Marker-free image registration of electron tomography tilt-series.

BACKGROUND: Tilt 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. RESULTS: In 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. CONCLUSION: Our 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.

Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria.

Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex). We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex. Tom40 is organized in a high molecular mass complex of approximately 350 kD. It forms a high conductance channel. Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner. The secondary structure of Tom40 comprises approximately 31% beta-sheet, 22% alpha-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy. Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of approximately 2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly.

The TOM core complex: the general protein import pore of the outer membrane of mitochondria.

Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of approximately 2.1 nm and a height of approximately 7 nm. Tom40 is the key structural element of the TOM core complex.

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.

Functional significance of symmetrical versus asymmetrical GroEL-GroES chaperonin complexes.

The Escherichia coli chaperonin GroEL and its regulator GroES are thought to mediate adenosine triphosphate-dependent protein folding as an asymmetrical complex, with substrate protein bound within the GroEL cylinder. In contrast, a symmetrical complex formed between one GroEL and two GroES oligomers, with substrate protein binding to the outer surface of GroEL, was recently proposed to be the functional chaperonin unit. Electron microscopic and biochemical analyses have now shown that unphysiologically high magnesium concentrations and increased pH are required to assemble symmetrical complexes, the formation of which precludes the association of unfolded polypeptide. Thus, the functional significance of GroEL:(GroES)2 particles remains to be demonstrated.

Aberration-corrected microscopy for structural biology applications.

This study explores the potential of a C(s)-corrected transmission electron microscope for structural studies of biological samples, in particular isolated macromolecular complexes. A 300-kV transmission electron microscope, equipped with a C(s) corrector was employed to record sets of images at different defocus and C(s) settings. The experiments were designed to determine whether imaging with large defocus benefits from C(s) correction. Defocus contrast in biological imaging has a stronger influence on image resolution than any other parameter. We find the results are in good agreement with theoretical framework, verifying that the typical imaging conditions required for biological investigations are not affected by C(s) correction.

Three-dimensional reconstruction and averaging of 30 S ribosomal subunits of Escherichia coli from electron micrographs.

From the micrographs of a tilt series, several particles of negatively stained 30 S ribosomal subunits of Escherichia coli were three-dimensionally reconstructed. Three of them showing similar orientation with respect to the supporting foil were averaged after alignment by newly developed three-dimensional correlation methods. As a main result we found a stained channel-like structure inside the particle. We tentatively propose that this corresponds, at least partially, to positively stained segments of the 16 S RNA.

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.

Electron microscopy and subunit-subunit interaction studies reveal a first architecture of COP9 signalosome.

The COP9 signalosome is involved in signal transduction, whereas the 26 S proteasome lid is a regulatory subcomplex of the 26 S proteasome responsible for degradation of ubiquitinated proteins. COP9 signalosome and lid possess significant sequence homologies among their eight core subunits and are likely derived from a common ancestor. Surprisingly, from our two-dimensional electron microscopy data, a common architectural plan for the two complexes could not be deduced. None-the-less, the two particles have structural features in common. Both COP9 signalosome and lid lack any symmetry in subunit arrangement and exhibit a central groove, possibly qualified for scaffolding functions.Filter-binding assays with recombinant COP9 signalosome components revealed a multitude of subunit-subunit interactions, supporting the asymmetrical appearance of the complex in electron microscopy. On the basis of two-dimensional images and subunit interaction studies, a first architectural model of COP9 signalosome was created. The fact that four distinct classes of particle views were identified and that only 50 % of the selected particles could be classified indicates a high degree of heterogeneity in electron microscopic images. Different orientations with respect to the viewing axis and conformational variety, presumably due to different grades of phosphorylation, are possible reasons for the heterogeneous appearance of the complex. Our biochemical data show that recombinant COP9 signalosome subunits 2 and 7 are phosphorylated by the associated kinase activity. The modification of COP9 signalosome subunit 2 might be essential for c-Jun phosphorylation. Dephosphorylation does not inactivate the associated kinase activity. Although substrate phosphorylation by COP9 signalosome is significantly decreased by lambda protein phosphatase treatment, "autophosphorylation" is increased.

The EM Program Package: A Platform for Image Processing in Biological Electron Microscopy

EM is a software package developed for image processing in the field of biological electron microscopy. After a brief summary of its history, an outline of the package is given, by describing some characteristic features rather than by a complete report of all facilities. New developments, connected with the introduction of a UNIX version and the advent of automated electron microscopy, are described in more detail.

Electron Tomography of Single Ice-Embedded Macromolecules: Three-Dimensional Alignment and Classification

From 3-D reconstructions of automatically recorded tilt series of ice-embedded macromolecules, several hundred 3-D images of single particles can be extracted. Here we describe correlation-based techniques to align the particles with respect to translation and orientation in 3-D and the calculation of an averaged reconstruction after application of the correct weighting function to the particle projections. Multivariate statistical analysis and classification are applied to the set of three-dimensionally reconstructed particles to investigate interimage variations on the 3-D level. Copyright 1997 Academic Press. Copyright 1997 Academic Press

ATPase cycle controls the conformation of an archaeal chaperonin as visualized by cryo-electron microscopy.

Chaperonins are double-ring protein folding machines fueled by ATP binding and hydrolysis. Conformational rearrangements upon ATPase cycling of the group I chaperonins, typified by the Escherichia coli GroEL/GroES system, have been thoroughly investigated by cryo-electron microscopy and X-ray crystallography. For archaeal group II chaperonins, however, these methods have so far failed to provide a correlation between the structural and the functional states. Here, we show that the conformation of the native alphabeta-thermosome of Thermoplasma acidophilum in vitrified ice is strictly regulated by adenine nucleotides.

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.

Electron microscopy and image analysis of the multicatalytic proteinase.

One 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.

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.

Structure of VAT, a CDC48/p97 ATPase homologue from the archaeon Thermoplasma acidophilum as studied by electron tomography.

Valosine-containing protein-like ATPase from Thermoplasma acidophilum is a member of the superfamily of ATPases associated with a diversity of cellular activities and is closely related to CDC48 from yeast and p97 from higher eukaryotes and more distantly to N-ethylmaleimide-sensitive fusion protein. We have used electron tomography to obtain low-resolution (2-2.5 nm) three-dimensional maps of both the whole 500 kDa complex and the N-terminally truncated valosine-containing protein-like ATPase from T. acidophilum complex lacking the putative substrate binding domain.

The molecular chaperone TF55. Assessment of symmetry.

TF55-like factor from Sulfolobus solfataricus was purified to homogeneity and analyzed by electron microscopy and image analysis to determine the symmetries of these particles. Three different procedures were used to analyze the electron micrographs: (1) fuzzy-set based classification of the particles according to their rotational power spectra; (2) multivariate statistical analysis based on singular value decomposition; (3) circular harmonic analysis. Averages obtained from the three methods show unequivocally that the TF55-like complex presents a 9-fold symmetry.

Wavelet transform filtering and nonlinear anisotropic diffusion assessed for signal reconstruction performance on multidimensional biomedical data.

Computer tomography (CT) techniques are the most widely applicable noninvasive methods for obtaining two- and three-dimensional insights into biological objects. They comprise CT for medical applications, as well as electron tomography used for investigating macromolecular and cellular specimens. Recent advances in the recording schemes improve the speed and resolution frontiers and provide new insights into structural organizations of different objects. However, many data sets suffer from a poor signal-to-noise ratio, which severely hinders the application of methods for automated data analysis, such as feature extraction, segmentation, and visualization. We propose the multidimensional implementation of two powerful signal reconstruction techniques, namely invariant wavelet filtering and nonlinear anisotropic diffusion. We establish quantitative measures to assess the signal reconstruction performance on synthetic data and biomedical images. The appropriate multidimensional implementations of wavelet and diffusion techniques allow for a superior performance over conventional noise-reduction methods. We derive the conditions for the choice between wavelet and diffusion techniques with respect to an optimal signal reconstruction performance. Results of applying the proposed methods in two very different imaging domains-molecular biology and clinical research-are provided.

The "EM" program system.

"EM" is a computer program system concerned with the processing of electron micrographs. It provides facilities for two- and three-dimensional image reconstruction, correlation, filtering, etc. as well as for storage and display of image data. A description of the scientific aims, the system design, and the possibilities to display pictures is given. The handling of the command language is illustrated by some examples.