Journal of Structural Biology

Cryo-electron tomography and 3-D analysis of the intact flagellum in Trypanosoma brucei.

Publication Date: 2012 Jan 25 PMID: 22285651
Authors: Hoog, J. L. - Bouchet-Marquis, C. - McIntosh, R. J. - Hoenger, A. - Gull, K.
Journal: J Struct Biol

Trypanosoma brucei is a uni-cellular protist that causes African sleeping sickness. These parasites have a flagellum that is attached to the cell body and is indispensible for its motility. The flagellum consists of a canonical 9+2 axoneme and a paraflagellar rod (PFR), an intricate tripartite, fibrous structure that is connected to the axoneme. In this paper we describe results from cryo-electron tomography of unperturbed flagella. This method revealed novel structures that are likely involved in attaching the flagellum to the cell. We also show the first cryo-electron tomographic images of a basal body in situ, revealing electron dense structures inside its triplet microtubules. Sub-tomogram averaging of the PFR revealed that its distal region is organized as an orthorhombic crystal.

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Direct electron detection yields cryo-EM reconstructions at resolutions beyond 3/4 Nyquist frequency.

Publication Date: 2012 Jan 21 PMID: 22285189
Authors: Bammes, B. E. - Rochat, R. H. - Jakana, J. - Chen, D. H. - Chiu, W.
Journal: J Struct Biol

One limitation in electron cryo-microscopy (cryo-EM) is the inability to recover high-resolution signal from the image-recording media at the full-resolution limit of the transmission electron microscope. Direct electron detection using CMOS-based sensors for digitally recording images has the potential to alleviate this shortcoming. Here, we report a practical performance evaluation of a Direct Detection Device (DDD) for biological cryo-EM at two different microscope voltages: 200 and 300kV. Our DDD images of amorphous and graphitized carbon show strong per-pixel contrast with image resolution near the theoretical sampling limit of the data. Single-particle reconstructions of two frozen-hydrated bacteriophages, P22 and epsilon15, establish that the DDD is capable of recording usable signal for 3-D reconstructions at about 4/5 of the Nyquist frequency, which is a vast improvement over the performance of conventional imaging media. We anticipate the unparalleled performance of this digital recording device will dramatically benefit cryo-EM for routine tomographic and single-particle structural determination of biological specimens.

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Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging.

Publication Date: 2012 Jan 18 PMID: 22273540
Authors: Schneider, G. - Guttmann, P. - Rehbein, S. - Werner, S. - Follath, R.
Journal: J Struct Biol

X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating monochromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spectral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochromator close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed.

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Special focused issue on X-ray microscopy of biological materials.

Publication Date: 2012 Jan 18 PMID: 22270178
Authors: Carrascosa, J. L. - Glaeser, R. M.
Journal: J Struct Biol

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Structure, stability and dynamics of norovirus P domain derived protein complexes studied by native mass spectrometry.

Publication Date: 2012 Jan 12 PMID: 22266117
Authors: Bereszczak, J. Z. - Barbu, I. M. - Tan, M. - Xia, M. - Jiang, X. - van Duijn, E. - Heck, A. J.
Journal: J Struct Biol

Expression of the protruding (P) domain of the norovirus capsid protein, in vitro, results in the formation of P dimers and larger oligomers, 12-mer and 24-mer P particles. All these P complexes retain the authentic antigenicity and carbohydrate-binding function of the norovirus capsid. They have been used as tools to study norovirus-host interactions, and the 24-mer P particle has been proposed as a vaccine and vaccine platform against norovirus and other pathogens. In view of their pharmaceutical interest it is important to characterise the structure, stability and dynamics of these protein complexes. Here we use a native mass spectrometric approach. We analyse the P particles under both non-reducing and reducing conditions, as it is known that the macromolecular assemblies are stabilised by inter-subunit disulphide bonding. A novel 18-mer complex is identified, and we show that under reducing conditions the 24-mer dissociates into P dimers that reassemble into the 12-mer small P particle and another novel 36-mer complex. The collisional cross-sections of the 12-mer and 24-mer P particles determined by ion mobility MS are in good agreement with theoretical predictions and electron microscopy data. We propose a model structure for the 18-mer based on ion mobility experiments. Our results demonstrate the interchangeable nature and dynamic relationship of all P domain complexes and confirm their binding activity to the host receptors - human histo blood group antigens (HBGAs). These findings, together with the identification of the 18-mer and 36-mer P complexes add new information to the intriguing interactions of the norovirus P domain.

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Removing high contrast artifacts via digital inpainting in cryo-electron tomography: An application of compressed sensing.

Publication Date: 2012 Jan 10 PMID: 22248454
Authors: Song, K. - Comolli, L. R. - Horowitz, M.
Journal: J Struct Biol

To cope with poor quality in cryo-electron tomography images, electron-dense markers, such as colloidal goldbeads, are often used to assist image registration and analysis algorithms. However, these markers can create artifacts that occlude a specimen due to their high contrast, which can also cause failure of some image processing algorithms. One way of reducing these artifacts is to replace high contrast objects with pixel densities that blend into the surroundings in the projection domain before volume reconstruction. In this paper, we propose digital inpainting via compressed sensing (CS) as a new method to achieve this goal. We show that cryo-ET projections are sparse in the discrete cosine transform (DCT) domain, and, by finding the sparsest DCT domain decompositions given uncorrupted pixels, we can fill in the missing pixel values that are occluded by high contrast objects without discontinuities. Our method reduces visual artifacts both in projections and in tomograms better than conventional algorithms, such as polynomial interpolation and random noise inpainting.

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Structural and dynamical analysis of an engineered FhuA channel protein embedded into a lipid bilayer or a detergent belt.

Publication Date: 2012 Jan 11 PMID: 22248453
Authors: Rodriguez-Ropero, F. - Fioroni, M.
Journal: J Struct Biol

Engineered channel proteins are promising nano-components with applications in nanodelivery and nanoreactors technology. Because few of the engineered channel proteins have been crystallized, solution studies based on Neutron Scattering, Circular Dichroism and NMR play a major role. Consequently, the understanding of membrane proteins dynamics in water/detergent solutions or when embedded in a lipid membrane, can clarify how the environment affects protein behavior. In this study, molecular dynamics simulations of the FhuA Escherichia coli outer membrane channel protein and its engineered FhuA Delta1-159 variant have been performed in two different environments: a DNPC (1,2-dinervonyl-sn-glycero-3-phosphocholine) lipid bilayer and a water/OES (N-octyl-2-hydroxyethyl sulfoxide) detergent solution. Furthermore the FhuA Delta1-159 variant has been simulated in the open and closed states, the last induced by the presence of six 3-(2-pyridyldithio)-propionic-acid in the channel inner core. Differences in protein structural and dynamical behavior between the two environments have been found. Considering the FhuA protein characterized by an elliptical-cylindrical symmetry: (a) neither variations on the secondary structure nor axial deformation have been observed in any of the systems; (b) the ellipticity of the channel section (open state) and its fluctuations are enhanced in presence of water/OES, while diminished or suppressed in the DNPC bilayer; (c) the insertion of hydrophobic pyridyl groups into the FhuA Delta1-159 channel (closed state) induces a higher ellipticity in water/OES solution, while shifting to a circular section in the DNPC membrane; (d) the cork domain represented by the first 159 amino acids does not play a major role for protein stability.

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Three-dimensional structure of the shell plate assembly of the chiton Tonicella marmorea and its biomechanical consequences.

Publication Date: 2012 Jan 10 PMID: 22248452
Authors: Connors, M. J. - Ehrlich, H. - Hog, M. - Godeffroy, C. - Araya, S. - Kallai, I. - Gazit, D. - Boyce, M. - Ortiz, C.
Journal: J Struct Biol

This study investigates the three-dimensional structure of the eight plate exoskeletal (shell) assembly of the chiton Tonicella marmorea. X-ray micro-computed tomography and 3D printing elucidate the mechanism of conformational change from a passive (slightly curved, attached to surface) to a defensive (rolled, detached from surface) state of the plate assembly. The passive and defensive conformations exhibited differences in longitudinal curvature index (0.43 vs. 0.70), average plate-to-plate overlap ( approximately 62% vs. approximately 48%), cross-sectional overlap heterogeneity (60-82.5% vs. 0-90%, fourth plate), and plate-to-plate separation distance (100% increase in normalized separation distance between plates 4 and 5), respectively. The plate-to-plate interconnections consist of two rigid plates joined by a compliant, actuating muscle, analogous to a geometrically structured shear lap joint. This work provides an understanding of how T. marmorea achieves the balance between mobility and protection. In the passive state, the morphometry of the plates and plate-to-plate interconnections results in an approximately continuous curvature and constant armor thickness, resulting in limited mobility but maximum protection. In the defensive state, the underlying soft tissues gain protection and the chiton gains mobility through tidal flow, but regions of vulnerability open dorsally, due to the increase in plate-to-plate separation and decrease in plate-to-plate overlap. Lastly, experiments using optical and scanning electron microscopy, mercury porosimetry, and Fourier-transform infrared spectroscopy explore the microstructure and spatial distribution of the six layers within the intermediate plates, the role of multilayering in resisting predatory attacks, and the detection of chitin as a major component of the intra-plate organic matrix and girdle.

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Hydrogen-bond network and pH sensitivity in transthyretin: Neutron crystal structure of human transthyretin.

Publication Date: 2012 Jan 11 PMID: 22248451
Authors: Yokoyama, T. - Mizuguchi, M. - Nabeshima, Y. - Kusaka, K. - Yamada, T. - Hosoya, T. - Ohhara, T. - Kurihara, K. - Tomoyori, K. - Tanaka, I. - Niimura, N.
Journal: J Struct Biol

Transthyretin (TTR) is a tetrameric protein associated with human amyloidosis. In vitro, the formation of amyloid fibrils by TTR is known to be promoted by low pH. Here we show the neutron structure of TTR, focusing on the hydrogen bonds, protonation states and pH sensitivities. A large crystal was prepared at pD 7.4 for neutron protein crystallography. Neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. The neutron structure solved at 2.0A resolution revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is composed of Thr75, Trp79, His88, Ser112, Pro113, Thr118-B and four water molecules, and is involved in both monomer-monomer and dimer-dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. In addition, the comparison with X-ray structure at pH 4.0 indicated that the protonation occurred to Asp74, His88 and Glu89 at pH 4.0. Our neutron model provides insights into the molecular stability of TTR related to the hydrogen-bond network, the pH sensitivity and the CH...O weak hydrogen bond.

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Computational separation of conformational heterogeneity using cryo-electron tomography and 3D sub-volume averaging.

Publication Date: 2012 Jan 10 PMID: 22248450
Authors: Frank, G. A. - Bartesaghi, A. - Kuybeda, O. - Borgnia, M. J. - White, T. A. - Sapiro, G. - Subramaniam, S.
Journal: J Struct Biol

We have previously used cryo-electron tomography combined with sub-volume averaging and classification to obtain 3D structures of macromolecular assemblies in cases where a single dominant species was present, and applied these methods to the analysis of a variety of trimeric HIV-1 and SIV envelope glycoproteins (Env). Here, we extend these studies by demonstrating automated, iterative, missing wedge-corrected 3D image alignment and classification methods to distinguish multiple conformations that are present simultaneously. We present a method for measuring the spatial distribution of the vector elements representing distinct conformational states of Env. We identify data processing strategies that allow clear separation of the previously characterized closed and open conformations, as well as unliganded and antibody liganded states of Env when they are present in mixtures. We show that identifying and removing spikes with the lowest signal-to-noise ratios improves the overall accuracy of alignment between individual Env sub-volumes, and that alignment accuracy, in turn, determines the success of image classification in assessing conformational heterogeneity in heterogeneous mixtures. We validate these procedures for computational separation by successfully separating and reconstructing distinct 3D structures for unliganded and antibody-liganded as well as open and closed conformations of Env present simultaneously in mixtures.

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Real-space processing of helical filaments in SPARX.

Publication Date: 2012 Jan 11 PMID: 22248449
Authors: Behrmann, E. - Tao, G. - Stokes, D. L. - Egelman, E. H. - Raunser, S. - Penczek, P. A.
Journal: J Struct Biol

We present a major revision of the iterative helical real-space refinement (IHRSR) procedure and its implementation in the SPARX single particle image processing environment. We built on over a decade of experience with IHRSR helical structure determination and we took advantage of the flexible SPARX infrastructure to arrive at an implementation that offers ease of use, flexibility in designing helical structure determination strategy, and high computational efficiency. We introduced the 3D projection matching code which now is able to work with non-cubic volumes, the geometry better suited for long helical filaments, we enhanced procedures for establishing helical symmetry parameters, and we parallelized the code using distributed memory paradigm. Additional features include a graphical user interface that facilitates entering and editing of parameters controlling the structure determination strategy of the program. In addition, we present a novel approach to detect and evaluate structural heterogeneity due to conformer mixtures that takes advantage of helical structure redundancy.

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Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting.

Publication Date: 2012 Jan 8 PMID: 22245778
Authors: Meier, C. - Brookings, D. C. - Ceska, T. A. - Doyle, C. - Gong, H. - McMillan, D. - Saville, G. P. - Mushtaq, A. - Knight, D. - Reich, S. - Pearl, L. H. - Powell, K. A. - Savva, R. - Allen, R. A.
Journal: J Struct Biol

Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments. Here we describe the application of one such fragmentation method, combinatorial domain hunting (CDH), to a target which historically was difficult to express, human MEK-1. We show how CDH was used to identify a fragment which covers the kinase domain of MEK-1 and which expresses and crystallizes significantly better than designed expression constructs, and we report the crystal structure of this fragment which explains some of its superior properties. Gene fragmentation methods, such as CDH, thus hold great promise for tackling difficult-to-express target proteins.

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Chemical mapping of mammalian cells by atom probe tomography.

Publication Date: 2012 Jan 8 PMID: 22245777
Authors: Narayan, K. - Prosa, T. - Fu, J. - Kelly, T. F. - Subramaniam, S.
Journal: J Struct Biol

In atom probe tomography (APT), a technique that has been used to determine 3D maps of ion compositions of metals and semiconductors at sub-nanometer resolutions, controlled emissions of ions can be induced from needle-shaped specimens in the vicinity of a strong electric field. Detection of these ions in the plane of a position sensitive detector provides two-dimensional compositional information while the sequence of ion arrival at the detector provides information in the third dimension. However, the applicability of APT to imaging unstained cells has not been explored. Here, we report the use of APT to obtain 3D spatial distributions of cellular ions and metabolites from unstained, freeze-dried mammalian cells. Multiple peaks were reliably obtained in the mass spectrum from tips with diameters of approximately 50nm and heights of approximately 200nm, with mass-to-charge ratios (m/z) ranging from 1 to 80. Peaks at m/z 12, 23, 28 and 39, corresponding to carbon, sodium, carbonyl and potassium ions respectively, showed distinct patterns of spatial distribution within the cell. Our studies establish that APT could become a powerful tool for mapping the sub-cellular distribution of atomic species, such as labeled metabolites, at 3D spatial resolutions as high as approximately 1nm.

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Crystal structure of JlpA, a surface-exposed lipoprotein adhesin of Campylobacter jejuni.

Publication Date: 2012 Jan 8 PMID: 22245776
Authors: Kawai, F. - Paek, S. - Choi, K. J. - Prouty, M. - Kanipes, M. I. - Guerry, P. - Yeo, H. J.
Journal: J Struct Biol

The Campylobacter jejuni JlpA protein is a surface-exposed lipoprotein that was discovered as an adhesin promoting interaction with host epithelium cells, an early critical step in the pathogenesis of C. jejuni disease. Increasing evidence ascertained that JlpA is antigenic, indicating a role of JlpA in immune response during the infectious process. Here, we report the crystal structure of JlpA at 2.7A resolution, revealing a catcher's mitt shaped unclosed half beta-barrel. Although the apparent architecture of JlpA is somewhat reminiscent of other bacterial lipoproteins such as LolB, the topology of JlpA is unique among the bacterial surface proteins reported to date and therefore JlpA represents a novel bacterial cell surface lipoprotein. The concave face of the structure results in an unusually large hydrophobic basin with a localized acidic pocket, suggesting a possibility that JlpA may accommodate multiple ligands. Therefore, the structure provides framework for determining the molecular function of JlpA and new strategies for the rational design of small molecule inhibitors efficiently targeting JlpA.

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Nucleotide-dependent conformational changes in the N-Ethylmaleimide Sensitive Factor (NSF) and their potential role in SNARE complex disassembly.

Publication Date: 2012 Jan 5 PMID: 22245547
Authors: Moeller, A. - Zhao, C. - Fried, M. G. - Wilson-Kubalek, E. M. - Carragher, B. - Whiteheart, S. W.
Journal: J Struct Biol

Homohexameric, N-Ethylmaleimide Sensitive Factor (NSF) disassembles Soluble NSF Attachment Protein Receptor (SNARE) complexes after membrane fusion, an essential step in vesicular trafficking. NSF contains three domains (NSF-N, NSF-D1, and NSF-D2), each contributing to activity. We combined electron microscopic (EM) analysis, analytical ultracentrifugation (AU) and functional mutagenesis to visualize NSF's ATPase cycle. 3D density maps show that NSF-D2 remains stable, whereas NSF-N undergoes large conformational changes. NSF-Ns splay out perpendicular to the ADP-bound hexamer and twist upwards upon ATP binding, producing a more compact structure. These conformations were confirmed by hydrodynamic, AU measurements: NSF-ATP sediments faster with a lower frictional ratio (f/f(0)). Hydrodynamic analyses of NSF mutants, with specific functional defects, define the structures underlying these conformational changes. Mapping mutations onto our 3D models allows interpretation of the domain movement and suggests a mechanism for NSF binding to and disassembly of SNARE complexes.

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Dynamo: A flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments.

Publication Date: 2012 Jan 8 PMID: 22245546
Authors: Castano-Diez, D. - Kudryashev, M. - Arheit, M. - Stahlberg, H.
Journal: J Struct Biol

Dynamo is a new software package for subtomogram averaging of cryo Electron Tomography (cryo-ET) data with three main goals: first, Dynamo allows user-transparent adaptation to a variety of high-performance computing platforms such as GPUs or CPU clusters. Second, Dynamo implements user-friendliness through GUI interfaces and scripting resources. Third, Dynamo offers user-flexibility through a plugin API. Besides the alignment and averaging procedures, Dynamo includes native tools for visualization and analysis of results and data, as well as support for third party visualization software, such as Chimera UCSF or EMAN2. As a demonstration of these functionalities, we studied bacterial flagellar motors and showed automatically detected classes with absent and present C-rings. Subtomogram averaging is a common task in current cryo-ET pipelines, which requires extensive computational resources and follows a well-established workflow. However, due to the data diversity, many existing packages offer slight variations of the same algorithm to improve results. One of the main purposes behind Dynamo is to provide explicit tools to allow the user the insertion of custom designed procedures - or plugins - to replace or complement the native algorithms in the different steps of the processing pipeline for subtomogram averaging without the burden of handling parallelization. Custom scripts that implement new approaches devised by the user are integrated into the Dynamo data management system, so that they can be controlled by the GUI or the scripting capacities. Dynamo executables do not require licenses for third party commercial software. Sources, executables and documentation are freely distributed on http://www.dynamo-em.org.

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