The three-dimensional structure of bovine rhodopsin determined by electron cryomicroscopy

G-protein-coupled receptors are integral membrane proteins that respond to environmental signals and initiate signal transduction pathways, which activate cellular processes. Rhodopsin, a well known member of the G-protein-coupled receptor family, is located in the disk membranes of the rod outer segment, where it is responsible for the visualization of dim light. Rhodopsin is the most extensively studied G-protein-coupled receptor, and knowledge about its structure serves as a template for other related receptors. We have gained detailed structural knowledge from the crystal structure (1), which was solved by x-ray crystallography in 2000 using three-dimensional crystals. Here we report a three-dimensional density map of bovine rhodopsin determined by electron cryomicroscopy of two-dimensional crystals with p22(1)2(1) symmetry. The usage of relatively small and disordered crystals made the process of structure determination challenging. Special attention was paid to the extraction of amplitudes and phases, since usable raw data were limited to a maximum tilt of 45 degrees. In the refinement process, an improved unbending procedure was applied. This led to a final resolution of 5.5 A in the membrane plane and approximately 13 A perpendicular to it, making our electron density map the most accurate map of a G-protein-coupled receptor currently available by electron microscopy. Most important is the information we gain about the center of the membrane plane and the orientation of the molecule relative to the bilayer. This information cannot be retrieved from the three-dimensional crystals. In our electron density map, all seven transmembrane helices were identified, and their arrangement is in agreement with the arrangement known from the crystal structure (1). In the retinal binding pocket, a density peak adjacent to helix 3 suggests the position of the beta-ionine ring of the chromophore, and in its vicinity several of the bigger amino acids can be identified.     

Applications of three-dimensional display techniques in medical imaging

This paper outlines some of the current uses of three-dimensional techniques in medical imaging applications and their potential for the future. As an example, three-dimensional imaging is described using a CT scanner, as it applies to a case involving craniofacial surgery. This includes defining the relationship between the requirements on the data acquisition system, as well as the specification of the hardware and software for the display. A currently used algorithm is described for the display of surfaces as a function of local position, orientation of the surface and the position of a virtual light source. This includes the use of transparency and cut plane greyscale techniques, in addition to the display of the surfaces. A speculation is made regarding the use of a three-dimensional display as the standard viewing mode in CT, with slice and multiplanar imaging as submodes.     

Probing the three-dimensional structure of human calreticulin

Calreticulin (CRT) is an abundant soluble protein of the endoplasmic reticulum lumen that functions as a molecular chaperone for nascent glycoproteins. We have probed the three-dimensional structure of human CRT using a series of biochemical and biophysical approaches in an effort to understand the molecular basis of its chaperone function. Sedimentation analysis and chemical cross-linking experiments showed that CRT is monodisperse and monomeric in solution with a molecular mass (MW) of 46 +/- 1 kDa. This MW value together with a sedimentation coefficient, s(o)(20,w), of 2.71 S yielded a frictional ratio, f/f(0), of 1.65. Assuming CRT to be a prolate ellipsoid, we calculated an apparent length of 29.8 nm and diameter of 2.44 nm consistent with an asymmetric elongated molecule. These hydrodynamic dimensions account for the apparent anomalous elution position of CRT on gel filtration columns. Far-UV circular dichroism experiments showed that CRT has a cooperative thermal denaturation transition with a midpoint temperature of 42.5 degrees C suggesting a marginally stable structure. Proteolysis experiments showed that the highly acidic segment at the C-terminus of CRT is most susceptible to digest, consistent with the absence of a well-defined polypeptide backbone structure in this region of the protein. Temperature-dependent proteolysis with thermolysin revealed a stable core region within the N- and P-domains. A stable fragment encompassing most of the P-domain was also identified in the thermolytic mixture. Collectively, our results suggest that CRT is likely to be a flexible molecule in solution which may be important for its chaperone function.     

东亚飞蝗消化道的三维重建方法研究

本文主要研究东亚飞蝗Locutsta migratoria manilansis(Meyen)消化道可视化模型三维重建方法.采用冰冻切片技术将冰冻包埋剂(OCT)包埋后的飞蝗成虫做连续切片,进行截面图像信息采集,建立数据集;通过Photoshop、Image-Pro Plus(IPP)软件对消化道截面图像进行分割、处理...     

An adjustable-threshold algorithm for the identification of objects in three-dimensional images

MOTIVATION:To develop a highly accurate, practical and fast automated segmentation algorithm for three-dimensional images containing biological objects. To test the algorithm on images of the Drosophila brain, and identify, count and determine the locations of neurons in the images. RESULTS: A new adjustable-threshold algorithm was developed to efficiently segment fluorescently labeled objects contained within three-dimensional images obtained from laser scanning confocal microscopy, or two-photon microscopy. The result of the test segmentation with Drosophila brain images showed that the algorithm is extremely accurate and provided detailed information about the locations of neurons in the Drosophila brain. Centroids of each object (nucleus of each neuron) were also recorded into an algebraic matrix that describes the locations of the neurons. AVAILABILITY: Interested parties should send their request for the NeuronMapper(TM) program with the segmentation algorithm to artemp@bcm.tmc.edu.     

A method for reconstructing three-dimensional positions of swarming mosquitoes

We developed a new field method for reconstructing the three-dimensional positions of swarming mosquitoes. This method overcame certain inherent difficulties accompanied by conventional stereoscopic methods and is applicable to three-dimensional measurements of other insect species. Firstly, we constructed a probabilistic model for stereoscopy; if mosquitoes and six reference points with known coordinates were photographed simultaneously from two or more perspectives, then from the positions of images of mosquitoes and the reference points on the photographs, 1) the position of each camera with respect to the reference points is estimated; 2) stereo images which correspond to an identical real mosquito are matched; and 3) the spatial positions of the mosquitoes are determined. We automated the processes 1), 2) and 3), developing computer programs based on our model. We then constructed a portable system for three-dimensional measurements of swarming mosquitoes in the field. Initial data that illustrate the application of our method to studying mosquito swarming were presented.     

Possible three-dimensional models for the polypeptide backbone structure of alamethicin

Based on an empirical procedure of predicting polypeptide backbone structures of proteins, an attempt was made to construct possible three-dimensional models for the secondary structure of alamethicin. Two chemical properties were used to limit the number of possible models: the presence of seven to eight α-aminoisobutyric acid residues whose conformations were quite restricted, and a covalent bond between the proline residue at position 1 and the glutamic acid residue at position 17. The predicted structures appear very compact and show features in agreement with circular dichroism and optical rotatory dispersion results, nuclear magnetic resonance measurements, and compressed monolayer studies.     

The effect of three-dimensional postural change on shear elastic modulus of the iliotibial band

To understand and treat iliotibial band (ITB) syndrome, caused by excessive compression between the ITB and lateral femoral condyle, it is important to identify factors contributing to an increase in ITB stiffness. The purpose of this study was to clarify the factors that contribute to an increase in ITB stiffness by examining the relationship between three-dimensional postural changes and ITB stiffness. Fourteen healthy individuals performed one-leg standing under 7 conditions (including normal one-leg standing as a control condition) in which the pelvic position was changed in three planes. The shear elastic modulus in the ITB was measured using shear-wave elastography, as a measure of ITB stiffness. The three-dimensional joint angles and external joint moments in the hip and knee joints were also measured to confirm the changes in joint angles and external load. Compared to the normal one-leg standing condition, ITB stiffness was significantly increased in the pelvic posterior tilted position (i.e. hip extension), contralateral pelvic dropped position (i.e. hip adduction), and contralateral pelvic posterior rotated position (i.e. hip external rotation). The findings suggest that interventions to reduce hip extension, adduction, and external rotation might be useful if these excessive positional changes are detected in patients with ITB syndrome.     

A rigid body model of the forearm

In this article the forearm, with its complex, continuous motion of masses during pronation/supination, was approximated by a rigid body model consisting of a radial segment rotating around an ulnar segment. The method used to obtain the model parameters is based on three-dimensional voxel data that include velocity information. We propose a criterion that allows the voxels to be attributed to either of the two segments. It is based on the notion that the rotational kinetic energy determined from the voxel data equals the kinetic energy of the rigid body model. To obtain a three-dimensional smoothing we further propose a parameterization of the shape of both segments. These shapes can then be used to determine the dynamic integrals of the segments, i.e. mass, center of mass, and inertia. Using this approach we determined all model parameters for a human forearm from three series of MRI scans in a supinated, a pronated, and an intermediate position. In the appendix, a procedure is described that allows the dynamic quantities to be scaled homogeneously without recalculation of the integrals. Thus, this article provides all essential parameters required for three-dimensional dynamic simulations of general movements of the forearm.     

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out-of-locus light and provides a greater resolution than the wide-field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction-limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two-dimensional image. Thus, confocal scanning optical microscopy allows accurate non-invasive optical sectioning and further three-dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three-dimensional imaging.     

Analysis of three-dimensional ciliary beating by means of high-speed stereomicroscopy.

Results are presented on the analysis of three-dimensional motion of compound cilia or cirri in voltage-clamped specimens of the protozoan Stylonychia mytilus. Time series of three-dimensional data were obtained by using the anaxial illumination method for simultaneous recording of stereoscopic video images. Data processing involved the following steps: determination of a reference coordinate system based solely on features present in each stereo-pair; tracing of cirral axes in digitized images, conversion to parameter curves by means of least-squares polynomial approximation, conversion of pairs of two-dimensional data to a series of three-dimensional data; correction for distortion due to projective shortening and conversion to a series of polynomial triplets, and analysis of the periodical components of the motion pattern in the frequency domain. Reconstructed beating cycles show typical differences between hyperpolarization-induced ciliary activity and depolarization-induced ciliary activity. Reconstructions of the motion of the basal segment of a cirrus are in agreement with existing data. Analysis of the curvature and torsion of a cirral axis during beating does not reveal any simple pattern of propagated activity within the axoneme. The return stroke may be subdivided into two phases. First, a curvature peak develops proximally. Secondly, a region with increased torsion arises more distally and spreads out in proximal direction. Both curvature and torsion return to minimal values by the beginning of the power stroke.     

Use of dual Euler angles to quantify the three-dimensional joint motion and its application to the ankle joint complex

This paper presents a modified Euler angles method, dual Euler angles approach, to describe general spatial human joint motions. In dual Euler angles approach, the three-dimensional joint motion is considered as three successive screw motions with respect to the axes of the moving segment coordinate system; accordingly, the screw motion displacements are represented by dual Euler angles. The algorithm for calculating dual Euler angles from coordinates of markers on the moving segment is also provided in this study. As an example, the proposed method is applied to describe motions of ankle joint complex during dorsiflexion–plantarflexion. A Flock of Birds electromagnetic tracking device (FOB) was used to measure joint motion in vivo. Preliminary accuracy tests on a gimbal structure demonstrate that the mean errors of dual Euler angles evaluated by using source data from FOB are less than 1° for rotations and 1 mm for translations, respectively. Based on the pilot study, FOB is feasible for quantifying human joint motions using dual Euler angles approach.     

Computer-aided three-dimensional reconstruction of nematode embryos from EM serial sections

Embryos of the nematode Caenorhabditis elegans were serially sectioned and photographed in the electron microscope (EM). The micrographs were used to produce three-dimensional (3D) reconstructions. Size and position of each nucleus were entered into a computer, displayed as spheres, and were color-coded to indicate lineage membership. Location in space and position in the cell cycle are generally adequate criteria to identify cells. The reconstructions allow visualization of lineage-related topographic patterns and ultrastructural analysis of differently determined cells.     

Troponin organization on relaxed and activated thin filaments revealed by electron microscopy and three-dimensional reconstruction

The steric model of muscle regulation holds that at low Ca(2+) concentration, tropomyosin strands, running along thin filaments, are constrained by troponin in an inhibitory position that blocks myosin-binding sites on actin. Ca(2+) activation, releasing this constraint, allows tropomyosin movement, initiating actin-myosin interaction and contraction. Although the different positions of tropomyosin on the thin filament are well documented, corresponding information on troponin has been lacking and it has therefore not been possible to test the model structurally. Here, we show that troponin can be detected on thin filaments and demonstrate how its changing association with actin can control tropomyosin position in response to Ca(2+). To accomplish this, thin filaments were reconstituted with an engineered short tropomyosin, creating a favorable troponin stoichiometry and symmetry for three-dimensional analysis. We demonstrate that in the absence of Ca(2+), troponin bound to both tropomyosin and actin can act as a latch to constrain tropomyosin in a position on actin that inhibits actomyosin ATPase. In addition, we find that on Ca(2+) activation the actin-troponin connection is broken, allowing tropomyosin to assume a second position, initiating actomyosin ATPase and thus permitting contraction to proceed.     

The accuracy of DLT extrapolation in three-dimensional film analysis

An analysis of errors arising from the Direct Linear Transformation (DLT) approach to three-dimensional reconstructions from two-dimensional images has been undertaken, the principal factor studied being the number and distribution of control points used in the calibration procedure. Significantly increased error was found to be associated with extrapolation to unknown points outside the control point distribution space. Differences in accuracy between two camera position set-ups and 11 vs 12 DLT parameter solutions were also examined.     

Vinculin regulates directionality and cell polarity in two- and three-dimensional matrix and three-dimensional microtrack migration

During metastasis, cells can use proteolytic activity to form tube-like “microtracks” within the extracellular matrix (ECM). Using these microtracks, cells can migrate unimpeded through the stroma. To investigate the molecular mechanisms of microtrack migration, we developed an in vitro three-dimensional (3D) micromolded collagen platform. When in microtracks, cells tend to migrate unidirectionally. Because focal adhesions are the primary mechanism by which cells interact with the ECM, we examined the roles of several focal adhesion molecules in driving unidirectional motion. Vinculin knockdown results in the repeated reversal of migration direction compared with control cells. Tracking the position of the Golgi centroid relative to the position of the nucleus centroid reveals that vinculin knockdown disrupts cell polarity in microtracks. Vinculin also directs migration on two-dimensional (2D) substrates and in 3D uniform collagen matrices, as indicated by reduced speed, shorter net displacement, and decreased directionality in vinculin-deficient cells. In addition, vinculin is necessary for focal adhesion kinase (FAK) activation in three dimensions, as vinculin knockdown results in reduced FAK activation in both 3D uniform collagen matrices and microtracks but not on 2D substrates, and, accordingly, FAK inhibition halts cell migration in 3D microtracks. Together these data indicate that vinculin plays a key role in polarization during migration.     

三维城市景观生态研究

城市景观元素的三维分析是城市景观研究发展的重要趋势之一。城市形态的结构特征与功能的空间作用不仅体现在二维空间中,同时在三维向度上表现出城市社会经济发展态势与空间演变的相互作用,因此在城市空间格局、过程与功能的讨论中,不能局限于二维平面。首先由三维视角提出城市景观格局及功能网络特征,并结合对航空摄影测量、卫星遥测、机载激光扫描等三维信息获取方法的认识,介绍目前城市景观三维信息提取的相关方法;其次,在三维城市景观生态研究内容方面,重点介绍城市景观要素提取、格局特征及其动态变化、生态环境效应和城市景观功能等相关研究,探讨城市内部的三维建成景观以及绿地景观要素提取与应用,并在传统景观格局分析方法基础上,提出了三维城市景观格局特征测量方法、动态变化监测方法以及动态模拟等相关内容,进一步对三维城市景观在生物多样性、局部气候变化等生态环境效应方面的研究进行了归纳总结;最后借助构建三维景观功能网络的思路,以作为三维城市景观功能优化研究的切入点。     

Mutational studies using a cation-conducting GABAA receptor reveal the selectivity determinants of the Cys-loop family of ligand-gated ion channels

The present study evaluates how four key amino acid residue positions (- 4' to - 1') within the M1-M2 linker of the GABA(A) receptor beta subunit influences ion selectivity of a cation-conducting GABA receptor. Cation selectivity was found to be highly dependent on the side-chains of the amino acid residues present. The critical factor for cation selectivity was the presence of a negatively charged Glu or Asp residue in the -1' position. Receptors containing the neutral amino acids Gln or Asn or a positively charged Arg residue were anion selective. In the presence of a -1' Glu residue, the amino acids in adjacent positions were also found to be important determinants of cation selectivity. Moreover, the length of the M1-M2 linker as well as the presence of a Pro residue within this segment also affected ion selectivity, suggesting that the local environment and three-dimensional position of the -1' Glu are essential determinants of cation permeation. Conversely, no specific amino acid residues were found to be essential for anion selectivity, suggesting that the basic architecture of the selectivity segment of this class of receptor channels is optimally suited for anion conduction.     

东亚飞蝗雄性生殖系统组织结构观察及三维可视化数字模型

本文研究东亚飞蝗Locusta migratoria manilensis(Meyen)雄性生殖系统组织解剖结构及三维可视化数字模型的构建。采用石蜡切片技术对东亚飞蝗进行组织切片,脱水、透明、HE染色和拍照;并应用冰冻切片技术将冰冻包埋剂包埋后的飞蝗雄性个体进行连续切片,进行截面图像信息采集,建立数据集;通过Photoshop、Image-Pro Plus(IPP)软件对雄性生殖器官截面图像进行分割、处理、序列化和三维重建。通过试验观察分析东亚飞蝗雄性生殖器官精巢、附腺、输精管、精球囊和射精囊及交配器的组织构造;并成功构建了飞蝗雄性生殖系统的三维结构可视化数字模型。该模型可以任意旋转,能从不同角度观察,该试验为研究和教学提供了理论基础。     

Gait analysis using gravitational acceleration measured by wearable sensors

A novel method for measuring human gait posture using wearable sensor units is proposed. The sensor units consist of a tri-axial acceleration sensor and three gyro sensors aligned on three axes. The acceleration and angular velocity during walking were measured with seven sensor units worn on the abdomen and the lower limb segments (both thighs, shanks and feet). The three-dimensional positions of each joint are calculated from each segment length and joint angle. Joint angle can be estimated mechanically from the gravitational acceleration along the anterior axis of the segment. However, the acceleration data during walking includes three major components; translational acceleration, gravitational acceleration and external noise. Therefore, an optimization analysis was represented to separate only the gravitational acceleration from the acceleration data. Because the cyclic patterns of acceleration data can be found during constant walking, a FFT analysis was applied to obtain some characteristic frequencies in it. A pattern of gravitational acceleration was assumed using some parts of these characteristic frequencies. Every joint position was calculated from the pattern under the condition of physiological motion range of each joint. An optimized pattern of the gravitational acceleration was selected as a solution of an inverse problem. Gaits of three healthy volunteers were measured by walking for 20 s on a flat floor. As a result, the acceleration data of every segment was measured simultaneously. The characteristic three-dimensional walking could be shown by the expression using a stick figure model. In addition, the trajectories of the knee joint in the horizontal plane could be checked by visual imaging on a PC. Therefore, this method provides important quantitive information for gait diagnosis.