Low-Resolution Density Maps from Atomic Models: How Stepping “Back” Can Be a Step “Forward”

Atomic-resolution structures have had a tremendous impact on modern biological science. Much useful information also has been gleaned by merging and correlating atomic-resolution structural details with lower-resolution (15–40 Å), three-dimensional (3D) reconstructions computed from images recorded with cryo-transmission electron microscopy (cryoTEM) procedures. One way to merge these structures involves reducing the resolution of an atomic model to a level comparable to a cryoTEM reconstruction. A low-resolution density map can be derived from an atomic-resolution structure by retrieving a set of atomic coordinates editing the coordinate file, computing structure factors from the model coordinates, and computing the inverse Fourier transform of the structure factors. This method is a useful tool for structural studies primarily in combination with 3D cryoTEM reconstructions. It has been used to assess the quality of 3D reconstructions, to determine corrections for the phase-contrast transfer function of the transmission electron microscope, to calibrate the dimensions and handedness of 3D reconstructions, to produce difference maps, to model features in macromolecules or macromolecular complexes, and to generate models to initiate model-based determination of particle orientation and origin parameters for 3D reconstruction.     

High-resolution electron cryomicroscopy of macromolecular assemblies

Electron cryomicroscopy is a high-resolution imaging technique that is particularly appropriate for the structural determination of large macromolecular assemblies, which are difficult to study by X-ray crystallography or NMR spectroscopy. For some biological molecules that form two-dimensional crystals, the application of electron cryomicroscopy and image reconstruction can help elucidate structures at atomic resolution. In instances where crystals cannot be formed, atomic-resolution information can be obtained by combining high-resolution structures of individual components determined by X-ray crystallography or NMR with image-derived reconstructions at moderate resolution. This can provide unique and crucial information on the mechanisms of these complexes. Finally, image reconstructions can be used to augment X-ray studies by providing initial models that facilitate phasing of crystals of large macromolecular machines such as ribosomes and viruses.     

ResLog plots as an empirical metric of the quality of cryo-EM reconstructions

Compared to the field of X-ray crystallography, the field of single particle three-dimensional electron microscopy has few reliable metrics for assessing the quality of 3D reconstructions. New metrics are needed that can determine whether a given 3D reconstruction accurately reflects the structure of the particles from which it was derived or instead depicts a plausible though incorrect structure due to coarse misalignment of particles. Here an empirical procedure is presented for differentiating between a reconstruction with well-aligned particles and a reconstruction with grossly misclassified particles. For a given dataset, 3D reconstructions are computed from subsets of particles with decreasing numbers of particles contributing to the reconstruction. A plot of inverse resolution vs. the logarithm of the number of particles (a “ResLog” plot) provides metrics for the reliability of the reconstruction and the overall quality of the dataset and processing. Specifically, the y-intercept of a regression line provides a measure of the relative accuracy of the particle alignment and classification, and the slope is an indicator of the overall data quality including the imaging conditions and processing steps. ResLog plots can also be used to optimize conditions for data collection and reconstruction parameters. Although resolution estimates can vary by method of calculation, ResLog-derived parameters are consistent whether calculated by Fourier shell correlation or Fourier neighbor correlation, or a new coordinate-based metric that serves as a yardstick for structures where atomic coordinates are available. ResLog plots could become part of a standard set of parameters to be included in 3D reconstruction reports.     

Electron tomography reveals diverse conformations of integrin alphaIIbbeta3 in the active state

We used electron tomography to determine the three-dimensional (3D) structure of integrin alphaIIbbeta3 in the active state. We found that we obtained better density maps when we reconstructed a 3D volume for each individual particle in the tilt series rather than to extract the particle-containing subvolumes from a 3D reconstruction of the entire specimen area. The 3D tomographic reconstructions of 100 particles revealed that activated alphaIIbbeta3 adopts many different conformations. An average of all the individual 3D reconstructions nicely accommodated the crystal structure of the alphaVbeta3 headpiece, confirming the locations assigned to the alpha- and beta-subunit in the density map. The most striking finding of our study is the structural flexibility of the lower leg of the beta-subunit as opposed to the conformational stability of the leg of the alpha-subunit. The good fit of the atomic structure of the betaI domain and the hybrid domain in the active state showed that the hybrid domain swings out, and most particles used for tomography are in the active state. Multivariate statistical analysis and classification applied to the set of 3D reconstructions revealed that more than 90% reconstructions are grouped into the classes that show the active state. Our results demonstrate that electron tomography can be used to classify complexes with a flexible structure such as integrins.     

Structural and functional insights into the interaction of echoviruses and decay-accelerating factor

Many enteroviruses bind to the complement control protein decay-accelerating factor (DAF) to facilitate cell entry. We present here a structure for echovirus (EV) type 12 bound to DAF using cryo-negative stain transmission electron microscopy and three-dimensional image reconstruction to 16-A resolution, which we interpreted using the atomic structures of EV11 and DAF. DAF binds to a hypervariable region of the capsid close to the 2-fold symmetry axes in an interaction that involves mostly the short consensus repeat 3 domain of DAF and the capsid protein VP2. A bulge in the density for the short consensus repeat 3 domain suggests that a loop at residues 174-180 rearranges to prevent steric collision between closely packed molecules at the 2-fold symmetry axes. Detailed analysis of receptor interactions between a variety of echoviruses and DAF using surface plasmon resonance and comparison of this structure (and our previous work; Bhella, D., Goodfellow, I. G., Roversi, P., Pettigrew, D., Chaudhry, Y., Evans, D. J., and Lea, S. M. (2004) J. Biol. Chem. 279, 8325-8332) with reconstructions published for EV7 bound to DAF support major differences in receptor recognition among these viruses. However, comparison of the electron density for the two virus.receptor complexes (rather than comparisons of the pseudo-atomic models derived from fitting the coordinates into these densities) suggests that the dramatic differences in interaction affinities/specificities may arise from relatively subtle structural differences rather than from large-scale repositioning of the receptor with respect to the virus surface.     

Electron tomography of biological samples

Electron tomography allows computing three-dimensional (3D) reconstructions of objects from their projections recorded at several angles. Combined with transmission electron microscopy, electron tomography has contributed greatly to the understanding of subcellular structures and organelles. Performed on frozen-hydrated samples, electron tomography has yielded useful information about complex biological structures. Combined with energy filtered transmission electron microscopy (EFTEM) it can be used to analyze the spatial distribution of chemical elements in biological or material sciences samples. In the present review, we present an overview of the requirements, applications, and perspectives of electron tomography in structural biology.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1497–1505.Original Russian Text Copyright © 2004 by Marco, Boudier, Messaoudi, Rigaud.     

Toolbox for non-intrusive structural and functional analysis of recombinant VLP based vaccines: a case study with hepatitis B vaccine

Background

Fundamental to vaccine development, manufacturing consistency, and product stability is an understanding of the vaccine structure-activity relationship. With the virus-like particle (VLP) approach for recombinant vaccines gaining popularity, there is growing demand for tools that define their key characteristics. We assessed a suite of non-intrusive VLP epitope structure and function characterization tools by application to the Hepatitis B surface antigen (rHBsAg) VLP-based vaccine.

Methodology

The epitope-specific immune reactivity of rHBsAg epitopes to a given monoclonal antibody was monitored by surface plasmon resonance (SPR) and quantitatively analyzed on rHBsAg VLPs in-solution or bound to adjuvant with a competitive enzyme-linked immunosorbent assay (ELISA). The structure of recombinant rHBsAg particles was examined by cryo transmission electron microscopy (cryoTEM) and in-solution atomic force microscopy (AFM).

Principal Findings

SPR and competitive ELISA determined relative antigenicity in solution, in real time, with rapid turn-around, and without the need of dissolving the particulate aluminum based adjuvant. These methods demonstrated the nature of the clinically relevant epitopes of HBsAg as being responsive to heat and/or redox treatment. In-solution AFM and cryoTEM determined vaccine particle size distribution, shape, and morphology. Redox-treated rHBsAg enabled 3D reconstruction from CryoTEM images – confirming the previously proposed octahedral structure and the established lipid-to-protein ratio of HBsAg particles. Results from these non-intrusive biophysical and immunochemical analyses coalesced into a comprehensive understanding of rHBsAg vaccine epitope structure and function that was important for assuring the desired epitope formation, determinants for vaccine potency, and particle stability during vaccine design, development, and manufacturing.

Significance

Together, the methods presented here comprise a novel suite of non-intrusive VLP structural and functional characterization tools for recombinant vaccines. Key VLP structural features were defined and epitope-specific antigenicity was quantified while preserving epitope integrity and particle morphology. These tools should facilitate the development of other VLP-based vaccines.     

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.     

Symmetry-adapted spherical harmonics method for high-resolution 3D single-particle reconstructions

Three-dimensional (3D) reconstruction is the last and an essential step toward high-resolution structural determination in single-particle cryo-electron microscopy (cryoEM). We have implemented a new algorithm for reconstructing 3D structures of macromolecular complexes with icosahedral symmetry from cryoEM images. Icosahedral symmetry-adapted functions (ISAFs) are used to interpolate structural factors in the reciprocal space to generate a 3D reconstruction in spherical coordinates. In our implementation, we introduced a recursive method for deriving higher order ISAFs from three lower order seed functions. We demonstrate improvements of our new method in both the noise suppression and the effective resolution in 3D reconstruction over the commonly used Fourier-Bessel synthesis method introduced by Crowther et al. three decades ago. Our 3D reconstruction method can be extended to macromolecular complexes with other symmetry types and is thus likely to impact future high-resolution cryoEM single-particle reconstruction efforts in general.     

Single particle reconstruction of membrane proteins: A tool for understanding the 3D structure of disease-related macromolecules

Membrane proteins play important roles in cell functions such as neurotransmission, muscle contraction, and hormone secretion, but their structures are mostly undetermined. Several techniques have been developed to elucidate the structure of macromolecules; X-ray or electron crystallography, nuclear magnetic resonance spectroscopy, and high-resolution electron microscopy. Electron microscopy-based single particle reconstruction, a computer-aided structure determination method, reconstructs a three-dimensional (3D) structure from projections of monodispersed protein. A large number of particle images are picked up from EM films, aligned and classified to generate two-dimensional (2D) averages, and, using the Euler angle of each 2D average, reconstructed into a 3D structure. This method is challenging due to the necessity for close collaboration between classical biochemistry and innovative information technology, including parallel computing. However, recent progress in electron microscopy, mathematical algorithms, and computational ability has greatly increased the subjects that are considered to be primarily addressable using single particle reconstruction. Membrane proteins are one of these targets to which the single particle reconstruction is successfully applied for understanding of their structures. In this paper, we will introduce recently reconstructed channel-related proteins and discuss the applicability of this technique in understanding molecular structures and their roles in pathology.     

Toward understanding the structure and interactions of microtubules and motor proteins

To obtain an overall three-dimensional picture of the interaction between microtubules and the motor proteins of the kinesin family it will be necessary to take account of both atomic resolution structures obtained by X-ray crystallography and medium resolution reconstructions obtained by electron cryomicroscopy. We examine the problems associated with obtaining the required structural information from electron micrographs of vitreous ice-embedded microtubules decorated with motor domains. We find that the minus-end directed motor, ncd, decorates microtubules with an 80 Å periodicity as for kinesin. Our theoretical analysis and experiments with ncd illustrate the difficulty in determining unambiguously the surface lattice organization by diffraction analysis of micrographs. 3D reconstructions of decorated microtubules are required to accurately locate the motor domains. Helical diffraction theory is not usually applicable because microtubules are cylindrical structures that rarely have complete helical symmetry. We propose using a back-projection method based on the long pitch helices formed by individual protofilaments. Model reconstructions show that this approach is feasible. © 1995 Wiley-Liss, Inc.     

Applications of a bilateral denoising filter in biological electron microscopy

Due to the sensitivity of biological sample to the radiation damage, the low dose imaging conditions used for electron microscopy result in extremely noisy images. The processes of digitization, image alignment, and 3D reconstruction also introduce additional sources of noise in the final 3D structure. In this paper, we investigate the effectiveness of a bilateral denoising filter in various biological electron microscopy applications. In contrast to the conventional low pass filters, which inevitably smooth out both noise and structural features simultaneously, we found that bilateral filter holds a distinct advantage in being capable of effectively suppressing noise without blurring the high resolution details. In as much, we have applied this technique to individual micrographs, entire 3D reconstructions, segmented proteins, and tomographic reconstructions.     

Real space refinement of acto-myosin structures from sectioned muscle

We have adapted a real space refinement protocol originally developed for high-resolution crystallographic analysis for use in fitting atomic models of actin filaments and myosin subfragment 1 (S1) to 3-D images of thin-sectioned, plastic-embedded whole muscle. The rationale for this effort is to obtain a refinement protocol that will optimize the fit of the model to the density obtained by electron microscopy and correct for poor geometry introduced during the manual fitting of a high-resolution atomic model into a lower resolution 3-D image. The starting atomic model consisted of a rigor acto-S1 model obtained by X-ray crystallography and helical reconstruction of electron micrographs. This model was rebuilt to fit 3-D images of rigor insect flight muscle at a resolution of 7 nm obtained by electron tomography and image averaging. Our highly constrained real space refinement resulted in modest improvements in the agreement of model and reconstruction but reduced the number of conflicting atomic contacts by 70% without loss of fit to the 3-D density. The methodology seems to be well suited to the derivation of stereochemically reasonable atomic models that are consistent with experimentally determined 3-D reconstructions computed from electron micrographs.     

An electron microscopy journey in the study of microtubule structure and dynamics

Structural characterization of microtubules has been the realm of three‐dimensional electron microscopy and thus has evolved hand in hand with the progress of this technique, from the initial 3D reconstructions of stained tubulin assemblies, and the first atomic model of tubulin by electron crystallography of 2D sheets of protofilaments, to the ever more detailed cryoelectron microscopy structures of frozen‐hydrated microtubules. Most recently, hybrid helical and single particle image processing techniques, and the latest detector technology, have lead to atomic models built directly into the density maps of microtubules in different functional states, shading new light into the critical process of microtubule dynamic instability.     

A novel canonical dual computational approach for prion AGAAAAGA amyloid fibril molecular modeling

Many experimental studies have shown that the prion AGAAAAGA palindrome hydrophobic region (113-120) has amyloid fibril forming properties and plays an important role in prion diseases. However, due to the unstable, noncrystalline and insoluble nature of the amyloid fibril, to date structural information on AGAAAAGA region (113-120) has been very limited. This region falls just within the N-terminal unstructured region PrP (1-123) of prion proteins. Traditional X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy experimental methods cannot be used to get its structural information. Under this background, this paper introduces a novel approach of the canonical dual theory to address the 3D atomic-resolution structure of prion AGAAAAGA amyloid fibrils. The novel and powerful canonical dual computational approach introduced in this paper is for the molecular modeling of prion AGAAAAGA amyloid fibrils, and that the optimal atomic-resolution structures of prion AGAAAAGA amyloid fibils presented in this paper are useful for the drive to find treatments for prion diseases in the field of medicinal chemistry. Overall, this paper presents an important method and provides useful information for treatments of prion diseases.     

The structure of the F-actin filament and the actin molecule.

A consensus view on the three-dimensional structure of the F-actin filament and the relative strength of the intersubunit contacts in the filament has been established from an atomic filament model and recent three-dimensional reconstructions from electron micrographs of F-actin filaments. Functional implications of recent structural and biochemical data indicating a rather dynamic filament structure are discussed.     

Cellular Architecture of Treponema pallidum: Novel Flagellum, Periplasmic Cone, and Cell Envelope as Revealed by Cryo Electron Tomography

High-resolution cryo electron tomography (cryo-ET) was utilized to visualize Treponema pallidum, the causative agent of syphilis, at the molecular level. Three-dimensional (3D) reconstructions from 304 infectious organisms revealed unprecedented cellular structures of this unusual member of the spirochetal family. High-resolution cryo-ET reconstructions provided detailed structures of the cell envelope, which is significantly different from that of Gram-negative bacteria. The 4-nm lipid bilayer of both outer membrane and cytoplasmic membrane resolved in 3D reconstructions, providing an important marker for interpreting membrane-associated structures. Abundant lipoproteins cover the outer leaflet of the cytoplasmic membrane, in contrast to the rare outer membrane proteins visible by scanning probe microscopy. High-resolution cryo-ET images also provided the first observation of T. pallidum chemoreceptor arrays, as well as structural details of the periplasmically located cone-shaped structure at both ends of the bacterium. Furthermore, 3D subvolume averages of periplasmic flagellar motors and flagellar filaments from living organisms revealed the novel flagellar architectures that may facilitate their rotation within the confining periplasmic space. Our findings provide the most detailed structural understanding of periplasmic flagella and the surrounding cell envelope, which enable this enigmatic bacterium to efficiently penetrate tissue and to escape host immune responses.     

Prions

The prion hypothesis^1-3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.^{4, 5} Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.^{6, 7} Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and – most probably - a partially ordered prion domain.⁶ For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.     

Atomic model of the papillomavirus capsid

Papillomaviruses propagate in differentiating skin cells, and certain types are responsible for the onset of cervical cancer. We have combined image reconstructions from electron cryomicroscopy (cryoEM) of bovine papillomavirus at 9 A resolution with coordinates from the crystal structure of small virus-like particles of the human papillomavirus type 16 L1 protein to generate an atomic model of the virion. The overall fit of the L1 model into the cryoEM map is excellent, but residues 402-446 in the 'C-terminal arm' must be rebuilt. We propose a detailed model for the structure of this arm, based on two constraints: the presence of an intermolecular disulfide bond linking residues 175 and 428, and the clear identification of a feature in the image reconstruction corresponding to an alpha-helix near the C-terminus of L1. We have confirmed the presence of the disulfide bond by mass spectrometry. Our 'invading arm' model shows that papilloma- and polyomaviruses have a conserved capsid architecture. Most of the rebuilt C-terminal arm is exposed on the viral surface; it is likely to have a role in infection and in immunogenicity.     

Single particle reconstructions of the transferrin-transferrin receptor complex obtained with different specimen preparation techniques

The outcome of three-dimensional (3D) reconstructions in single particle electron microscopy (EM) depends on a number of parameters. We have used the well-characterized structure of the transferrin (Tf)-transferrin receptor (TfR) complex to study how specimen preparation techniques influence the outcome of single particle EM reconstructions. The Tf-TfR complex is small (290kDa) and of low symmetry (2-fold). Angular reconstitution from images of vitrified specimens does not reliably converge on the correct structure. Random conical tilt reconstructions from negatively stained specimens are reliable, but show variable degrees of artifacts depending on the negative staining protocol. Alignment of class averages from vitrified specimens to a 3D negative stain reference model using FREALIGN largely eliminated artifacts in the resulting 3D maps, but not completely. Our results stress the need for critical evaluation of structures determined by single particle EM.