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ABSTRACT: Cryo-electron tomography of vitreous sections is currently the only method for visualizing the eukaryotic ultrastructure at close to native state with molecular resolution. Here, we describe the detailed procedure of how to prepare suitable vitreous sections from mammalian skin for cryo-electron tomography, how to align the projection images of the tilt-series, and finally how to perform sub-tomogram averaging on macromolecular complexes with periodic arrangement such as desmosomes.
Methods in molecular biology (Clifton, N.J.) 01/2013; 961:97-117.
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Helen R Saibil,
Anja Seybert,
Anja Habermann,
Juliane Winkler,
Mikhail Eltsov,
Mario Perkovic,
Daniel Castaño-Diez,
Margot P Scheffer,
Uta Haselmann,
Petr Chlanda,
Susan Lindquist,
Jens Tyedmers, Achilleas S Frangakis
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ABSTRACT: Yeast prions constitute a "protein-only" mechanism of inheritance that is widely deployed by wild yeast to create diverse phenotypes. One of the best-characterized prions, [PSI(+)], is governed by a conformational change in the prion domain of Sup35, a translation-termination factor. When this domain switches from its normal soluble form to an insoluble amyloid, the ensuing change in protein synthesis creates new traits. Two factors make these traits heritable: (i) the amyloid conformation is self-templating; and (ii) the protein-remodeling factor heat-shock protein (Hsp)104 (acting together with Hsp70 chaperones) partitions the template to daughter cells with high fidelity. Prions formed by several other yeast proteins create their own phenotypes but share the same mechanistic basis of inheritance. Except for the amyloid fibril itself, the cellular architecture underlying these protein-based elements of inheritance is unknown. To study the 3D arrangement of prion assemblies in their cellular context, we examined yeast [PSI(+)] prions in the native, hydrated state in situ, taking advantage of recently developed methods for cryosectioning of vitrified cells. Cryo-electron tomography of the vitrified sections revealed the prion assemblies as aligned bundles of regularly spaced fibrils in the cytoplasm with no bounding structures. Although the fibers were widely spaced, other cellular complexes, such as ribosomes, were excluded from the fibril arrays. Subtomogram image averaging, made possible by the organized nature of the assemblies, uncovered the presence of an additional array of densities between the fibers. We suggest these structures constitute a self-organizing mechanism that coordinates fiber deposition and the regulation of prion inheritance.
Proceedings of the National Academy of Sciences 08/2012; 109(37):14906-11. · 9.68 Impact Factor
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ABSTRACT: In this study, electron tomograms of plunge-frozen isolated chromatin in both open and compacted form were recorded. We have resolved individual nucleosomes in these tomograms in order to provide a 3D view of the arrangement of nucleosomes within chromatin fibers at different compaction states. With an optimized template matching procedure we obtained accurate positions and orientations of nucleosomes in open chromatin in "low-salt" conditions (5 mM NaCl). The mean value of the planar angle between three consecutive nucleosomes is 70°, and the mean center-to-center distance between consecutive nucleosomes is 22.3 nm. Since the template matching approach was not effective in crowded conditions, for nucleosome detection in compact fibers (40 mM NaCl and 1 mM MgCl(2)) we developed the nucleosome detection procedure based on the watershed algorithm, followed by sub-tomogram alignment, averaging, and classification by Principal Components Analysis. We find that in compact chromatin the nucleosomes are arranged with a predominant face-to-face stacking organization, which has not been previously shown for native isolated chromatin. Although the path of the DNA cannot be directly seen in compact conditions, it is evident that the nucleosomes stack with their dyad axis aligned in forming a "double track" conformation which is a consequence of DNA joining adjacent nucleosome stacks. Our data suggests that nucleosome stacking is an important mechanism for generating chromatin compaction in vivo.
Journal of Structural Biology 11/2011; 178(2):207-14. · 3.41 Impact Factor
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ABSTRACT: Chromatin folding in eukaryotes fits the genome into the limited volume of the cell nucleus. Formation of higher-order chromatin structures attenuates DNA accessibility, thus contributing to the control of essential genome functions such as transcription, DNA replication, and repair. The 30-nm fiber is thought to be the first hierarchical level of chromatin folding, but the nucleosome arrangement in the compact 30-nm fiber was previously unknown. We used cryoelectron tomography of vitreous sections to determine the structure of the compact, native 30-nm fiber of avian erythrocyte nuclei. The predominant geometry of the 30-nm fiber revealed by subtomogram averaging is a left-handed two-start helix with approximately 6.5 nucleosomes per 11 nm, in which the nucleosomes are juxtaposed face-to-face but are shifted off their superhelical axes with an axial translation of approximately 3.4 nm and an azimuthal rotation of approximately 54°. The nucleosomes produce a checkerboard pattern when observed in the direction perpendicular to the fiber axis but are not interdigitated. The nucleosome packing within the fibers shows larger center-to-center internucleosomal distances than previously anticipated, thus excluding the possibility of core-to-core interactions, explaining how transcription and regulation factors can access nucleosomes.
Proceedings of the National Academy of Sciences 10/2011; 108(41):16992-7. · 9.68 Impact Factor
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ABSTRACT: The cytoplasmic surface of intercellular junctions is a complex network of molecular interactions that link the extracellular region of the desmosomal cadherins with the cytoskeletal intermediate filaments. Although 3D structures of the major plaque components are known, the overall architecture remains unknown. We used cryoelectron tomography of vitreous sections from human epidermis to record 3D images of desmosomes in vivo and in situ at molecular resolution. Our results show that the architecture of the cytoplasmic surface of the desmosome is a 2D interconnected quasiperiodic lattice, with a similar spatial organization to the extracellular side. Subtomogram averaging of the plaque region reveals two distinct layers of the desmosomal plaque: a low-density layer closer to the membrane and a high-density layer further away from the membrane. When combined with a heuristic, allowing simultaneous constrained fitting of the high-resolution structures of the major plaque proteins (desmoplakin, plakophilin, and plakoglobin), it reveals their mutual molecular interactions and explains their stoichiometry. The arrangement suggests that alternate plakoglobin-desmoplakin complexes create a template on which desmosomal cadherins cluster before they stabilize extracellularly by binding at their N-terminal tips. Plakophilins are added as a molecular reinforcement to fill the gap between the formed plaque complexes and the plasma membrane.
Proceedings of the National Academy of Sciences 04/2011; 108(16):6480-5. · 9.68 Impact Factor
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ABSTRACT: Classification of electron sub-tomograms is a challenging task, due the missing-wedge and the low signal-to-noise ratio of the data. Classification algorithms tend to classify data according to their orientation to the missing-wedge, rather than to the underlying signal. Here we use a neural network approach, called the Kernel Density Estimator Self-Organizing Map (KerDenSOM3D), which we have implemented in three-dimensions (3D), also having compensated for the missing-wedge, and we comprehensively compare it to other classification methods. For this purpose, we use various simulated macromolecules, as well as tomographically reconstructed in vitro GroEL and GroEL/GroES molecules. We show that the performance of this classification method is superior to previously used algorithms. Furthermore, we show how this algorithm can be used to provide an initial cross-validation of template-matching approaches. For the example of sub-tomogram classification extracted from cellular tomograms of Mycoplasma pneumonia and Spiroplasma melliferum cells, we show the bias of template-matching, and by using differing search and classification areas, we demonstrate how the bias can be significantly reduced.
Journal of Structural Biology 03/2011; 174(3):494-504. · 3.41 Impact Factor
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ABSTRACT: Complexes of OprM and MexA, two proteins of the MexA-MexB-OprM multidrug efflux pump from Pseudomonas aeruginosa, an opportunistic Gram-negative bacterium, were reconstituted into proteoliposomes by detergent removal. Stacks of protein layers with a constant height of 21nm, separated by lipid bilayers, were obtained at stoichiometry of 1:1 (w/w). Using cryo-electron microscopy and tomography, we showed that these protein layers were composed of MexA-OprM complexes self-assembled into regular arrays. Image processing of extracted sub-tomograms depicted the architecture of the bipartite complex sandwiched between two lipid bilayers, representing an environment close to that of the native whole pump (i.e. anchored between outer and inner membranes of P. aeruginosa). The MexA-OprM complex appeared as a cylindrical structure in which we were able to identify the OprM molecule and the MexA moiety. MexA molecules have a cylindrical shape prolonging the periplasmic helices of OprM, and widening near the lipid bilayer. The flared part is likely composed of two MexA domains adjacent to the lipid bilayer, although their precise organization was not reachable mainly due to their flexibility. Moreover, the intermembrane distance of 21nm indicated that the height of the bipartite complex is larger than that of the tripartite AcrA-AcrB-TolC built-up model in which TolC and AcrB are docked into contact. We proposed a model of MexA-OprM taking into account features of previous models based on AcrA-AcrB-TolC and our structural results providing clues to a possible mechanism of tripartite system assembly.
Biochimica et Biophysica Acta 10/2010; 1798(10):1953-60. · 4.66 Impact Factor
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ABSTRACT: The robust alignment of tilt-series collected for cryo-electron tomography in the absence of fiducial markers, is a problem that, especially for tilt-series of vitreous sections, still represents a significant challenge. Here we present a complete software package that implements a cross-correlation-based procedure that tracks similar image features that are present in several micrographs and explores them implicitly as substitutes for fiducials like gold beads and quantum dots. The added value compared to previous approaches, is that the algorithm explores a huge number of random positions, which are tracked on several micrographs, while being able to identify trace failures, using a cross-validation procedure based on the 3D marker model of the tilt-series. Furthermore, this method allows the reliable identification of areas which behave as a rigid body during the tilt-series and hence addresses specific difficulties for the alignment of vitreous sections, by correcting practical caveats. The resulting alignments can attain sub-pixel precision at the local level and is able to yield a substantial number of usable tilt-series (around 60%). In principle, the algorithm has the potential to run in a fully automated fashion, and could be used to align any tilt-series directly from the microscope. Finally, we have significantly improved the user interface and implemented the source code on the graphics processing unit (GPU) to accelerate the computations.
Journal of Structural Biology 04/2010; 170(1):117-26. · 3.41 Impact Factor
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ABSTRACT: The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re-localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re-targeted to alternative sites such as the inner membrane in the presence of site-specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage-free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.
The EMBO Journal 03/2010; 29(5):910-23. · 9.20 Impact Factor
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Seán I O'Donoghue,
David S Goodsell, Achilleas S Frangakis,
Fabrice Jossinet,
Roman A Laskowski,
Michael Nilges,
Helen R Saibil,
Andrea Schafferhans,
Rebecca C Wade,
Eric Westhof,
Arthur J Olson
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ABSTRACT: Structural biology is rapidly accumulating a wealth of detailed information about protein function, binding sites, RNA, large assemblies and molecular motions. These data are increasingly of interest to a broader community of life scientists, not just structural experts. Visualization is a primary means for accessing and using these data, yet visualization is also a stumbling block that prevents many life scientists from benefiting from three-dimensional structural data. In this review, we focus on key biological questions where visualizing three-dimensional structures can provide insight and describe available methods and tools.
Nature Methods 03/2010; 7(3 Suppl):S42-55. · 19.28 Impact Factor
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ABSTRACT: The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re-localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re-targeted to alternative sites such as the inner membrane in the presence of site-specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage-free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.
The EMBO Journal 01/2010; 29(5):910-923. · 9.20 Impact Factor
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Sebastian Kühner,
Vera van Noort,
Matthew J Betts,
Alejandra Leo-Macias,
Claire Batisse,
Michaela Rode,
Takuji Yamada,
Tobias Maier,
Samuel Bader,
Pedro Beltran-Alvarez, [......],
Alexander Schmidt,
Eva Yus,
Ruedi Aebersold,
Richard Herrmann,
Bettina Böttcher, Achilleas S Frangakis,
Robert B Russell,
Luis Serrano,
Peer Bork,
Anne-Claude Gavin
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ABSTRACT: The genome of Mycoplasma pneumoniae is among the smallest found in self-replicating organisms. To study the basic principles of bacterial proteome organization, we used tandem affinity purification-mass spectrometry (TAP-MS) in a proteome-wide screen. The analysis revealed 62 homomultimeric and 116 heteromultimeric soluble protein complexes, of which the majority are novel. About a third of the heteromultimeric complexes show higher levels of proteome organization, including assembly into larger, multiprotein complex entities, suggesting sequential steps in biological processes, and extensive sharing of components, implying protein multifunctionality. Incorporation of structural models for 484 proteins, single-particle electron microscopy, and cellular electron tomograms provided supporting structural details for this proteome organization. The data set provides a blueprint of the minimal cellular machinery required for life.
Science 11/2009; 326(5957):1235-40. · 31.20 Impact Factor
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ABSTRACT: Although the formation of 30-nm chromatin fibers is thought to be the most basic event of chromatin compaction, it remains controversial because high-resolution imaging of chromatin in living eukaryotic cells had not been possible until now. Cryo-electron microscopy of vitreous sections is a relatively new technique, which enables direct high-resolution observation of the cell structures in a close-to-native state. We used cryo-electron microscopy and image processing to further investigate the presence of 30-nm chromatin fibers in human mitotic chromosomes. HeLa S3 cells were vitrified by high-pressure freezing, thin-sectioned, and then imaged under the cryo-electron microscope without any further chemical treatment or staining. For an unambiguous interpretation of the images, the effects of the contrast transfer function were computationally corrected. The mitotic chromosomes of the HeLa S3 cells appeared as compact structures with a homogeneous grainy texture, in which there were no visible 30-nm fibers. Power spectra of the chromosome images also gave no indication of 30-nm chromatin folding. These results, together with our observations of the effects of chromosome swelling, strongly suggest that, within the bulk of compact metaphase chromosomes, the nucleosomal fiber does not undergo 30-nm folding, but exists in a highly disordered and interdigitated state, which is, on the local scale, comparable with a polymer melt.
Proceedings of the National Academy of Sciences 01/2009; 105(50):19732-7. · 9.68 Impact Factor
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ABSTRACT: The graphics processing unit (GPU), which originally was used exclusively for visualization purposes, has evolved into an extremely powerful co-processor. In the meanwhile, through the development of elaborate interfaces, the GPU can be used to process data and deal with computationally intensive applications. The speed-up factors attained compared to the central processing unit (CPU) are dependent on the particular application, as the GPU architecture gives the best performance for algorithms that exhibit high data parallelism and high arithmetic intensity. Here, we evaluate the performance of the GPU on a number of common algorithms used for three-dimensional image processing. The algorithms were developed on a new software platform called "CUDA", which allows a direct translation from C code to the GPU. The implemented algorithms include spatial transformations, real-space and Fourier operations, as well as pattern recognition procedures, reconstruction algorithms and classification procedures. In our implementation, the direct porting of C code in the GPU achieves typical acceleration values in the order of 10-20 times compared to a state-of-the-art conventional processor, but they vary depending on the type of the algorithm. The gained speed-up comes with no additional costs, since the software runs on the GPU of the graphics card of common workstations.
Journal of Structural Biology 08/2008; 164(1):153-60. · 3.41 Impact Factor
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ABSTRACT: Three-dimensional image analysis includes image processing, segmentation and visualization operations, which facilitate the interpretation of data. We have developed a toolbox for three-dimensional (3D) electron microscopy (EM) in Amira, which is a commercial software package, used by many laboratories. Our toolbox integrates a number of established procedures specifically tailored for 3D EM. These include input-output, filtering, segmentation, visualization and ray-tracing functions, which can be accessed directly from a user-friendly pop-up menu. They allow performing denoising and segmentation tasks directly in Amira, without the need of other programs, and ultimately allow the visualization of the results at photo-realistic quality with ray-tracing. They also allow a direct interaction with the data, such that, e.g., sub-tomograms can be directly extracted, or segmentation areas can be interactively selected. The implemented functions are fast, reliable and intuitive, yielding a comprehensive package for visualization in EM.
Journal of Structural Biology 06/2008; 164(1):161-5. · 3.41 Impact Factor
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ABSTRACT: Desmosomes are cadherin-based intercellular junctions that primarily provide mechanical stability to tissues such as epithelia and cardiac muscle. Desmosomal cadherins, which are Ca(2+)-dependent adhesion molecules, are of central importance in mediating direct intercellular interaction. The close association of these proteins, with intracellular components of desmosomes ultimately linked to the cytoskeleton, is believed to play an important role in tissue morphogenesis during development and wound healing. Elucidation of the binding mechanism of adhesive interfaces between the extracellular domains of cadherins has been approached by structural, biophysical and biochemical methods. X-ray crystal structures of isolated extracellular domains of cadherins have provided compelling evidence of the mutual binding of the highly conserved N-terminal residue, Trp(2), from opposing proteins. This binding interface was also implicated by biochemical and cell-adhesion assays and mutagenesis data to be the primary adhesive interface between cells. Recent results based on electron tomography of epidermal desmosomes were consistent with this view, showing cadherin molecules interacting at their N-terminal tips. An integrative structural approach involving X-ray crystallography, cryo-electron tomography and immuno-electron microscopy should give the complete picture of the architecture of this important junction; identifying its various proteins and showing their arrangements and binding interfaces under native conditions. Together with these 'static' approaches, live-cell imaging of cultured keratinocytes should provide important insights into the dynamic property of the assembly and disassembly of desmosomes.
Biochemical Society Transactions 05/2008; 36(Pt 2):181-7. · 3.71 Impact Factor
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ABSTRACT: Cryo-electron tomography of vitreous sections is currently the most promising technique for visualizing arbitrary regions of eukaryotic cells or tissue at molecular resolution. Despite significant progress in the sample preparation techniques over the past few years, the three dimensional reconstruction using electron tomography is not as simple as in plunge frozen samples for various reasons, but mainly due to the effects of irradiation on the sections and the resulting poor alignment. Here, we present a new algorithm, which can provide a useful three-dimensional marker model after investigation of hundreds to thousands of observations calculated using local cross-correlation throughout the tilt series. The observations are chosen according to their coherence to a particular model and assigned to virtual markers. Through this type of measurement a merit figure can be calculated, precisely estimating the quality of the reconstruction. The merit figures of this alignment method are comparable to those obtained with plunge frozen samples using fiducial gold markers. An additional advantage of the algorithm is the implicit detection of areas in the sections that behave as rigid bodies and can thus be properly reconstructed.
Journal of Structural Biology 04/2008; 161(3):249-59. · 3.41 Impact Factor
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ABSTRACT: Cryo-electron tomography (CET) is currently the only three-dimensional imaging technique capable of visualizing macromolecules in their cellular context at close-to-native conditions with a resolution in the nanometer range. An important component for the analysis of the data is their classification, which should discriminate among various macromolecules, conformational changes and interaction partners. Missing structure factors, typically in a wedge-shaped region in Fourier space if single-axis tilting is performed, hamper classification of cryo-electron tomographic data. Here, we describe a classification method for three-dimensional (3D) sub-tomograms extracted from cryo-electron tomograms, which takes the missing wedge into account and provides reliable results. The similarity of the individually aligned sub-tomograms is scored by constrained correlation. Subsequently, they are clustered based on their pairwise correlation values. In order to demonstrate the feasibility of this approach, we apply the proposed method to simulated tomographic data of the chaperone thermosome in different conformations. By comparison of the principal components of the resulting matrix we show that the proposed metric is significantly less prone to the orientation of the missing wedge compared to the unconstrained correlation. Moreover, we apply our classification method to an experimental dataset of GroEL with and without GroES, where we achieve a distinct discrimination between the putative GroEL and GroEL/GroES complexes.
Journal of Structural Biology 04/2008; 161(3):276-86. · 3.41 Impact Factor
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ABSTRACT: Desmosomes are cadherin-based adhesive intercellular junctions, which are present in tissues such as heart and skin. Despite considerable efforts, the molecular interfaces that mediate adhesion remain obscure. Here we apply cryo-electron tomography of vitreous sections from human epidermis to visualize the three-dimensional molecular architecture of desmosomal cadherins at close-to-native conditions. The three-dimensional reconstructions show a regular array of densities at approximately 70 A intervals along the midline, with a curved shape resembling the X-ray structure of C-cadherin, a representative 'classical' cadherin. Model-independent three-dimensional image processing of extracted sub-tomograms reveals the cadherin organization. After fitting the C-cadherin atomic structure into the averaged sub-tomograms, we see a periodic arrangement of a trans W-like and a cis V-like interaction corresponding to molecules from opposing membranes and the same cell membrane, respectively. The resulting model of cadherin organization explains existing two-dimensional data and yields insights into a possible mechanism of cadherin-based cell adhesion.
Nature 01/2008; 450(7171):832-7. · 36.28 Impact Factor
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ABSTRACT: Cryo-electron tomography of vitreous sections is currently the most promising technique for visualizing arbitrary regions of eukaryotic cells or tissue at molecular resolution. Despite significant progress in the sample preparation techniques over the past few years, the three dimensional reconstruction using electron tomography is not as simple as in plunge frozen samples for various reasons, but mainly due to the effects of irradiation on the sections and the resulting poor alignment. Here, we present a new algorithm, which can provide a useful three-dimensional marker model after investigation of hundreds to thousands of observations calculated using local cross-correlation throughout the tilt series. The observations are chosen according to their coherence to a particular model and assigned to virtual markers. Through this type of measurement a merit figure can be calculated, precisely estimating the quality of the reconstruction. The merit figures of this alignment method are comparable to those obtained with plunge frozen samples using fiducial gold markers. An additional advantage of the algorithm is the implicit detection of areas in the sections that behave as rigid bodies and can thus be properly reconstructed.
Journal of Structural Biology 10/2007; 159(3):413-23. · 3.41 Impact Factor
