducting ?1 subunit is a single peptide chain with four homologous but non-identical repeats. Here we have determined the first near atomic resolution structure of a eukaryotic Cav channel Cav1.1 by cryo electron microscopy (cryoEM). The structure reveals the pseudo fourfold symmetric ?1 subunit and its interactions with the auxiliary ?2?, ? and ? subunits. 3D Classification of the particles yielded two reconstructions that reveal pronounced conformational changes in the intracellular segments of the protein. Combining with the structural studies in RyR1 from skeletal muscles, our work has suggested a mechanistic understanding of excitation–contraction coupling. The Cav1.1 structure provides a three-dimensional template for molecular interpretations of the functions and disease mechanisms of Cav and related Nav channels and also provides the basis for structure-guided drug design.
Z. Hong Zhou
“In situ structures of viral genome-protein complexes in action”
Direct electron-counting cryoEM combined with advanced computational processing now offers new opportunities to determine structures of genomes and genome replication/transcription in action. Towards this end, viruses are the subject of choice as they are the simplest replicating machines that package their DNA or RNA genomes with a minimal set of proteins to sustain survival and spread. A prominent example is dsRNA viruses in the Reoviridae that carry out endogenous mRNA synthesis through a transcriptional enzyme complex (TEC). I will present the organization of the dsRNA genome inside quiescent cytoplasmic polyhedrosis virus CPV (q-CPV) and the in situ atomic structures of TEC within CPV in the quiescent and transcribing (t-CPV) states. We show that the 10 segmented dsRNAs are organized with 10 TECs in a specific, non-symmetric manner, with a dsRNA segment attached directly to each TEC. TEC consists of two extensively-interacting subunits: an RNA-dependent RNA polymerase (RdRP) subunit and an NTPase subunit, VP4. By comparing the TEC structures in q-CPV and t-CPV, we find that the bracelet domain of RdRP undergoes significant conformational change, leading to formation of the RNA template entry channel and access to the polymerase active site. The N-terminal helix from each of the two subunits of the capsid shell protein (CSP) interacts with VP4 and RdRP. These findings establish the missing link between sensing of external cues by the CSP and activation of endogenous RNA transcription by the TEC inside the virus. The work highlights new opportunities offered by electron-counting cryoEM to obtain in situ structures of viral RNA and DNA genomes and genome replication for viruses and other organisms.