Biomedical subjects

Reiner Hegerl

Publications and source records attributed to Reiner Hegerl.

5 recordsLinked to original sources

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↗