Achilleas S Frangakis

Goethe-Universität Frankfurt am Main, Frankfurt, Hesse, Germany

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Publications (66)461.53 Total impact

  • Michael Kunz · Zhou Yu · Achilleas S. Frangakis
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    ABSTRACT: The M-free score is a heuristic to measure the reference bias in applications such as template matching and sub-tomogram averaging. In the original formulation the mask typically used in these applications had to be separated into a working and a testing area. Here we present a variant of the calculation of the M-free score, which under certain conditions does not require adapting the mask used during the processing. This is made possible by a modified algorithm that allows for arbitrary variances in the testing and in the working area. Consequently, the reference bias can be estimated with knowledge of only the starting reference, the final average and the mask used for processing. We show that the new formulation of the M-free score gives a reliable measure of the reference bias for any sub-tomogram average that has ancillary data, such as when the averaged structure contains density in the periphery, when a complex is attached to a membrane (membrane-associated complexes) or when one subunit is attached to others (e.g. in viruses). Further, we show that in contrast to correlation-based measurements, the M-free score is sensitive to wrong-alignments and contaminations present in the data set. The scope of this new calculation of the M-free score is to reduce the constraints of the previous approach and in certain cases to avoid an adaptation of the mask. The M-free score gives a separate reliability measure for sub-tomogram averaging and template matching. Copyright © 2015. Published by Elsevier Inc.
    No preview · Article · Aug 2015 · Journal of Structural Biology
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    Full-text · Dataset · Jul 2015
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    ABSTRACT: The closure of epidermal openings is an essential biological process that causes major developmental problems such as spina bifida in humans if it goes awry. At present, the mechanism of closure remains elusive. Therefore, we reconstructed a model closure event, dorsal closure in fly embryos, by large-volume correlative electron tomography. We present a comprehensive, quantitative analysis of the cytoskeletal reorganization, enabling separated epidermal cells to seal the epithelium. After establishing contact through actin-driven exploratory filopodia, cells use a single lamella to generate 'roof tile'-like overlaps. These shorten to produce the force, 'zipping' the tissue closed. The shortening overlaps lack detectable actin filament ensembles but are crowded with microtubules. Cortical accumulation of shrinking microtubule ends suggests a force generation mechanism in which cortical motors pull on microtubule ends as for mitotic spindle positioning. In addition, microtubules orient filopodia and lamellae before zipping. Our 4D electron microscopy picture describes an entire developmental process and provides fundamental insight into epidermal closure.
    No preview · Article · Apr 2015 · Nature Cell Biology
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    ABSTRACT: The host endolysosomal compartment is often manipulated by intracellular bacterial pathogens. Salmonella (Salmonella enterica serovar Typhimurium) secrete numerous effector proteins, including SifA, through a specialized type III secretion system to hijack the host endosomal system and generate the Salmonella-containing vacuole (SCV). To form this replicative niche, Salmonella targets the Rab7 GTPase to recruit host membranes through largely unknown mechanisms. We show that Pleckstrin homology domain-containing protein family member 1 (PLEKHM1), a lysosomal adaptor, is targeted by Salmonella through direct interaction with SifA. By binding the PLEKHM1 PH2 domain, Salmonella utilize a complex containing PLEKHM1, Rab7, and the HOPS tethering complex to mobilize phagolysosomal membranes to the SCV. Depletion of PLEKHM1 causes a profound defect in SCV morphology with multiple bacteria accumulating in enlarged structures and significantly dampens Salmonella proliferation in multiple cell types and mice. Thus, PLEKHM1 provides a critical interface between pathogenic infection and the host endolysosomal system. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Dec 2014 · Cell Host & Microbe
  • Michael Kunz · Achilleas S Frangakis
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    ABSTRACT: In tomography, the quality of the reconstruction is essential because the complete cascade of the subsequent analysis is based on it. To date, weighted back-projection (WBP) has been the most commonly used technique due to its versatility and performance in sub-tomogram averaging. Here we present super-sampling SART that is based on the simultaneous algebraic reconstruction technique. While algebraic reconstruction techniques typically produce better contrast and lately showed a significant improvement in terms of processing speed, sub-tomogram averages derived from those reconstructions were inferior in resolution compared to those derived from WBP data. Super-sampling SART, however, outperforms both in term of contrast and the resolution achieved in sub-tomogram averaging several other tested methods and in particular WBP. The main feature of super-sampling SART, as the name implies, is the super-sampling option - by which parameter-based up-sampling and down-sampling are used to reduce artifacts. In particular, the aliasing that is omnipresent in the reconstruction can be practically eliminated without a significant increase in the computational time. Furthermore, super-sampling SART reaches convergence within a single iteration, making the processing time comparable to WBP, and eliminating the ambiguity of parameter-controlled convergence times. We find that grouping of projections increases the contrast, while when projections are used individually the resolution can be maximized. Using sub-tomogram averaging of ribosomes as a test case, we show that super-sampling SART achieves equal or better sub-tomogram averaging results than WBP, which is of particular importance in cryo-electron tomography.
    No preview · Article · Oct 2014 · Journal of Structural Biology
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    Zhou Yu · Achilleas S. Frangakis
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    ABSTRACT: Cryo-electron tomography provides a snapshot of the cellular proteome. With template matching, the spatial positions of various macromolecular complexes within their native cellular context can be detected. However, the growing awareness of the reference bias introduced by the cross-correlation based approaches, and more importantly the lack of a reliable confidence measurement in the selection of these macromolecular complexes, has restricted the use of these applications. Here we propose a heuristic, in which the reference bias is measured in real space in an analogous way to the R-free value in X-ray crystallography. We measure the reference bias within the mask used to outline the area of the template, and do not modify the template itself. The heuristic works by splitting the mask into a working and a testing area in a volume ratio of 9:1. While the working area is used during the calculation of the cross-correlation function, the information from both areas is explored to calculate the M-free score. We show using artificial data, that the M-free score gives a reliable measure for the reference bias. The heuristic can be applied in template matching and in sub-tomogram averaging. We further test the applicability of the heuristic in tomograms of purified macromolecules, and tomograms of whole Mycoplasma cells.
    Preview · Article · Jul 2014 · Journal of Structural Biology
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    ABSTRACT: Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x-y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.
    Full-text · Article · May 2014 · Journal of Structural Biology
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    ABSTRACT: The centromeric histone H3 variant centromeric protein A (CENP-A), whose sequence is the least conserved among all histone variants, is responsible for specifying the location of the centromere. Here, we present a comprehensive study of CENP-A nucleosome arrays by cryo-electron tomography. We see that CENP-A arrays have different biophysical properties than canonical ones under low ionic conditions, as they are more condensed with a 20% smaller average nearest-neighbor distance and a 30% higher nucleosome density. We find that CENP-A nucleosomes have a predominantly crossed DNA entry/exit site that is narrowed on average by 8°, and they have a propensity to stack face to face. We therefore propose that CENP-A induces geometric constraints at the nucleosome DNA entry/exit site to bring neighboring nucleosomes into close proximity. This specific property of CENP-A may be responsible for generating a fundamental process that contributes to increased chromatin fiber compaction that is propagated under physiological conditions to form centromeric chromatin.
    Full-text · Article · Feb 2014 · Biophysical Journal
  • Ashraf Al-Amoudi · Achilleas Frangakis
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    ABSTRACT: Intercellular adhesion junctions are fundamental for the function and development of multi-cellular organisms. Desmosomes and adherens junctions (AJs) represent major categories of these junctions. Both desmosomes and AJs are present in epithelia but desmosomes are more abundant in tissues prone to mechanical stress such as heart and skin. The intercellular space of desmosomes and AJs relies on the associations between members of Ca2+- dependent adhesion molecules called cadherins which share high sequence similarity among them. Recently, the first crystal structure of the full extracellular domains of C-cadherin (a representative of classical cadherins) showed that the cadherin molecules adopt a stable, curved conformation [1]. In this crystal structure, the neighbouring molecules are oriented in antiparallel and engaged through a mutual exchange of the tryptophan 2 (Trp2) forming a W-like shape. Such Trp2 trans-interactions were supported by mutagenesis data and cell-adhesion assays and are now considered to be physiologically relevant [2].
    No preview · Article · Nov 2013
  • Ashraf Al-Amoudi · Achilleas S Frangakis
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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.
    No preview · Article · Jan 2013 · Methods in molecular biology (Clifton, N.J.)
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    ABSTRACT: The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology.
    Full-text · Article · Dec 2012 · PLoS ONE
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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.
    Full-text · Article · Aug 2012 · Proceedings of the National Academy of Sciences
  • F Porrati · E Begun · M Winhold · Ch H Schwalb · R Sachser · A S Frangakis · M Huth
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    ABSTRACT: CoPt-C binary alloys have been fabricated by focused-electron-beam-induced deposition by the simultaneous use of Co₂(CO)₈ and (CH₃)₃CH₃C₅H₄Pt as precursor gases. The alloys are made of CoPt nanoparticles embedded in a carbonaceous matrix. TEM investigations show that as-grown samples are in an amorphous phase. By means of a room temperature low-energy electron irradiation treatment the CoPt nanoparticles transform into face-centered tetragonal L1₀ nanocrystallites. In parallel, the system undergoes a transition from a superparamagnetic to a ferromagnetic state at room temperature. By variation of the post-growth irradiation dose the electrical and magneto-transport properties of the alloy can be continuously tuned.
    No preview · Article · Apr 2012 · Nanotechnology
  • Ohad Medalia · Achilleas Frangakis

    No preview · Article · Mar 2012 · Journal of Structural Biology
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    ABSTRACT: How a long strand of genomic DNA is compacted into a mitotic chromosome remains one of the basic questions in biology. The nucleosome fibre, in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fibre and further hierarchical regular structures to form mitotic chromosomes, although the actual existence of these regular structures is controversial. Here, we show that human mitotic HeLa chromosomes are mainly composed of irregularly folded nucleosome fibres rather than 30-nm chromatin fibres. Our comprehensive and quantitative study using cryo-electron microscopy and synchrotron X-ray scattering resolved the long-standing contradictions regarding the existence of 30-nm chromatin structures and detected no regular structure >11 nm. Our finding suggests that the mitotic chromosome consists of irregularly arranged nucleosome fibres, with a fractal nature, which permits a more dynamic and flexible genome organization than would be allowed by static regular structures.
    Full-text · Article · Feb 2012 · The EMBO Journal
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    ABSTRACT: The shear-responsive transcription factor Krüppel-like factor 2 (KLF2) is a critical regulator of endothelial gene expression patterns induced by atheroprotective flow. As microRNAs (miRNAs) post-transcriptionally control gene expression in many pathogenic and physiological processes, we investigated the regulation of miRNAs by KLF2 in endothelial cells. KLF2 binds to the promoter and induces a significant upregulation of the miR-143/145 cluster. Interestingly, miR-143/145 has been shown to control smooth muscle cell (SMC) phenotypes; therefore, we investigated the possibility of transport of these miRNAs between endothelial cells and SMCs. Indeed, extracellular vesicles secreted by KLF2-transduced or shear-stress-stimulated HUVECs are enriched in miR-143/145 and control target gene expression in co-cultured SMCs. Extracellular vesicles derived from KLF2-expressing endothelial cells also reduced atherosclerotic lesion formation in the aorta of ApoE(-/-) mice. Combined, our results show that atheroprotective stimuli induce communication between endothelial cells and SMCs through an miRNA- and extracellular-vesicle-mediated mechanism and that this may comprise a promising strategy to combat atherosclerosis.
    Full-text · Article · Feb 2012 · Nature Cell Biology
  • Margot P Scheffer · Mikhail Eltsov · Jan Bednar · Achilleas S Frangakis
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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.
    No preview · Article · Nov 2011 · Journal of Structural Biology
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    ABSTRACT: Binary systems of Pt-Si are prepared by electron-beam-induced deposition using the two precursors, trimethyl(methylcyclopentadienyl)platinum(IV) (MeCpPt(Me)(3)) and neopentasilane (Si(SiH(3))(4)), simultaneously. By varying the relative flux of the two precursors during deposition, we are able to study composites containing platinum and silicon in different ratios by means of energy-dispersive X-ray spectroscopy, atomic force microscopy, electrical transport measurements, and transmission electron microscopy. The results show strong evidence for the formation of a binary, metastable Pt(2)Si(3) phase, leading to a maximum in the conductivity for a Si/Pt ratio of 3:2.
    No preview · Article · Nov 2011 · ACS Nano
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    Margot P Scheffer · Mikhail Eltsov · Achilleas S Frangakis
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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.
    Full-text · Article · Oct 2011 · Proceedings of the National Academy of Sciences
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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.
    Full-text · Article · Apr 2011 · Proceedings of the National Academy of Sciences

Publication Stats

4k Citations
461.53 Total Impact Points


  • 2010-2015
    • Goethe-Universität Frankfurt am Main
      • • Institute of Physical and Theoretical Chemistry
      • • Institut für Biophysik
      Frankfurt, Hesse, Germany
  • 2014
    • Universität Basel
      Bâle, Basel-City, Switzerland
    • Buchmann Institute for Molecular Life Sciences
      Frankfurt, Hesse, Germany
  • 2013
    • Deutsches Zentrum für Neurodegenerative Erkrankungen
      Bonn, North Rhine-Westphalia, Germany
  • 2004-2012
    • European Molecular Biology Laboratory
      • Structural and Computational Biology Unit (Heidelberg)
      Heidelburg, Baden-Württemberg, Germany
  • 2002
    • California Institute of Technology
      Pasadena, California, United States
  • 1999-2002
    • Max Planck Institute of Biochemistry
      • Department of Molecular Structural Biology
      München, Bavaria, Germany