Reiner Hegerl

The multicatalytic proteinase (prosome) is ubiquitous from eukaryotes to archaebacteria

From the thermoacidophilic archaebacterium, Thermoplasma acidophilum, a proteolytically active particle has been isolated which is almost identical in size and shape with the multicatalytic proteinase (prosome) from rat. This result indicates that prosomes have been developed early in evolution and that they possibly serve functions common to all living cells.

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 ≈4 nm, macromolecules in the size range of 0.5–1 MDa can be identified with good fidelity.

Towards automatic electron tomography

Electron microscope control, that allows the automatic recording of tilt series for the 3D reconstruction of individual objects, has been realized. The experimental set-up includes a 200 kV TEM equipped with a 1K x 1K CCD camera, both controlled externally by a fast dedicated image-processing computer. For the goniometer control an accurate electronic readout of the tilt angle and a board driving the goniometer motor have been installed. For low-dose imaging, three to five different specimen areas are used: one (or two) for the determination of object displacements during tilting, one (or two) for autofocusing, and another one for recording the tilt series to be used for the 3D reconstruction. Tilt series can be recorded with a rather low total dose, the lower limit being set by the requirement that subsequent projection images have to be aligned by means of cross-correlation functions. The method has been tested with graphitized carbon particles on carbon film and with negatively stained proteasomes from the archaebacterium Thermoplasma acidophilum. Some future developments towards fully automatic electron tomography are discussed.

Electron microscopy and image analysis of the multicatalytic proteinase

On electron micrographs, negatively stained multicatalytic proteinase molecules are viewed end‐on (ring shaped) or side‐on (rectangular shaped). For aurothioglucose, ammonium molybdate‐ and phosphotungstate‐stained molecules, the dimensions measured are consistent. In contrast, uranyl acetate‐staining reveals ring‐shaped particles which vary in diameter between 12 and 16 nm. This is due to a partial collapse and substantial flattening of the structure. Digital image analysis of side‐on views of the particles reveals a tripartite, reel‐shaped structure. Within the ring‐like, end‐on projections of ammonium molybdate‐stained molecules six local centres of mass can be discerned; their position appears to depart, however, from a true six‐fold symmetry.

Three-dimensional structure of the regular surface layer (HPI layer) of Deinococcus radiodurans

The low-resolution structure of the regular surface layer of Deinococcus radiodurans has been determined from negatively stained specimens by three-dimensional electron microscopy. The layer has P6 symmetry, a lattice constant of 18 nm and a thickness of 6.5 nm. Three-dimensional reconstruction was performed by a hybrid real space/Fourier space approach that incorporates partial compensation of lattice distortions: The model obtained is discussed in the light of independent information about the surface structure of this layer, derived from metal shadowing and surface relief reconstruction. While agreement is quite satisfactory for the apparently more rigid inner surface, the outer surface shows severe flattening effects. The structure of the HPI layer is compared with other bacterial surface layers using a classification scheme that is outlined in the Appendix.

Tricorn protease - the core of a modular proteolytic system

Large macromolecular assemblies have evolved as a means of compartmentalizing reactions in organisms lacking membrane-bounded compartments. A tricorn-shaped protease was isolated from the archaeon Thermoplasma and was shown to form a multisubunit proteolytic complex. The 120-kilodalton monomer assembled to form a hexameric toroid that could assemble further into a capsid structure. Tricorn protease appeared to act as the core of a proteolytic system; when it interacted with several smaller proteins, it displayed multicatalytic activities.

Towards automatic electron tomography II. Implementation of autofocus and low-dose procedures

Abstract Automatic electron tomography involves the compensation for lateral specimen displacements and, at higher resolution, refocusing. Improved automatic procedures are described and the accuracy and reliability of the basic steps is assessed. It is demonstrated that the cumulative dose in collecting tomographic 3D data sets can be kept small enough for the investigation of ice-embedded specimens.

Marker-free image registration of electron tomography tilt-series

BackgroundTilt series are commonly used in electron tomography as a means of collecting three-dimensional information from two-dimensional projections. A common problem encountered is the projection alignment prior to 3D reconstruction. Current alignment techniques usually employ gold particles or image derived markers to correctly align the images. When these markers are not present, correlation between adjacent views is used to align them. However, sequential pairwise correlation is prone to bias and the resulting alignment is not always optimal.ResultsIn this paper we introduce an algorithm to find regions of the tilt series which can be tracked within a subseries of the tilt series. These regions act as landmarks allowing the determination of the alignment parameters. We show our results with synthetic data as well as experimental cryo electron tomography.ConclusionOur algorithm is able to correctly align a single-tilt tomographic series without the help of fiducial markers thanks to the detection of thousands of small image patches that can be tracked over a short number of images in the series.

The three-dimensional structure of proteasomes from Thermoplasma acidophilum as determined by electron microscopy using random conical tilting

The three‐dimensional structure of proteasomes from the archaebacterium Thermoplasma acidophilum has been determined to a resolution of approximately 2 nm from electron micrographs of negatively stained preparations using the method of random conical tilting. The particles turn out to be essentially cylinder‐shaped barrels, 15 nm long and 11 nm wide, enclosing a tripartite inner compartiment. An account is given of some of the present limitations which prevent to attain a higher resolution and possible ways to overcome these limitations are indicated.

Automated Segmentation of Electron Tomograms for a Quantitative Description of Actin Filament Networks

Cryo-electron tomography allows to visualize individual actin filaments and to describe the three-dimensional organization of actin networks in the context of unperturbed cellular environments. For a quantitative characterization of actin filament networks, the tomograms must be segmented in a reproducible manner. Here, we describe an automated procedure for the segmentation of actin filaments, which combines template matching with a new tracing algorithm. The result is a set of lines, each one representing the central line of a filament. As demonstrated with cryo-tomograms of cellular actin networks, these line sets can be used to characterize filament networks in terms of filament length, orientation, density, stiffness (persistence length), or the occurrence of branching points.