Biomedical subjects

Reiner Hegerl

Publications and source records attributed to Reiner Hegerl.

9 recordsLinked to original sources

PubMed2006

Structural analysis of the 26S proteasome by cryoelectron tomography.

The 26S proteasome is the key enzyme of intracellular protein degradation in eukaryotic cells. It is a multisubunit complex of 2.5 MDa confining the proteolytic action to an inner compartment with tightly controlled access. Structural studies of this intriguing molecular machine have been hampered by its intrinsic instability and its dynamics. Here we have used an unconventional approach to obtain a three-dimensional structure of the holocomplex uncompromised by preparation-induced alterations and unbiased by any starting model. We have performed a tomographic reconstruction, followed by averaging over approx. 150 individual reconstructions, of Drosophila 26S proteasomes suspended in a thin layer of amorphous ice.

Cryoelectron Microscopy↗

PubMed2005

Molecular architecture and assembly mechanism of Drosophila tripeptidyl peptidase II.

In eukaryotes, tripeptidyl peptidase II (TPPII) is a crucial component of the proteolytic cascade acting downstream of the 26S proteasome in the ubiquitin-proteasome pathway. It is an amino peptidase belonging to the subtilase family removing tripeptides from the free N terminus of oligopeptides. The 150-kDa subunits of Drosophila TPPII assemble into a giant proteolytic complex of 6 MDa with a remarkable architecture consisting of two segmented and twisted strands that form a spindle-shaped structure. A refined 3D model has been obtained by cryoelectron microscopy, which reveals details of the molecular architecture and, in conjunction with biochemical data, provides insight into the assembly mechanism. The building blocks of this complex are apparently dimers, within which the 150-kDa monomers are oriented head to head. Stacking of these dimers leads to the formation of twisted single strands, two of which comprise the fully assembled spindle. This spindle also forms when TPPII is heterologously expressed in Escherichia coli, demonstrating that no scaffolding protein is required for complex formation and length determination. Reciprocal interactions of the N-terminal part of subunits from neighboring strands are probably involved in the formation of the native quaternary structure, lending the TPPII spindle a stability higher than that of single strands.

Aminopeptidases↗

PubMed2005

Novel type of enzyme multimerization enhances substrate affinity of oat beta-glucosidase.

Oat beta-glucosidase in plastid hydrolyzes avenacosides to C26-desgluco-avenacosides to combat against fungal infections. The enzyme has a unique quaternary protein structure of a three-dimensionally radiated assembly of long fibrillae. We elucidated the fibrillar assembly of oat type 1 beta-glucosidase by means of cryo-electron microscopy, enzyme kinetics and chemical modification. It was assembled by linear stacking of hollow trimeric units and the resulting fibril had a long central tunnel connecting to the outer medium via regularly distributed side fenestrations. The enzyme active sites were located within the central tunnel. This unique multimer assembly increased enzyme affinity to avenacosides, in vivo substrates, and may function to discriminate avenacosides from many other kinds of beta-glucoside in oat. The fibrillar multimer of oat beta-glucosidase is a novel quaternary protein structure for enzyme supramolecular assembly that may have a functional role in the regulation of enzyme affinity.

Avena↗

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↗

PubMed2003

Pyrodictium cannulae enter the periplasmic space but do not enter the cytoplasm, as revealed by cryo-electron tomography.

The hyperthermophilic archaeon Pyrodictium grows in the form of a macroscopically visible network. It consists of cells entrapped in an extracellular matrix of hollow tubules, the "cannulae." Here, we present the three-dimensional structure of a single cell in conjunction with two extracellular cannulae, as determined by cryo-electron microscopy. To achieve this, the information from two independent tilt series of the same specimen was combined, with the specimen rotated in the second series. In the three-dimensional tomographic reconstruction, we were able to trace the two cannulae in their full length, in particular, also inside the cell. One cannula enters the periplasmic space, while the other cannula contacts the surface of the cell, the S-layer. This indicates that the cannulae interconnect individual cells with each other on the level of their periplasmic space; we do not, however, have evidence that they enter the cytoplasm of the cells. The implications of these data for possible functions of the cannulae are discussed.

Cryoelectron Microscopy↗

PubMed2003

An approach to examining model dependence in EM reconstructions using cross-validation.

Reference bias refers to a common problem in fitting experimental data to an initial model. Given enough free parameters, a good fit of any experimental data to the model can be obtained, even if the experimental data contain only noise. Reference-based alignment methods used in electron microscopy (EM) are subject to this type of bias, in that images containing pure noise can regenerate the reference. Cross-validation is based on the idea that the experimental data used to assess the validity of the fitting should not be the same data as were used to do the fitting. Here we present the application of cross-validation to one form of reference-based alignment: 3D-projection matching in single-particle reconstructions. Our results show that reference bias is indeed present in reconstructions, but that the effect is small for real data compared to that for random noise, and that this difference in behavior is magnified, rather than diminished, during iterative refinement.

Escherichia coli↗

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↗

PubMedDate not supplied

Cryoelectron microscopy and cryoelectron tomography of the nuclear pre-mRNA processing machine.

Large nuclear ribonucleoprotein particles, which can be viewed as the naturally assembled precursor messenger RNA (pre-mRNA) processing machine, were analyzed in frozen-hydrated preparations by cryoelectron microscopy. A general and reproducible strategy for preparing ice-embedded large nuclear ribonucleoprotein (lnRNP) particles at sufficiently high concentration was developed. Taking advantage of their negatively charged components, the lnRNP particles are adsorbed and thus concentrated on a positively charged lipid monolayer while preserving their native structure. Using this approach we carried out cryoelectron tomography and three-dimensional image reconstruction of individual lnRNP particles. The study revealed a structure similar to that of negatively stained particles studied previously, yet with additional features. The small additional domain visualized in negative stain appeared to be larger in the ice preparations. In addition, using image restoration from focus series of ice-embedded lnRNP particles, new features such as holes within the subunits were visualized in two dimensions, and it was shown that the subunits are interconnected via a fiber, very likely formed by the pre-mRNA. This finding supports the model that each subunit represents a spliceosome that splices out the intron wound around it.

Cell Nucleus Structures↗

PubMedDate not supplied

Segmentation of two- and three-dimensional data from electron microscopy using eigenvector analysis.

An automatic image segmentation method is used to improve processing and visualization of data obtained by electron microscopy. Exploiting affinity criteria between pixels, e.g., proximity and gray level similarity, in conjunction with an eigenvector analysis, the image is subdivided into areas which correspond to objects or meaningful regions. Extending a proposal by Shi and Malik (1997, Proceedings of the IEEE conference on Computer Vision and Pattern Recognition, pp. 731-737) the approach was adapted to the field of electron microscopy, especially to three-dimensional application as needed by electron tomography. Theory, implementation, parameter setting, and results obtained with a variety of data are presented and discussed. The method turns out to be a powerful tool for visualization with the potential for further improvement by developing and tuning new affinity.

Algorithms↗