High-resolution cryo-EM Structures Research Articles

Sub-3 Å Cryo-EM Structures of Necrosis Virus Particles via the Use of Multipurpose TEM with Electron Counting Camera.

During this global pandemic, cryo-EM has made a great impact on the structure determination of COVID-19 proteins. However, nearly all high-resolution results are based on data acquired on state-of-the-art microscopes where their availability is restricted to a number of centers across the globe with the studies on infectious viruses being further regulated or forbidden. One potential remedy is to employ multipurpose microscopes. Here, we investigated the capability of 200 kV multipurpose microscopes equipped with a direct electron camera in determining the structures of infectious particles. We used 30 nm particles of the grouper nerve necrosis virus as a test sample and obtained the cryo-EM structure with a resolution as high as ∼2.7 Å from a setting that used electron counting. For comparison, we tested a high-end cryo-EM (Talos Arctica) using a similar virus (Macrobrachium rosenbergii nodavirus) to obtain virtually the same resolution. Those results revealed that the resolution is ultimately limited by the depth of field. Our work updates the density maps of these viruses at the sub-3Å level to allow for building accurate atomic models from de novo to provide structural insights into the assembly of the capsids. Importantly, this study demonstrated that multipurpose TEMs are capable of the high-resolution cryo-EM structure determination of infectious particles and is thus germane to the research on pandemics.

Abstract P-29: Cryoem Study of the Inhibition of Bacterial Ribosomes by Madumycin II

Background: The efficiency of widely used antibiotics is limited by continuous improvement of resistance mechanisms. Thus, the research of poorly studied drugs that have not received practical use until now becomes relevant again. Protein translation is one of the major targets for antibiotics. Madumycin II (MADU) is an antibiotic of the streptogramin A class that binds to the peptidyl transferase center of the initiated bacterial 70S ribosome inhibiting the first cycle of peptide bond formation (I.A. Osterman et al. Nucleic Acids Res., 2017). The ability of MADU to interfere with translating ribosome is an open question that we address by investigation of high-resolution cryo-EM structures of MADU bound 70S ribosome complexes from Escherichia coli. Methods: Purified initiated and translating ribosome complexes preincubated with MADU were applied onto freshly glow discharged carbon-coated grids (Quantifoil R 1.2/1.3) and flash-frozen in the liquid ethane pre-cooled by liquid nitrogen in the Vitrobot Mark IV. Frozen grids were transferred into an in-house Titan Krios microscope. Data were collected using EPU software. Movie stacks were preprocessed in Warp software. For image processing, we have used several software packages: Relion 3.1, CryoSPARC, and CisTEM. The model was built in Coot. Results: We have obtained high-resolution cryo-EM structures of two ribosomal complexes with MADU before and after the first cycle of peptide bond formation with an average resolution of 2.3 Å. Preliminary analysis of the structures shows no major differences in the MADU binding mode to the ribosomal complexes under study suggesting that the quantity of amino acid residues attached to the P-site tRNA does not impact MADU bonding. Moreover, in both cases, we observed similar destabilization of the CCA-ends of A- and P-site tRNAs underlining the comparable influence of MADU on the ribosomal complexes. Conclusion: Our results suggest that although MADU binding site is located in the peptidyl transferase center, the presence of the second amino acid residue on the P-site tRNA does not preclude antibiotic binding. We assume that further elongation of the polypeptide chain would not have any impact either. High conformational lability of the CCA-ends of tRNA at the A and P sites upon binding of MADU obviously plays an important role in the inhibition mechanism of the bacterial ribosome. The further structural and biochemical analysis will be necessary to shed more light on the detailed mechanism of MADU action.

Practical considerations for using K3 cameras in CDS mode for high-resolution and high-throughput single particle cryo-EM

Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed standard (nonCDS) mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 Å with a 1.346 Å physical pixel size). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834 Å physical pixel size) we achieved a resolution of 1.65 Å within 8-h of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 Å resolution with 5 min of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.

Retinal IMP Dehydrogenase Resist GTP Feedback Inhibition by Filamentous Polymerization

Dynamic assembly of metabolic enzymes is an evolutionarily conserved mechanism for the physical organization of metabolism. IMP dehydrogenase (IMPDH), is a highly-conserved enzyme that catalyzes the first committed step in GTP synthesis and forms dynamic filamentous assemblies in vertebrate cells. Humans have two isoforms of the protein: IMPDH2 which is upregulated in dividing cells and IMPDH1 which is expressed at low levels as the housekeeping enzyme in most cells. IMPDH2 assembles into filaments to tune feedback inhibition by the downstream product GTP, however the consequences of IMPDH1 polymerization are unclear. This question is made particularly compelling because there are 9 mutations in IMPDH1 that lead to blindness in humans, 8 of which cluster around the nucleotide binding sites that mediate filament assembly in IMPDH2. Moreover, IMPDH1 is produced as 2 unique splice variants in the retina, and their influence on assembly and regulation remain unexplored. Here we show that IMPDH1 polymerization does not confer increased resistance to GTP-inhibition, unlike the previously-studied isoform IMPDH2. We present high-resolution cryo-electron microscopy (cryoEM) structures that show IMPDH1 forms a novel filament when bound to GTP, providing a structural explanation for this difference in isoforms. Using kinetic assays we demonstrate that IMPDH1 retinal splice variants are four times less sensitive to GTP-inhibition than canonical IMPDH1, an effect that is specific to the retinal protein being assembled into a filament. We present a high-resolution cryoEM structure of one of the IMPDH1 retinal variants bound to GTP that shows it forms a filament identical to IMPDH2, explaining why polymerization increases resistance to GTP inhibition. We anticipate these findings to provide a foundation for understanding why mutations in IMPDH1 lead to blindness in humans and a more robust investigation of splice variants of other metabolic enzymes that form dynamic assemblies.

How to build a proteasome.

The biogenesis of eukaryotic 20S proteasomes requires the accurate assembly of 14 closely related protein subunits and occurs in a complex series of chaperone-dependent steps. Important insights into this pathway are now provided by the high-resolution cryo-EM structures of two 20S proteasome assembly intermediates.

High-resolution cryo-EM structure of photosystem II reveals damage from high-dose electron beams

Photosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallography has revealed its atomic structure and some intermediate structures. However, these structures are in the crystalline state and its final state structure has not been solved. Here we analyzed the structure of PSII in solution at 1.95 Å resolution by single-particle cryo-electron microscopy (cryo-EM). The structure obtained is similar to the crystal structure, but a PsbY subunit was visible in the cryo-EM structure, indicating that it represents its physiological state more closely. Electron beam damage was observed at a high-dose in the regions that were easily affected by redox states, and reducing the beam dosage by reducing frames from 50 to 2 yielded a similar resolution but reduced the damage remarkably. This study will serve as a good indicator for determining damage-free cryo-EM structures of not only PSII but also all biological samples, especially redox-active metalloproteins.

Structural insights into the assembly and substrate selectivity of human SPT-ORMDL3 complex.

Human serine palmitoyltransferase (SPT) complex catalyzes the initial and rate-limiting step in the de novo biosynthesis of all sphingolipids. ORMDLs regulate SPT function, with human ORMDL3 being related to asthma. Here we report three high-resolution cryo-EM structures: the human SPT complex, composed of SPTLC1, SPTLC2 and SPTssa; the SPT-ORMDL3 complex; and the SPT-ORMDL3 complex bound to two substrates, PLP-L-serine (PLS) and a non-reactive palmitoyl-CoA analogue. SPTLC1 and SPTLC2 form a dimer of heterodimers as the catalytic core. SPTssa participates in acyl-CoA coordination, thereby stimulating the SPT activity and regulating the substrate selectivity. ORMDL3 is located in the center of the complex, serving to stabilize the SPT assembly. Our structural and biochemical analyses provide a molecular basis for the assembly and substrate selectivity of the SPT and SPT-ORMDL3 complexes, and lay a foundation for mechanistic understanding of sphingolipid homeostasis and for related therapeutic drug development.

Cryo-EM structures reveal the molecular basis of receptor-initiated coxsackievirus uncoating

Enterovirus uncoating receptors bind at the surface depression ("canyon") that encircles each capsid vertex causing the release of a host-derived lipid called "pocket factor" that is buried in a hydrophobic pocket formed by the major viral capsid protein, VP1. Coxsackievirus and adenovirus receptor (CAR) is a universal uncoating receptor of group B coxsackieviruses (CVB). Here, we present five high-resolution cryoEM structures of CVB representing different stages of virus infection. Structural comparisons show that the CAR penetrates deeper into the canyon than other uncoating receptors, leading to a cascade of events: collapse of the VP1 hydrophobic pocket, high-efficiency release of the pocket factor and viral uncoating and genome release under neutral pH, as compared with low pH. Furthermore, we identified a potent therapeutic antibody that can neutralize viral infection by interfering with virion-CAR interactions, destabilizing the capsid and inducing virion disruption. Together, these results define the structural basis of CVB cell entry and antibody neutralization.

GOLEM: Automated and Robust Cryo-EM-Guided Ligand Docking with Explicit Water Molecules

A detailed understanding of ligand-protein interactions underpins rational drug-design strategies. In recent years, technological advances in cryo-electron microscopy (cryo-EM) brought a new era for the structural determination of biological macromolecules and assemblies at a high resolution, marking cryo-EM a promising tool for studying ligand-protein interactions. However, even in high-resolution cryo-EM structures, the bound ligand's densities are often of lower quality due to its dynamic nature, frustrating an accurate coordinate assignment. Computational methods have been offering complementary efforts to aid structure modeling, such as MDFF (molecular dynamics flexible fitting) to fit atomic structures of macromolecules into corresponding cryo-EM densities. To address the challenge of ligand modeling in cryo-EM maps, here we report the development and validation of GOLEM (Genetic Optimization of Ligand in Experimental Maps), an automated and robust ligand docking method that models the ligand's optimal pose and conformation in cryo-EM maps. GOLEM employs a Lamarckian genetic algorithm, which performs a hybrid global/local search to explore the ligand's conformational, orientational, and positional space, with explicit consideration of water displacement and bridging water molecules' position and orientation. GOLEM takes into account both energetics and the correlation with the electron density maps in its scoring function, which is a combination of the system's energy and the energy of the ligand in a cryo-EM-derived external potential. GOLEM is validated in multiple cryo-EM structures with a wide range of map resolutions and ligand types, producing ligand poses in excellent agreement with the densities. As a VMD plugin, GOLEM is free-of-charge and accessible to the community. With these features, we believe GOLEM will be a valuable tool for ligand modeling in cryo-EM efforts towards drug discoveries.

High Resolution Cryo-Em Structure of the EspA Filament from EPEC

Antimicrobial resistance is a growing concern for the global population, predominantly in animal husbandry, hospitals, and developing countries. Several pathogens are of concern, including Escherichia coli. The rise in resistance has contributed to the decline in development of novel antibiotics, thus pushing us closer to a pre-antibiotic era. Virulence mechanisms that bacteria use are often not required for their survival and therefore are promising targets for the development of anti-virulence compounds. The T3SS is a proteinaceous channel that spans the inner and outer membranes of the bacterial cell, projecting into the extracellular medium where it interacts with the host cell membrane to deliver virulence factors. The main components of the T3SS are: (1) the basal body, spanning the inner and outer membranes of the bacterial cell; (2) the needle that projects from the basal body, and (3) the tip and pore-forming translocon that completes the channel from bacteria to host. Entero-pathogenic and -hemorrhagic E. coli (EPEC/EHEC) possess a distinct T3SS from that of other species; EPEC/EHEC possess a needle extension, or filament, in place of the ‘tip’ component, called EspA. The function of EspA is required for colonization of EPEC within the gut and espA EPEC knockouts are associated with decreased virulence. Here we describe the cryo-EM structure of natively-sheared EspA filaments from EPEC, determined to 3.6 Å resolution. Within the filament lumen, a pattern of positively charged residues adjacent to a hydrophobic groove lines the lumen of the filament in a spiral manner, suggesting a mechanism of translocation through the channel mediated via electrostatics. Using structure-guided mutagenesis of these residues, in vivo studies corroborate the role of these residues in secretion function. The high-resolution structure of the EspA filament will aid in structure-guided drug design of novel antivirulence therapeutics.

Role of Individual Zinc Binding Sites in the Cation Diffusion Facilitator YIIP

YiiP is a dimeric, zinc/proton bacterial antiporter that belongs to the family of Cation Diffusion Facilitators. YiiP contributes to zinc homeostasis by exporting Zn(II) from the cytosol to the periplasm. YiiP has three Zn(II) binding sites: site a in the membrane domain, site B in an intracellular loop between transmembrane helices M2 and M3, and site C harboring two ions at the dimer interface between the C-terminal domains (CTD) of the homodimer. Here we present high-resolution cryoEM structures of YiiP in complex with a Fab antibody fragment in the presence and absence of Zn(II). Comparison of these structures reveals a dramatic conformational change that affects the accessibility of the transport sites. This conformational change includes bending of M2 and M5 helices, disordering of the M2-M3 loop, and a ∼25° kink between the CTD and the membrane domain. These results are consistent with Molecular Dynamics simulations that show how the M2-M3 loop becomes flexible without the stabilizing effect of Zn(II) bound in site B. To further characterize the role of individual Zn(II) sites in these structural changes, single-site mutations were introduced at each site: D51A at site A, D70A at site B, and D287A at site C. All mutations reduced the stoichiometry of Zn(II) binding, measured by atomic absorption spectroscopy, and abolished Zn(II) transport. Cryo-EM structures of the three mutants were obtained from detergent solubilized YiiP-Fab complexes that were fully loaded with Zn(II). Structures of D51A and D287A mutants revealed conformations comparable to the holo state. In contrast, the D70A mutant produced a structure that resembled the apo state. These findings suggest that binding of Zn(II) to site B induces a conformational change that allows Zn(II) to bind to the transport sites in advance of the transport process.

A Pipeline for Integrative Structural Modelling of Very Complex Molecular Complexes

Although electron microscopy (EM) allows determining the structure of macromolecular complexes at near-atomic resolution, high resolution is still difficult to achieve for complexes that are flexible, heterogenous, or imaged in cells with cryo-electron tomography. A state-of-the-art methodology to obtain structures of such complexes is integrative structural modelling, which combines data from complementary techniques, such as X-ray crystallography, EM, NMR, SAXS or cross-linking mass spectrometry. The current integrative modelling software, however, is either limited to complexes of simple architectures or requires custom modifications, and thus a major expertise, when applied to more complex architectures. Thus, we developed a versatile modelling pipeline for integrative modelling of protein complexes with very complex architecture in an accessible and efficient way. Our pipeline is implemented using Integrative Modeling Platform library, on top of which it implements custom algorithms for efficient sampling of the conformational space, versatile configuration language and graphical interface for input preparation, modelling and analysis, and additional restraints. Flexible and symmetrical modeling is also supported. The pipeline enables modeling of very complex architectures, for example, with multiple symmetries or with EM maps for alternative conformational states. I will present the pipeline and how we applied it to model complexes ranging from the 1 MDa Elongator to the 110 MDa human nuclear pore complex (NPC). For the Elongator complex, I will present how our published model agrees with the subsequent high-resolution cryo-EM structure. For the NPC, I will show our most recent results of how the pipeline allowed building structural models of NPCs from yeast S. cerevisiae and S. pombe, including a novel multi-state modelling protocol for modelling based on multiple EM maps simultaneously. The new NPC models reveal surprisingly different architectures and depict conformational changes of NPCs in response to cellular states.

Refinement of a cryo-EM structure of hERG: Bridging structure and function

The human-ether-a-go-go-related gene (hERG) encodes the voltage-gated potassium channel (KCNH2 or Kv11.1, commonly known as hERG). This channel plays a pivotal role in the stability of phase 3 repolarization of the cardiac action potential. Although a high-resolution cryo-EM structure is available for its depolarized (open) state, the structure surprisingly did not feature many functionally important interactions established by previous biochemical and electrophysiology experiments. Using molecular dynamics flexible fitting (MDFF), we refined the structure and recovered the missing functionally relevant salt bridges in hERG in its depolarized state. We also performed electrophysiology experiments to confirm the functional relevance of a novel salt bridge predicted by our refinement protocol. Our work shows how refinement of a high-resolution cryo-EM structure helps to bridge the existing gap between the structure and function in the voltage-sensing domain (VSD) of hERG.

Single-Particle Cryo-EM of Membrane Proteins.

In the recent years, the protein databank has been fueled by the exponential growth of high-resolution electron cryo-microscopy (cryo-EM) structures. This trend will be further accelerated through the continuous software and method developments and the increasing availability of imaging centers, which will open cryo-EM to a wide array of researchers with their diverse scientific goals and questions. Especially for structural biology of membrane proteins, cryo-EM offers significant advantages as it can overcome multiple limitations of classical methods. Most importantly, in cryo-EM, the sample is prepared as a vitrified suspension, which abolishes the need for crystallization, reduces the required sample amount and allows usage of a wide arsenal of hydrophobic environments. Despite recent improvements, high-resolution cryo-EM still poses some significant challenges, and standardized procedures, especially for the characterization of membrane proteins, are missing. While there can be no ultimate recipe toward a high-resolution cryo-EM structure for every membrane protein, certain factors seem to be universally relevant. Here, we share the protocols that have been successfully used in our laboratory. We hope that this may be a useful resource to other researchers in the field and may increase their chances of success.

High-resolution structures of multiple 5-HT3AR-setron complexes reveal a novel mechanism of competitive inhibition.

Serotonin receptors (5-HT3AR) play a crucial role in regulating gut movement, and are the principal target of setrons, a class of high-affinity competitive antagonists, used in the management of nausea and vomiting associated with radiation and chemotherapies. Structural insights into setron-binding poses and their inhibitory mechanisms are just beginning to emerge. Here, we present high-resolution cryo-EM structures of full-length 5-HT3AR in complex with palonosetron, ondansetron, and alosetron. Molecular dynamic simulations of these structures embedded in a fully-hydrated lipid environment assessed the stability of ligand-binding poses and drug-target interactions over time. Together with simulation results of apo- and serotonin-bound 5-HT3AR, the study reveals a distinct interaction fingerprint between the various setrons and binding-pocket residues that may underlie their diverse affinities. In addition, varying degrees of conformational change in the setron-5-HT3AR structures, throughout the channel and particularly along the channel activation pathway, suggests a novel mechanism of competitive inhibition.

High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex

Heart contraction depends on a complicated array of interactions between sarcomeric proteins required to convert chemical energy into mechanical force. Cyclic interactions between actin and myosin molecules, controlled by troponin and tropomyosin, generate the sliding force between the actin-based thin and myosin-based thick filaments. Alterations in this sophisticated system due to missense mutations can lead to cardiovascular diseases. Numerous structural studies proposed pathological mechanisms of missense mutations at the myosin-myosin, actin-tropomyosin, and tropomyosin-troponin interfaces. However, despite the central role of actomyosin interactions a detailed structural description of the cardiac actomyosin interface remained unknown. Here, we report a cryo-EM structure of a cardiac actomyosin complex at 3.8Å resolution. The structure reveals the molecular basis of cardiac diseases caused by missense mutations in myosin and actin proteins.

High-resolution cryo-EM structure of urease from the pathogen Yersinia enterocolitica

Urease converts urea into ammonia and carbon dioxide and makes urea available as a nitrogen source for all forms of life except animals. In human bacterial pathogens, ureases also aid in the invasion of acidic environments such as the stomach by raising the surrounding pH. Here, we report the structure of urease from the pathogen Yersinia enterocolitica at 2 Å resolution from cryo-electron microscopy. Y. enterocolitica urease is a dodecameric assembly of a trimer of three protein chains, ureA, ureB and ureC. The high data quality enables detailed visualization of the urease bimetal active site and of the impact of radiation damage. The obtained structure is of sufficient quality to support drug development efforts.

Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state

Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the relative position of the N-terminal α-helices in the three LFs determines which factor is translocated first.

High-resolution cryo-EM structure of the EspA filament from EPEC: revealing the mechanism of effector translocation in the type 3 secretion system

High-resolution cryo-EM structure of the EspA filament from EPEC: revealing the mechanism of effector translocation in the type 3 secretion system

Practical Considerations of Using the K3 Camera in CDS Mode for High-resolution and High-throughput Single Particle Cryo-EM

Abstract Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed standard (nonCDS) mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 A with a 1.346 A physical pixel). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834 A physical pixel size) we achieved a resolution of 1.65 A within 9 hours of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 A resolution with 5 minutes of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.