Individual-particle Electron Tomography Research Articles

Facile hermetic TEM grid preparation for molecular imaging of hydrated biological samples at room temperature

Although structures of vitrified supramolecular complexes have been determined at near-atomic resolution, elucidating in situ molecular structure in living cells remains a challenge. Here, we report a straightforward liquid cell technique, originally developed for real-time visualization of dynamics at a liquid-gas interface using transmission electron microscopy, to image wet biological samples. Due to the scattering effects from the liquid phase, the micrographs display an amplitude contrast comparable to that observed in negatively stained samples. We succeed in resolving subunits within the protein complex GroEL imaged in a buffer solution at room temperature. Additionally, we capture various stages of virus cell entry, a process for which only sparse structural data exists due to their transient nature. To scrutinize the morphological details further, we used individual particle electron tomography for 3D reconstruction of each virus. These findings showcase this approach potential as an efficient, cost-effective complement to other microscopy technique in addressing biological questions at the molecular level.

LoTToR: An Algorithm for Missing-Wedge Correction of the Low-Tilt Tomographic 3D Reconstruction of a Single-Molecule Structure

A single-molecule three-dimensional (3D) structure is essential for understanding the thermal vibrations and dynamics as well as the conformational changes during the chemical reaction of macromolecules. Individual-particle electron tomography (IPET) is an approach for obtaining a snap-shot 3D structure of an individual macromolecule particle by aligning the tilt series of electron tomographic (ET) images of a targeted particle through a focused iterative 3D reconstruction method. The method can reduce the influence on the 3D reconstruction from large-scale image distortion and deformation. Due to the mechanical tilt limitation, 3D reconstruction often contains missing-wedge artifacts, presented as elongation and an anisotropic resolution. Here, we report a post-processing method to correct the missing-wedge artifact. This low-tilt tomographic reconstruction (LoTToR) method contains a model-free iteration process under a set of constraints in real and reciprocal spaces. A proof of concept is conducted by using the LoTToR on a phantom, i.e., a simulated 3D reconstruction from a low-tilt series of images, including that within a tilt range of ±15°. The method is validated by using both negative-staining (NS) and cryo-electron tomography (cryo-ET) experimental data. A significantly reduced missing-wedge artifact verifies the capability of LoTToR, suggesting a new tool to support the future study of macromolecular dynamics, fluctuation and chemical activity from the viewpoint of single-molecule 3D structure determination.

3D Reconstruction and Structural Study of IgG1 Antibody by Individual-particle Electron Tomography

Author(s): Xing, Zhang; Yu-Heng, Liao; Hui-Min, Tong; Lei, Zhang; Sheng-Li, Zhang; Gang, Ren

Single-Molecule 3D Images of \u201cHole-Hole\u201d IgG1 Homodimers by Individual-Particle Electron Tomography

The engineering of immunoglobulin-G molecules (IgGs) is of wide interest for improving therapeutics, for example by modulating the activity or multiplexing the specificity of IgGs to recognize more than one antigen. Optimization of engineered IgG requires knowledge of three-dimensional (3D) structure of synthetic IgG. However, due to flexible nature of the molecules, their structural characterization is challenging. Here, we use our reported individual-particle electron tomography (IPET) method with optimized negative-staining (OpNS) for direct 3D reconstruction of individual IgG hole-hole homodimer molecules. The hole-hole homodimer is an undesired variant generated during the production of a bispecific antibody using the knob-into-hole heterodimer technology. A total of 64 IPET 3D density maps at ~15 Å resolutions were reconstructed from 64 individual molecules, revealing 64 unique conformations. In addition to the known Y-shaped conformation, we also observed an unusual X-shaped conformation. The 3D structure of the X-shaped conformation contributes to our understanding of the structural details of the interaction between two heavy chains in the Fc domain. The IPET approach, as an orthogonal technique to characterize the 3D structure of therapeutic antibodies, provides insight into the 3D structural variety and dynamics of heterogeneous IgG molecules.

Hybrid Analysis of Mab Flexibility by Electron Microscopy and Scattering

The biopharmaceutical industry is one of the most valuable business sectors of the US economy. In 2016, ten of the 15 top-selling drugs were protein biologics; and of these, five were monoclonal antibodies (mAbs). Biopharmaceuticals also represent at least 40% of the medicines in development today, and the global market for protein biologics in 2022 is projected to be over $200B. A key measurement gap in the biopharmaceutical industry is the inability of current methods to fully characterize the structure of highly flexible, protein-based drugs, such as mAbs. Crystallization is generally not successful due to their high flexibility; and even when successful, captures only a single conformational snapshot of the molecule. Similarly, the continuum of conformational states does not allow conventional multi-particle averaging in CryoEM. Using the NISTmAb (RM8671), a model drug-like monoclonal antibody reference material, we have developed hybrid methods to combine EM with small-angle x-ray and neutron scattering in order to build more complete models of antibody structure and flexibility in solution. In this methodology, Individual Particle Electron Tomography (IPET) is initially used to build a collection of conformational states of the antibody. These states can then be used to model scattering data under physiologically or pharmaceutically relevant solution conditions (e.g., different formulations). We have applied these methods to the NISTmAb reference material itself, as well as to soluble aggregates naturally present in small amounts in the product, and to a NISTmAb/antigen complex. Ultimately, the ability to measure the detailed conformational distribution of biotherapeutics may allow this behavior to be correlated with attributes of pharmaceutical significance, such as stability, efficacy, and immune tolerance. This understanding could allow for the more rational design of stabile and effective biopharmaceutical molecules and formulations.

Single-molecule 3D imaging of human plasma intermediate-density lipoproteins reveals a polyhedral structure

Intermediate-density lipoproteins (IDLs), the remnants of very-low-density lipoproteins via lipolysis, are rich in cholesteryl ester and are associated with cardiovascular disease. Despite pharmacological interest in IDLs, their three-dimensional (3D) structure is still undetermined due to their variation in size, composition, and dynamic structure. To explore the 3D structure of IDLs, we reconstructed 3D density maps from individual IDL particles using cryo-electron microscopy (cryo-EM) and individual-particle electron tomography (IPET, without averaging from different molecules). 3D reconstructions of IDLs revealed an unexpected polyhedral structure that deviates from the generally assumed spherical shape model (Frias et al., 2007; Olson, 1998; Shen et al., 1977). The polyhedral-shaped IDL contains a high-density shell formed by flat surfaces that are similar to those of very-low-density lipoproteins but have sharper dihedral angles between nearby surfaces. These flat surfaces would be less hydrophobic than the curved surface of mature spherical high-density lipoprotein (HDL), leading to a lower binding affinity of IDL to hydrophobic proteins (such as cholesteryl ester transfer protein) than HDL. This is the first visualization of the IDL 3D structure, which could provide fundamental clues for delineating the role of IDL in lipid metabolism and cardiovascular disease.

An Algorithm for Enhancing the Image Contrast of Electron Tomography

Three-dimensional (3D) reconstruction of a single protein molecule is essential for understanding the relationship between the structural dynamics and functions of the protein. Electron tomography (ET) provides a tool for imaging an individual particle of protein from a series of tilted angles. Individual-particle electron tomography (IPET) provides an approach for reconstructing a 3D density map from a single targeted protein particle (without averaging from different particles of this type of protein), in which the target particle was imaged from a series of tilting angles. However, owing to radiation damage limitations, low-dose images (high noise, and low image contrast) are often challenging to be aligned for 3D reconstruction at intermediate resolution (1–3 nm). Here, we propose a computational method to enhance the image contrast, without increasing any experimental dose, for IPET 3D reconstruction. Using an edge-preserving smoothing-based multi-scale image decomposition algorithm, this method can detect the object against a high-noise background and enhance the object image contrast without increasing the noise level or significantly decreasing the image resolution. The method was validated by using both negative staining (NS) ET and cryo-ET images. The successful 3D reconstruction of a small molecule (<100 kDa) indicated that this method can be used as a supporting tool to current ET 3D reconstruction methods for studying protein dynamics via structure determination from each individual particle of the same type of protein.

Structural Plasticity of Neurexin 1α: Implications for its Role as Synaptic Organizer

α-Neurexins are synaptic organizing molecules implicated in neuropsychiatric disorders. They bind and arrange an array of different partners in the synaptic cleft. The extracellular region of neurexin 1α (n1α) contains six LNS domains (L1–L6) interspersed by three Egf-like repeats. N1α must encode highly evolved structure–function relationships in order to fit into the narrow confines of the synaptic cleft, and also recruit its large, membrane-bound partners. Internal molecular flexibility could provide a solution; however, it is challenging to delineate because currently no structural methods permit high-resolution structure determination of large, flexible, multi-domain protein molecules. To investigate the structural plasticity of n1α, in particular the conformation of domains that carry validated binding sites for different protein partners, we used a panel of structural techniques. Individual particle electron tomography revealed that the N-terminally and C-terminally tethered domains, L1 and L6, have a surprisingly limited range of conformational freedom with respect to the linear central core containing L2 through L5. A 2.8-Å crystal structure revealed an unexpected arrangement of the L2 and L3 domains. Small-angle X-ray scattering and electron tomography indicated that incorporation of the alternative splice insert SS6 relieves the restricted conformational freedom between L5 and L6, suggesting that SS6 may work as a molecular toggle. The architecture of n1α thus encodes a combination of rigid and flexibly tethered domains that are uniquely poised to work together to promote its organizing function in the synaptic cleft, and may permit allosterically regulated and/or concerted protein partner binding.

IgG Antibody 3D Structures and Dynamics.

Antibodies are vital for human health because of their ability to function as nature’s drugs by protecting the body from infection. In recent decades, antibodies have been used as pharmaceutics for targeted therapy in patients with cancer, autoimmune diseases, and cardiovascular diseases. Capturing the dynamic structure of antibodies and characterizing antibody fluctuation is critical for gaining a deeper understanding of their structural characteristics and for improving drug development. Current techniques for studying three-dimensional (3D) structural heterogeneity and variability of proteins have limitations in ascertaining the dynamic structural behavior of antibodies and antibody-antigen complexes. Here, we review current techniques used to study antibody structures with a focus on the recently developed individual-particle electron tomography (IPET) technique. IPET, as a particle-by-particle methodology for 3D structural characterization, has shown advantages in studying structural variety and conformational changes of antibodies, providing direct imaging data for biomolecular engineering to improve development and clinical application of synthetic antibodies.

Three-dimensional structural dynamics of DNA origami Bennett linkages using individual-particle electron tomography

Scaffolded DNA origami has proven to be a powerful and efficient technique to fabricate functional nanomachines by programming the folding of a single-stranded DNA template strand into three-dimensional (3D) nanostructures, designed to be precisely motion-controlled. Although two-dimensional (2D) imaging of DNA nanomachines using transmission electron microscopy and atomic force microscopy suggested these nanomachines are dynamic in 3D, geometric analysis based on 2D imaging was insufficient to uncover the exact motion in 3D. Here we use the individual-particle electron tomography method and reconstruct 129 density maps from 129 individual DNA origami Bennett linkage mechanisms at ~ 6–14 nm resolution. The statistical analyses of these conformations lead to understanding the 3D structural dynamics of Bennett linkage mechanisms. Moreover, our effort provides experimental verification of a theoretical kinematics model of DNA origami, which can be used as feedback to improve the design and control of motion via optimized DNA sequences and routing.

Polyhedral 3D structure of human plasma very low density lipoproteins by individual particle cryo-electron tomography1

Human VLDLs assembled in the liver and secreted into the circulation supply energy to peripheral tissues. VLDL lipolysis yields atherogenic LDLs and VLDL remnants that strongly correlate with CVD. Although the composition of VLDL particles has been well-characterized, their 3D structure is elusive because of their variations in size, heterogeneity in composition, structural flexibility, and mobility in solution. Here, we employed cryo-electron microscopy and individual-particle electron tomography to study the 3D structure of individual VLDL particles (without averaging) at both below and above their lipid phase transition temperatures. The 3D reconstructions of VLDL and VLDL bound to antibodies revealed an unexpected polyhedral shape, in contrast to the generally accepted model of a spherical emulsion-like particle. The smaller curvature of surface lipids compared with HDL may also reduce surface hydrophobicity, resulting in lower binding affinity to the hydrophobic distal end of the N-terminal β-barrel domain of cholesteryl ester transfer protein (CETP) compared with HDL. The directional binding of CETP to HDL and VLDL may explain the function of CETP in transferring TGs and cholesteryl esters between these particles. This first visualization of the 3D structure of VLDL could improve our understanding of the role of VLDL in atherogenesis.

Individual-Particle Electron Tomography: A Method for Monitoring the 3D Structure of Low-Dimensional DNA-Based Nanodevice

DNA base-pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of the three-dimensional (3D) structure of the DNA-nanocrystal hybridized building blocks. Existing techniques do not allow for the structural determination of these flexible and heterogeneous samples. Here, we employed cryo-electron microscopy (cryo-EM) and negative-staining (NS) techniques to investigate the morphology of DNA-nanogold conjugates that were self-assembled from a mixture of an 84-base-pair double-stranded DNA (dsDNA) conjugated with two 5-nm nanogold particles for potential substrates in plasmon coupling experiments. Using NS electron tomography and the individual-particle electron tomography (IPET) reconstruction method, we obtained 3D reconstructed 14 electron density maps at a resolution of ~2 nm from each individual dsDNA-nanogold particle. Using these 3D density maps as constraints, we derived 14 conformations of dsDNA by projected a standard flexible dsDNA model onto the observed EM density maps using Molecular Dynamics (MD) simulations. The variation of the conformations was largely consistent with the variation from liquid solution. Moreover, the IPET approach provides the most complete experimental determination of the flexibility and fluctuation range of these directed nanocrystal assemblies to date. The general features revealed by these experiments can be expected to occur in a broad range of DNA-assembled nanostructures.

Three-dimensional structural dynamics and fluctuations of DNA-nanogold conjugates by individual-particle electron tomography.

DNA base pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of three-dimensional structure of DNA-nanocrystal-hybridized building blocks. Existing techniques do not allow for structural determination of these flexible and heterogeneous samples. Here we report cryo-electron microscopy and negative-staining electron tomography approaches to image, and three-dimensionally reconstruct a single DNA-nanogold conjugate, an 84-bp double-stranded DNA with two 5-nm nanogold particles for potential substrates in plasmon-coupling experiments. By individual-particle electron tomography reconstruction, we obtain 14 density maps at ∼2-nm resolution. Using these maps as constraints, we derive 14 conformations of dsDNA by molecular dynamics simulations. The conformational variation is consistent with that from liquid solution, suggesting that individual-particle electron tomography could be an expected approach to study DNA-assembling and flexible protein structure and dynamics.

Three Dimensional Dynamics and Fluctuations of DNA-Nanogold Dimers by Individual-Particle Electron Tomography

Organic-inorganic hybridized nanocrystals have been proven as an important additional tool for addressing many emerging challenges biological and material sciences. Nanogold and quantum dot conjugates have been used extensively as biomolecular markers, while DNA base pairing has directed the self-assembly of discrete groupings and arrays of organic and inorganic nanocrystals in formation of a network solid for electronic devices and memory components. The control of DNA-mediated assembly depends crucially upon the ability to exposure three-dimensional (3D) structure of DNA-nanocrystals hybridized building blocks. Current techniques limit in structure determination of flexible and heterogeneous sample. Here, we employed the electron tomography and negative-staining techniques to investigate the 3D structure of DNA-nanogold dimers that were self-assembled from a mixture of an 84-base-pair double-stranded DNA (dsDNA) conjugated with two 5-nm nanogold particles. We reconstructed a total of 14 electron density maps at a resolution of ∼2 nm from each individual dsDNA-nanogold particle using the individual-particle electron tomography (IPET) reconstruction method. Using these 3D density maps as constrain, we projected a standard flexible DNA model onto the observed EM density maps and derived 14 conformations of dsDNA by molecular dynamics simulations. By aligning these structures from one distal end nanogold, the distribution showed the DNA fluctuation and flexibility between nanogolds, which providing a blueprint research for studying of DNA-mediated assembly of complex inorganic nanostructures.

3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography.

Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.

Determination of the Dynamic Structures of Nacent Discoidal High-Density Lipoprotein (HDL) Bound to Lecithin Cholesterol Acyltransferase (LCAT) and Paraoxonase 1 (PON1)

The relative levels of plasma cholesterol in different lipoproteins are the major indicator for CVD and related diseases. Nascent discoidal HDL particles, which are then converted to spherical mature HDL by lecithin-cholesterol acyltransferase (LCAT), are integral in reverse cholesterol transport. Detailed knowledge of HDL structure is key to understanding its molecular mechanisms, including how Paraoxynase 1 (PON1) associates with the phospholipid surface of HDL helping prevent oxidation. Despite public interest in HDL structure related function, their highly dynamic nature has prevented precise structural determinations for nearly half a century. Here, we used our recently developed individual particle electron tomography (IPET) technique to study each individual complex of HDL-LCAT. The 3D reconstructions show, i) LCAT associates with HDL along a proposed mobile region of the apoA-1 backbone (res ∼122-142), where both monomers overlap antiparallel along the discoidal edge of HDL; ii) PON1 interacts with HDL through its hydrophobic tail, which binds into the phospholipid surface, and loosely associates with apoA-1. The structure provides novel information for modeling nascent discoidal HDL and further reveals the first view of HDL structural dynamics in three-dimensions. This result will benefit public knowledge and pharmaceutical drug design to control plasma HDL cholesterol level.

Determination of the Dynamic Structures of Igg Antibody by Individual-Particle Electron Tomography

Antibody, also known as immunoglobulin, functions as one of the key parts in human immune response. Studying antibodies provides benefits in treating immune system diseases, medical diagnostics utilizing particular antibodies, and applications such as monoclonal antibody therapy. Antibodies perform functions through identifying and neutralizing various foreign objects based on a unique Y-shaped highly dynamic structure. In antibody structure, two antigen-binding domains (Fabs) are connected to effector domain (Fc) via a flexible hinge-loop region allowing two antigen-binding sites to vary. Dynamics of the loop region allow antibodies to undergo large conformational change to search binding sites on various biological macromolecule surfaces, fitting the paratope to a targeted antigen. Conventional techniques, such as X-ray crystallography, nuclear magnetic resonance technique, or single particle TEM reconstruction method, in studying antibody structure are faced with difficulty in revealing structural dynamic properties in 3D, due to their requirement of averaging vast numbers of particles.Here, we use our recently developed individual-particle electron tomography (IPET) to obtain nearly a hundred 3D density maps each from an individual antibody. Each 3D map provided the spacing and orientations of the three domains of each antibody. By comparison of the domain spatial relation among different antibody molecules, the 3D distributions of the two Fab domains uncovered the dynamics and fluctuations of this antibody type in solution. These dynamic characteristics provide us insight into antibody functional expression, and show potential approaches to increase antibody binding affinity.

Peptide-Conjugation Induced Conformational Changes in Human IgG1 Observed by Optimized Negative-Staining and Individual-Particle Electron Tomography

Peptides show much promise as potent and selective drug candidates. Fusing peptides to a scaffold monoclonal antibody produces a conjugated antibody which has the advantages of peptide activity yet also has the pharmacokinetics determined by the scaffold antibody. However, the conjugated antibody often has poor binding affinity to antigens that may be related to unknown structural changes. The study of the conformational change is difficult by conventional techniques because structural fluctuation under equilibrium results in multiple structures co-existing. Here, we employed our two recently developed electron microscopy (EM) techniques: optimized negative-staining (OpNS) EM and individual-particle electron tomography (IPET). Two-dimensional (2D) image analyses and three-dimensional (3D) maps have shown that the domains of antibodies present an elongated peptide-conjugated conformational change, suggesting that our EM techniques may be novel tools to monitor the structural conformation changes in heterogeneous and dynamic macromolecules, such as drug delivery vehicles after pharmacological synthesis and development.

108 Individual-particle electron tomography: a method for studying macromolecule dynamics

In solution, macromolecules are naturally flexible and dynamic. Dynamic personalities and structural heterogeneities of macromolecules are essential to understanding their proper function (Karplus & Kuriyan, 2005). However, structural determination of dynamic/heterogeneous macromolecules is limited by current technology such as: X-ray crystallography, nuclear magnetic resonance spectrum, small angle scattering, and electron microscopy single-particle reconstruction. A common weakness of all current techniques is requiring an averaged signal from thousands to millions of different macromolecules. Using averaged “signal” must involve in an assumption that macromolecules remain in identical structures or few identical conformations. This assumption is a good estimate for some macromolecule that have a rigid body, but not for most macromolecules that have “soft”, flexible, and dynamic body, such as lipoproteins and antibodies. An ideal approach for structure determination regardless of macromolecular dynamics is to use non-averaged signal, i.e. the signal from a single macromolecule itself. We developed a ‘‘focused electron tomography reconstruction’’ (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance macromolecule (Zhang & Ren, 2012). FETR can tolerate certain levels of image distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. Since this approach can obtain the structure of a single-instance macromolecule, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single ‘‘snapshot’’ of molecule structure determination (Zhang & Ren, 2012). FETR/IPET provides a completely new opportunity for a single-macromolecule structure determination, and could be used to study the dynamic character, equilibrium fluctuation, to reveal macromolecular mechanism, and even to track the intermediate state of the reaction of macromolecules (Zhang et al., 2010; Zhang & Ren, 2010).

A 3-D Image of an Individual Protein

The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction because they require an average from thousands to millions different molecules. We developed a ‘‘focused electron tomography reconstruction’’ (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single ‘‘snapshot’’ molecule structure determination. FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.