Spectral signal-to-noise ratio and resolution assessment of 3D reconstructions

Measuring the quality of three-dimensional (3D) reconstructed biological macromolecules by transmission electron microscopy is still an open problem. In this article, we extend the applicability of the spectral signal-to-noise ratio (SSNR) to the evaluation of 3D volumes reconstructed with any reconstruction algorithm. The basis of the method is to measure the consistency between the data and a corresponding set of reprojections computed for the reconstructed 3D map. The idiosyncrasies of the reconstruction algorithm are taken explicitly into account by performing a noise-only reconstruction. This results in the definition of a 3D SSNR which provides an objective indicator of the quality of the 3D reconstruction. Furthermore, the information to build the SSNR can be used to produce a volumetric SSNR (VSSNR). Our method overcomes the need to divide the data set in two. It also provides a direct measure of the performance of the reconstruction algorithm itself; this latter information is typically not available with the standard resolution methods which are primarily focused on reproducibility alone.     

Small-angle X-ray scattering-based three-dimensional reconstruction of the immunogen KLH1 reveals different oxygen-dependent conformations

For decades the respiratory protein keyhole limpet hemocyanin (KLH1) from the marine gastropod Megathura crenulata has been used widely as a potent immunostimulant, useful hapten carrier, and valuable agent in the treatment of bladder carcinoma. Although much information on the immunological properties of KLH1 is available, biochemical and structural data are still incomplete. Small-angle x-ray scattering revealed the existence of two conformations, an oxy state being slightly more compact than the deoxy state. Based on small-angle scattering curves, a newly developed Monte Carlo algorithm delivered a surface representation of proteins. The massive changes of the surfaces of reconstructed didecameric KLH1 molecules are explained as a twist of the two non-covalently associated decameric half-molecules. Upon oxygenation, the KLH1 molecule becomes longer and skinnier. This study provides the first real evidence how a molluscan hemocyanin changes conformation during an allosteric transition.     

Small-angle neutron scattering reveals an oxygen-dependent conformational change of the immunogen keyhole limpet hemocyanin type 1 (KLH1)

The respiratory protein of the keyhole limpet, Megathura crenulata, the hemocyanin (KLH), commonly used as an immunogen, binds oxygen cooperatively, which implies the existence of different conformations. For the first time, two different conformations of KLH1 were detected upon oxygenation, a deoxy and an oxy state, using small-angle neutron scattering. Rearrangements in the quaternary structure of KLH1 were predicted from the different radii of gyration and the shifts of the minima and maxima in the scattering curves. Upon oxygenation, KLH1 becomes smaller and more compact. Model reconstruction of KLH1 indicates a hollow cylinder with two rings located close to both ends, which move slightly together upon oxygenation.     

冷冻电镜单颗粒三维重构密度掩模的自动生成方法研究

冷冻电镜单颗粒三维重构技术是用来解析生物大分子三维结构的常用方法.然而目前在单颗粒三维重构过程中,溶剂平滑操作还存在一定缺陷:没有一款主流的单颗粒三维重构程序能够自动寻找掩模(mask)三维密度图,使得三维重构过程难免受到噪音统计学模型计算偏差的干扰.为解决这一问题,本研究借鉴X射线晶体学中解析优化相位所广泛采用的溶剂平滑方法,采用高斯滤波、坎尼边缘检测、最小误差阈值处理等方法处理重构所得三维密度图,优化溶剂平滑操作,发展在单颗粒三维重构过程中自动寻找mask三维密度图的方法.运用三维密度图傅里叶壳层相关系数(fourier shell correlation,FSC)曲线图、模拟颗粒数据重构角度误差散点图等指标评估此方法的效果.结果表明,自动寻找mask密度图的方法能够较好地找到涵盖分子结构信号区域的mask密度图,较为明显提高三维重构所得密度图分辨率.     

UCSF tomography: an integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction

A real-time alignment and reconstruction scheme for electron microscopic tomography (EMT) has been developed and integrated within our UCSF tomography data collection software. This newly integrated software suite provides full automation from data collection to real-time reconstruction by which the three-dimensional (3D) reconstructed volume is immediately made available at the end of each data collection. Real-time reconstruction is achieved by calculating a weighted back-projection on a small Linux cluster (five dual-processor compute nodes) concurrently with the UCSF tomography data collection running on the microscope's computer, and using the fiducial-marker free alignment data generated during the data collection process. The real-time reconstructed 3D volume provides users with immediate feedback to fully asses all aspects of the experiment ranging from sample choice, ice thickness, experimental parameters to the quality of specimen preparation. This information can be used to guide subsequent data collections. Access to the reconstruction is especially useful in low-dose cryo EMT where such information is very difficult to obtain due to extraordinary low signal to noise ratio in each 2D image. In our environment, we generally collect 2048 x 2048 pixel images which are subsequently computationally binned four-fold for the on-line reconstruction. Based upon experiments performed with thick and cryo specimens at various CCD magnifications (50000x-80000x), alignment accuracy is sufficient to support this reduced resolution but should be refined before calculating a full resolution reconstruction. The reduced resolution has proven to be quite adequate to assess sample quality, or to screen for the best data set for full-resolution reconstruction, significantly improving both productivity and efficiency of system resources. The total time from start of data collection to a final reconstructed volume (512 x 512 x 256 pixels) is about 50 min for a +/-70 degrees 2k x 2k pixel tilt series acquired at every 1 degrees.     

Three-dimensional structure of T3 connector purified from overexpressing bacteria.

The bacteriophage T3 connector has been purified from overexpressed protein in Escherichia coli, harboring a plasmid containing the gene encoding p8 protein. The connector, which is composed of 12 copies of p8, has been crystallized in two-dimensional sheets and studied by electron microscopy from negatively stained specimens. A two-dimensional Fourier filtering and averaging procedure was performed with crystalline specimens. In addition, single particle averaging techniques were used with other preparations. The average images obtained from these two approaches gave similar results. A three-dimensional reconstruction from two-dimensional crystals of T3 connectors was obtained by collecting several sets of tilted views and using standard Fourier procedures. The resolution of the three-dimensional map was 1.65 nm. The reconstructed connector shows two main domains: a wider one with 12 small units in the periphery and with an external diameter of 14.9 nm, and a smaller one with 8.5 nm diameter. The height of the reconstructed connector has been determined to be around 8.5 nm. The reconstruction clearly shows an internal open channel running along the longitudinal axis of the particle and having an average diameter of 3.7 nm.     

Detecting particles in cryo-EM micrographs using learned features

A new learning-based approach is presented for particle detection in cryo-electron micrographs using the Adaboost learning algorithm. The approach builds directly on the successful detectors developed for the domain of face detection. It is a discriminative algorithm which learns important features of the particle's appearance using a set of training examples of the particles and a set of images that do not contain particles. The algorithm is fast (10 s on a 1.3 GHz Pentium M processor), is generic, and is not limited to any particular shape or size of the particle to be detected. The method has been evaluated on a publicly available dataset of 82 cryoEM images of keyhole lympet hemocyanin (KLH). From 998 automatically extracted particle images, the 3-D structure of KLH has been reconstructed at a resolution of 23.2 A which is the same resolution as obtained using particles manually selected by a trained user.     

Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy

We report on initial results of using a new direct detection device (DDD) for single particle reconstruction of vitreous ice embedded specimens. Images were acquired on a Tecnai F20 at 200 keV and a nominal magnification of 29,000×. This camera has a significantly improved signal to noise ratio and modulation transfer function (MTF) at 200 keV compared to a standard CCD camera installed on the same microscope. Control of the DDD has been integrated into Leginon, an automated data collection system. Using GroEL as a test specimen, we obtained images of ∼30 K particles with the CCD and the DDD from the same specimen sample using essentially identical imaging conditions. Comparison of the maps reconstructed from the CCD images and the DDD images demonstrates the improved performance of the DDD. We also obtained a 3D reconstruction from ∼70 K GroEL particles acquired using the DDD; the quality of the density map demonstrates the potential of this new recording device for cryoEM data acquisition.     

Keyhole limpet hemocyanin type 2 (KLH2): detection and immunolocalization of a labile functional unit h

Keyhole limpet hemocyanin (KLH) is a mixture of two hemocyanin isoforms, termed KLH1 and KLH2. Within KLH1 eight oxygen-binding functional units (FUs), 1-a to 1-h, have been identified, in contrast to KLH2, which was previously thought to be organized in seven FUs (2-a to 2-g). By limited proteolysis of KLH2 subunits, isolation of the polypeptide fragments, and N-terminal sequencing, we have now identified an eighth FU of type h, with a molecular mass of 43 kDa. This is unusually small for a FU h from a gastropodan hemocyanin. It is also shown that KLH2 didecamers can be split into a stable and homogeneous population of decamers by dialysis against 50 mM Tris/HCl, pH 7.5, in the absence of divalent cations. Electron microscopic immunolocalization using a specific monoclonal antibody reveals that FU KLH2-h is located at the collar of the decamer.     

Biomechanical Study on the Novel Biomimetic Hemi-Pelvis Prosthesis

A pelvic endoprosthesis is the primary means of pelvic reconstruction after internal hemipelvectomy. In this study, a novel biomimetic hemipelvic prosthesis, including an artificial ilium, an artificial acetabulum, and an artificial pubis, was developed. A Finite Element Method （FEM） was carried out to investigate the biomechanical performance of a pelvis reconstructed with biomimetic hemipelvic prosthesis. Two models, including the reconstructed pelvis and the original pelvis （control model）, were established according to the geometry from CT data of a human male patient with pelvic bone sarcomas. The FE models predict that the biomeehanical function of the pelvic ring can be reestablished using this prosthesis. Results show that the body force loaded on the S 1 vertebra is restored and transferred towards the sacro-iliac joint, and along the ilium onto the bearing surface of the artificial ilium, then to the artificial acetabulum and pubis. Von Mises stresses observed in this reconstructed pelvis model are still within a low and elastic range below the yielding strength of cortical bone and Ti6A14V. The values of deformation and strain of the reconstructed pelvis are close to the data obtained in the original pelvis. With the partial replacement of the pubis, little influence is found towards the pubis symphysis. However, the interface between the prosthesis and pelvic bone may become the critical part of the reconstructed pelvis due to the discontinuity in the material properties, which results in stress shielding and deformation constraining. So a biomimetic flexible connection or inter layer to release the deformation of pelvis is suggested in future designing.     

Reconstruction for 3D PET Based on Total Variation Constrained Direct Fourier Method

This paper presents a total variation (TV) regularized reconstruction algorithm for 3D positron emission tomography (PET). The proposed method first employs the Fourier rebinning algorithm (FORE), rebinning the 3D data into a stack of ordinary 2D data sets as sinogram data. Then, the resulted 2D sinogram are ready to be reconstructed by conventional 2D reconstruction algorithms. Given the locally piece-wise constant nature of PET images, we introduce the total variation (TV) based reconstruction schemes. More specifically, we formulate the 2D PET reconstruction problem as an optimization problem, whose objective function consists of TV norm of the reconstructed image and the data fidelity term measuring the consistency between the reconstructed image and sinogram. To solve the resulting minimization problem, we apply an efficient methods called the Bregman operator splitting algorithm with variable step size (BOSVS). Experiments based on Monte Carlo simulated data and real data are conducted as validations. The experiment results show that the proposed method produces higher accuracy than conventional direct Fourier (DF) (bias in BOSVS is 70% of ones in DF, variance of BOSVS is 80% of ones in DF).     

Alignment of 3D structures of macromolecular assemblies

MOTIVATION: A number of macromolecular assemblies are being reconstructed in 3D from electron micrographs. The analysis yields a 3D matrix representing the protein density map. In reconstruction processes and in comparing the results of different experiments, it is often necessary to obtain all models oriented the same way in three dimensions. The problem is not trivial since there exist no 3D counterpart of correlation analysis used for 2D images. It is usually solved by time consuming trial and error algorithms. RESULTS: 3D density distributions can be brought to a 'canonical' orientation. The tensor of inertia of the distribution is determined and its eigenvectors are oriented along the coordinate axes. The method is fast and essentially free of reference. It is suitable for structures whose inertial axes do not completely degenerate as they do in icosahedral viruses or if symmetry is cubic. Applications are presented for asymmetric objects and for molecules possessing symmetry axes higher than twofold. IMPLEMENTATION: The implementation simply requires the accumulation of the inertial tensor and its diagonalisation. Volume data rotation has been already illustrated in this journal by the authors.     

Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST)

We describe the development of quantitative electron spectroscopic tomography (QuEST), which provides 3-D distributions of elements on a nanometer scale. Specifically, it is shown that QuEST can be applied to map the distribution of phosphorus in unstained sections of embedded cells. A series of 2-D elemental maps is derived from images recorded in the energy filtering transmission electron microscope for a range of specimen tilt angles. A quantitative 3-D elemental distribution is then reconstructed from the elemental tilt series. To obtain accurate quantitative elemental distributions it is necessary to correct for plural inelastic scattering at the phosphorus L(2,3) edge, which is achieved by acquiring unfiltered and zero-loss images at each tilt angle. The data are acquired automatically using a cross correlation technique to correct for specimen drift and focus change between successive tilt angles. An algorithm based on the simultaneous iterative reconstruction technique (SIRT) is implemented to obtain quantitative information about the number of phosphorus atoms associated with each voxel in the reconstructed volume. We assess the accuracy of QuEST by determining the phosphorus content of ribosomes in a eukaryotic cell, and then apply it to estimate the density of nucleic acid in chromatin of the cell's nucleus. From our experimental data, we estimate that the sensitivity for detecting phosphorus is 20 atoms in a 2.7 nm-sized voxel.     

Accelerated Optical Projection Tomography Applied to In Vivo Imaging of Zebrafish

Optical projection tomography (OPT) provides a non-invasive 3-D imaging modality that can be applied to longitudinal studies of live disease models, including in zebrafish. Current limitations include the requirement of a minimum number of angular projections for reconstruction of reasonable OPT images using filtered back projection (FBP), which is typically several hundred, leading to acquisition times of several minutes. It is highly desirable to decrease the number of required angular projections to decrease both the total acquisition time and the light dose to the sample. This is particularly important to enable longitudinal studies, which involve measurements of the same fish at different time points. In this work, we demonstrate that the use of an iterative algorithm to reconstruct sparsely sampled OPT data sets can provide useful 3-D images with 50 or fewer projections, thereby significantly decreasing the minimum acquisition time and light dose while maintaining image quality. A transgenic zebrafish embryo with fluorescent labelling of the vasculature was imaged to acquire densely sampled (800 projections) and under-sampled data sets of transmitted and fluorescence projection images. The under-sampled OPT data sets were reconstructed using an iterative total variation-based image reconstruction algorithm and compared against FBP reconstructions of the densely sampled data sets. To illustrate the potential for quantitative analysis following rapid OPT data acquisition, a Hessian-based method was applied to automatically segment the reconstructed images to select the vasculature network. Results showed that 3-D images of the zebrafish embryo and its vasculature of sufficient visual quality for quantitative analysis can be reconstructed using the iterative algorithm from only 32 projections—achieving up to 28 times improvement in imaging speed and leading to total acquisition times of a few seconds.     

Sensitivity of breasts reconstructed with the autologous latissimus dorsi flap

The purpose of this study was to measure, both objectively and subjectively, the sensitivity of breasts reconstructed with the autologous latissimus dorsi flap and to compare these results with those of other reconstruction techniques, especially the transverse rectus abdominis myocutaneous flap. The study population included 50 patients with autologous latissimus dorsi flap breast reconstruction; these patients had an average age of 51 years and an average follow-up of 27 months. Patients answered a seven-item questionnaire that attempted to define the sensitivity of the reconstructed and opposite breasts. This sensitivity was then measured objectively using standard techniques for heat, cold, and tactile sensations. After statistical analysis, these results were compared with those published for other reconstruction techniques. Overall results were comparable or superior to those published for other techniques for autologous breast reconstruction. A total of 56 percent of patients had fine or very fine sensitivity, but 70 percent deemed this sensitivity to be less than that of the opposite breast. A total of 94 percent of patients perceived the reconstructed breast as integral to their body image. The superior medial part of the breast had the greatest sensitivity, both objectively and subjectively. Autologous latissimus dorsi breast reconstruction, a good technique with excellent aesthetic results, affords satisfactory sensitivity. This is yet another advantage of the technique.     

Identification and characterization of keyhole limpet hemocyanin N-glycans mediating cross-reactivity with Schistosoma mansoni

Keyhole limpet hemocyanin (KLH) of the mollusc Megathura crenulata is known to serologically cross-react with Schistosoma mansoni glycoconjugates in a carbohydrate-dependent manner. To elucidate the structural basis for this cross-reactivity, KLH glycans were released from tryptic glycopeptides and fluorescently labeled. Cross-reacting glycans were identified using a polyclonal antiserum reacting with soluble S. mansoni egg antigens, isolated by a three-dimensional fractionation scheme and analyzed by different mass spectrometric techniques as well as linkage analysis and exoglycosidase treatment. The results revealed that cross-reacting species comprise approximately 4.5% of released glycans. They all represent novel types of N-glycans with a Fuc(alpha1-3)GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc motif, which is known to occur also in schistosomal glycoconjugates. The tetrasaccharide unit is attached to the 3-linked antenna of a trimannosyl core, which can be further decorated by galactosyl residues, a xylose residue in 2-position of the central mannose and/or a fucose at the innermost N-acetylglucosamine. This study provides for the first time detailed structural data on the KLH carbohydrate entities responsible for cross-reactivity with glycoconjugates from S. mansoni.     

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy¹ and two-dimensional (2D) electron crystallography² have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method³, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments⁴, microtubules⁵, amyloid fibers⁶, tobacco mosaic viruses⁷, and bacteria flagella⁸, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid⁹ is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.     

A Toolbox for ab initio 3-D reconstructions in single-particle electron microscopy

Structure determination of a novel macromolecular complex via single-particle electron microscopy depends upon overcoming the challenge of establishing a reliable 3-D reconstruction using only 2-D images. There are a variety of strategies that deal with this issue, but not all of them are readily accessible and straightforward to use. We have developed a “toolbox” of ab initio reconstruction techniques that provide several options for calculating 3-D volumes in an easily managed and tightly controlled work-flow that adheres to standard conventions and formats. This toolbox is designed to streamline the reconstruction process by removing the necessity for bookkeeping, while facilitating transparent data transfer between different software packages. It currently includes procedures for calculating ab initio reconstructions via random or orthogonal tilt geometry, tomograms, and common lines, all of which have been tested using the 50S ribosomal subunit. Our goal is that the accessibility of multiple independent reconstruction algorithms via this toolbox will improve the ease with which models can be generated, and provide a means of evaluating the confidence and reliability of the final reconstructed map.     

Immunoregulatory abnormalities in autoimmune thyroid diseases

We have investigated the ability of lymphocytes from normal subjects and patients with autoimmune thyroid diseases to respond to a thyroidal antigen (human thyroglobulin, hTG) and a non-thyroidal antigen (Keyhole limpet hemocyanin, KLH) in vitro, using a hapten (trinitrophenol, TNP)-carrier system. This system is based on the concept that the T helper cells which respond to hTG or KLH should stimulate anti-TNP antibody producing B cells in the presence of TNP conjugated hTG (TNP-hTG) or KLH (TNP-KLH). After 5 or 6 days of culture of peripheral blood mononuclear cells with pokeweed mitogen (PWM), PWM and TNP-hTG, or PWM and TNP-KLH, IgM anti-TNP and IgM anti-sheep red blood cell (SRBC) plaque forming cells (PFC) were enumerated. The results showed that (1) in normal controls, hTG caused only suppression in both TNP and SRBC response, and KLH caused dose-related enhancement and suppression in TNP response without a change in SRBC response, and (2) in patients, both hTG and KLH resulted in dose-related enhancement in TNP response without a change in SRBC response. These data suggest that patients with autoimmune thyroid diseases have regulatory cell abnormalities confined to a thyroid antigen.