Biomedical subjects

Friedrich Förster

Publications and source records attributed to Friedrich Förster.

9 recordsLinked to original sources

PubMed2006

Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition.

Cryoelectron tomography (CET) combines the potential of three-dimensional (3D) imaging with a close-to-life preservation of biological samples. It allows the examination of large and stochastically variable structures, such as organelles or whole cells. At the current resolution it becomes possible to visualize large macromolecular complexes in their functional cellular environments. Pattern recognition methods can be used for a systematic interpretation of the tomograms; target molecules are identified and located based on their structural signature and their correspondence with a template. Here, we demonstrate that such an approach can be used to map 70S ribosomes in an intact prokaryotic cell (Spiroplasma melliferum) with high fidelity, in spite of the low signal-to-noise ratio (SNR) of the tomograms. At a resolution of 4.7 nm the average generated from the 236 ribosomes found in a tomogram is in good agreement with high resolution structures of isolated ribosomes as obtained by X-ray crystallography or cryoelectron microscopy. Under the conditions of the experiment (logarithmic growth phase) the ribosomes are evenly distributed throughout the cytosol, occupying approximately 5% of the cellular volume. A subset of about 15% is found in close proximity to and with a distinct orientation with respect to the plasma membrane. This study represents a first step towards generating a more comprehensive cellular atlas of macromolecular complexes.

Algorithms↗

PubMed2006

The mechanism of HIV-1 core assembly: insights from three-dimensional reconstructions of authentic virions.

Infectious HIV particles contain a characteristic cone-shaped core encasing the viral RNA and replication proteins. The core exhibits significant heterogeneity in size and shape, yet consistently forms a well-defined structure. The mechanism by which the core is assembled in the maturing virion remains poorly understood. Using cryo-electron tomography, we have produced three-dimensional reconstructions of authentic, unstained HIV-1. These reveal the viral morphology with unprecedented clarity and suggest the following mechanism for core formation inside the extracellular virion: core growth initiates at the narrow end of the cone and proceeds toward the distal side of the virion until limited by the viral membrane. Curvature and closure of the broad end of the core are then directed by the inner surface of the viral membrane. This mechanism accommodates significant flexibility in lattice growth while ensuring the closure of cores of variable size and shape.

Cryoelectron Microscopy↗

PubMed2005

Retrovirus envelope protein complex structure in situ studied by cryo-electron tomography.

We used cryo-electron tomography in conjunction with single-particle averaging techniques to study the structures of frozen-hydrated envelope glycoprotein (Env) complexes on intact Moloney murine leukemia retrovirus particles. Cryo-electron tomography allows 3D imaging of viruses in toto at a resolution sufficient to locate individual macromolecules, and local averaging of abundant complexes substantially improves the resolution. The averaging of repetitive features in electron tomograms is hampered by a low signal-to-noise ratio and anisotropic resolution, which results from the "missing-wedge" effect. We developed an iterative 3D averaging algorithm that compensates for this effect and used it to determine the trimeric structure of Env to a resolution of 2.7 nm, at which individual domains can be resolved. Strikingly, the 3D reconstruction is shaped like a tripod in which the trimer penetrates the membrane at three distinct locations approximately 4.5 nm apart from one another. The Env reconstruction allows tentative docking of the x-ray crystal structure of the receptor-binding domain. This study thus provides 3D structural information regarding the prefusion conformation of an intact unstained retrovirus surface protein.

Algorithms↗

PubMed2005

TOM software toolbox: acquisition and analysis for electron tomography.

Automated data acquisition procedures have changed the perspectives of electron tomography (ET) in a profound manner. Elaborate data acquisition schemes with autotuning functions minimize exposure of the specimen to the electron beam and sophisticated image analysis routines retrieve a maximum of information from noisy data sets. "TOM software toolbox" integrates established algorithms and new concepts tailored to the special needs of low dose ET. It provides a user-friendly unified platform for all processing steps: acquisition, alignment, reconstruction, and analysis. Designed as a collection of computational procedures it is a complete software solution within a highly flexible framework. TOM represents a new way of working with the electron microscope and can serve as the basis for future high-throughput applications.

Algorithms↗

PubMed2005

Morphological characterization of molecular complexes present in the synaptic cleft.

We obtained tomograms of isolated mammalian excitatory synapses by cryo-electron tomography. This method allows the investigation of biological material in the frozen-hydrated state, without staining, and can therefore provide reliable structural information at the molecular level. We developed an automated procedure for the segmentation of molecular complexes present in the synaptic cleft based on thresholding and connectivity, and calculated several morphological characteristics of these complexes. Extensive lateral connections along the synaptic cleft are shown to form a highly connected structure with a complex topology. Our results are essentially parameter-free, i.e., they do not depend on the choice of certain parameter values (such as threshold). In addition, the results are not sensitive to noise; the same conclusions can be drawn from the analysis of both nondenoised and denoised tomograms.

Animals↗

PubMed2005

Structural studies by electron tomography: from cells to molecules.

Electron tomography (ET) is uniquely suited to obtain three-dimensional reconstructions of pleomorphic structures, such as cells, organelles or supramolecular assemblies. Although the principles of ET have been known for decades, its use has gathered momentum only in recent years, thanks to technological advances and its combination with improved specimen preparation techniques. The rapid freezing/freeze-substitution preparation is applicable to whole cells and tissues, and it is the method of choice for ET investigations of cellular ultrastructure. The frozen-hydrated preparation provides the best possible structural preservation and allows the imaging of molecules, complexes, and supramolecular assemblies in their native state and their natural environment. Devoid of staining and chemical fixation artifacts, cryo-ET provides a faithful representation of both the surface and internal structure of molecules. In combination with advanced computational methods, such as molecular identification based on pattern recognition techniques, cryo-ET is currently the most promising approach to comprehensively map macromolecular architecture inside cellular tomograms.

Algorithms↗

PubMed2004

Nuclear pore complex structure and dynamics revealed by cryoelectron tomography.

Nuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. To analyze their structure in a close-to-life state, we studied transport-active, intact nuclei from Dictyostelium discoideum by means of cryoelectron tomography. Subvolumes of the tomograms containing individual NPCs were extracted in silico and subjected to three-dimensional classification and averaging, whereby distinct structural states were observed. The central plug/transporter (CP/T) was variable in volume and could occupy different positions along the nucleocytoplasmic axis, which supports the notion that it essentially represents cargo in transit. Changes in the position of the CP/T were accompanied by structural rearrangements in the NPC scaffold.

Active Transport, Cell Nucleus↗

PubMed2004

Computational exploration of structural information from cryo-electron tomograms.

Cryo-electron tomography aims to act as an interface between in vivo cell imaging and techniques achieving atomic resolution. This attempt to bridge the resolution gap is facilitated by recent software and hardware advances. Information provided by atomically resolved macromolecules and molecular interaction data need to be put into a common framework in order to create a hybrid multidimensional cellular image. A major partner in this enterprise is the development of regularization and pattern recognition techniques, which try to identify macromolecular complexes as a function of their structural signature in cryo-electron tomograms of living cells.

Animals↗

PubMed2002

Identification of macromolecular complexes in cryoelectron tomograms of phantom cells.

Electron tomograms of intact frozen-hydrated cells are essentially three-dimensional images of the entire proteome of the cell, and they depict the whole network of macromolecular interactions. However, this information is not easily accessible because of the poor signal-to-noise ratio of the tomograms and the crowded nature of the cytoplasm. Here, we describe a template matching algorithm that is capable of detecting and identifying macromolecules in tomographic volumes in a fully automated manner. The algorithm is based on nonlinear cross correlation and incorporates elements of multivariate statistical analysis. Phantom cells, i.e., lipid vesicles filled with macromolecules, provide a realistic experimental scenario for an assessment of the fidelity of this approach. At the current resolution of approximately 4 nm, macromolecules in the size range of 0.5-1 MDa can be identified with good fidelity.

Algorithms↗