Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits.
Piazza, I., Rutkowska, A., Ori, A., Walczak, M., Metz, J., Pelechano, V., Beck, M. & Haering, C.H.
Nat Struct Mol Biol. 2014 May 18. doi: 10.1038/nsmb.2831.
Condensin complexes have central roles in the three-dimensional organization of chromosomes during cell divisions, but how they interact with chromatin to promote chromosome segregation is largely unknown. Previous work has suggested that condensin, in addition to encircling chromatin fibers topologically within the ring-shaped structure formed by its SMC and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA formed by the two HEAT-repeat subunits of the Saccharomyces cerevisiae condensin complex. From detailed mapping data of the interfaces between the HEAT-repeat and kleisin subunits, we generated condensin complexes that lack one of the HEAT-repeat subunits and consequently fail to associate with chromosomes in yeast and human cells. The finding that DNA binding by condensin's HEAT-repeat subunits stimulates the SMC ATPase activity suggests a multistep mechanism for the loading of condensin onto chromosomes.
Prosurvival function of the cellular apoptosis susceptibility/importin-alpha1 transport cycle is repressed by p53 in liver cancer.
Winkler, J., Ori, A., Holzer, K., Sticht, C., Dauch, D., Eiteneuer, E.M., Pinna, F., Geffers, R., Ehemann, V., Andres-Pons, A., Breuhahn, K., Longerich, T., Lorenzo Bermejo, J., Gretz, N., Zender, L., Schirmacher, P., Beck, M. & Singer, S.
Hepatology. 2014 May 6. doi: 10.1002/hep.27207.
Proteins of the karyopherin superfamily including importins and exportins represent an essential part of the nucleo-cytoplasmic transport machinery. However, the functional relevance and regulation of karyopherins in hepatocellular carcinoma (HCC) is poorly understood. Here, we identified Cellular Apoptosis Susceptibility (CAS, exportin-2) and its transport substrate importin-alpha1 (imp-alpha1) among significantly up-regulated transport factor genes in HCC. Disruption of the CAS/imp-alpha1 transport cycle by RNAi in HCC cell lines resulted in decreased tumor cell growth and increased apoptosis. The apoptotic phenotype upon CAS depletion could be recapitulated by direct knockdown of the X-linked inhibitor of apoptosis (XIAP) and partially reverted by XIAP overexpression. In addition, XIAP and CAS mRNA expression levels were correlated in HCC patient samples (r = 0.463; p<0.01) supporting the in vivo relevance of our findings. Furthermore, quantitative mass spectrometry analyses of murine HCC samples (p53-/- vs. p53 +/+) indicated higher protein expression of CAS and imp-alpha1 in p53-/- tumors. Consistent with a role of p53 in regulating the CAS/imp-alpha1 transport cycle, we observed that both transport factors were repressed upon p53 induction in a p21-dependent manner. Conclusion: Our study indicates that the CAS/imp-alpha1 transport cycle is linked to XIAP and required to maintain tumor cell survival in HCC. Moreover, we found that CAS and imp-alpha1 are targets of p53-mediated repression, which represents a novel aspect of p53's ability to control tumor cell growth in hepatocarcinogenesis. (Hepatology 2014).
Toward understanding the structure of the vertebrate nuclear pore complex.
Beck, M. & Glavy, J.S.
This is a review article.
Nucleus. 2014 Apr 3;5(2).
Nuclear pore complexes are large macromolecular assemblies that facilitate the nucleocytoplasmic exchange of macromolecules. Because of their intricate composition, membrane association and sheer size, the integration of various, complementary structure determination approaches is a prerequisite for elucidating their structure. We have recently employed such an integrated strategy to analyze the scaffold structure of the cytoplasmic and nuclear rings of the human nuclear pore complex. In this extra view, we highlight two specific aspects of this work: the power of electron microscopy for bridging different resolution regimes and the importance of post-translational modifications for regulating nucleoporin interactions. We review recent technological developments and give a perspective toward future structure determination approaches.
The Use of Targeted Proteomics to Determine the Stoichiometry of Large Macromolecular Assemblies.
Ori, A., Andres-Pons, A. & Beck, M.
Methods Cell Biol. 2014;122C:117-146. doi: 10.1016/B978-0-12-417160-2.00006-0.
Accurate knowledge of the stoichiometry of protein complexes is a crucial prerequisite for understanding their structure and function. To purify or enrich large and intricate protein complexes such that their structure is preserved and to absolutely quantify all of their protein components is an enormous technical challenge. In this chapter, we describe how to purify nuclear envelopes from human tissue culture cells that are highly enriched for nuclear pore complexes. We use the nuclear pore as an example to discuss how the structural preservation of such preparations can be controlled. Furthermore, we give a practical guide how to develop and employ targeted proteomic assays for both, the absolute quantification of stoichiometries and the relative quantification of protein complex composition across multiple biological conditions. The concept discussed here is universally applicable to any protein complex.
Integrated structural analysis of the human nuclear pore complex scaffold.
Bui, K.H., von Appen, A., DiGuilio, A.L., Ori, A., Sparks, L., Mackmull, M.T., Bock, T., Hagen, W., Andres-Pons, A., Glavy, J.S. & Beck, M.
Cell. 2013 Dec 5;155(6):1233-43. doi: 10.1016/j.cell.2013.10.055.
The nuclear pore complex (NPC) is a fundamental component of all eukaryotic cells that facilitates nucleocytoplasmic exchange of macromolecules. It is assembled from multiple copies of about 30 nucleoporins. Due to its size and complex composition, determining the structure of the NPC is an enormous challenge, and the overall architecture of the NPC scaffold remains elusive. In this study, we have used an integrated approach based on electron tomography, single-particle electron microscopy, and crosslinking mass spectrometry to determine the structure of a major scaffold motif of the human NPC, the Nup107 subcomplex, in both isolation and integrated into the NPC. We show that 32 copies of the Nup107 subcomplex assemble into two reticulated rings, one each at the cytoplasmic and nuclear face of the NPC. This arrangement may explain how changes of the diameter are realized that would accommodate transport of huge cargoes.
Protein interfaces of the conserved Nup84 complex from Chaetomium thermophilum shown by crosslinking mass spectrometry and electron microscopy.
Thierbach, K., von Appen, A., Thoms, M., Beck, M., Flemming, D. & Hurt, E.
Structure. 2013 Sep 3;21(9):1672-82. doi: 10.1016/j.str.2013.07.004. Epub 2013Aug 15.
A key building block of the nuclear pore complex (NPC) is the Nup84 subcomplex that has been structurally analyzed predominantly in the yeast system. To expand this analysis and gain insight into the evolutionary conservation of its structure, we reconstituted an octameric Nup84 complex using the subunits from a thermophile, Chaetomium thermophilum (ct). This assembly carries Nup37 and Elys, which are characteristic subunits of the orthologous human Nup107-Nup160 complex but absent from the yeast Saccharomyces cerevisiae. We found that Elys binds cooperatively to the complex requiring both Nup37 and Nup120. Unexpectedly, the reconstituted ctNup84 complex formed a striking dimer structure with an unpredicted side-to-side arrangement of two molecules. Finally, crosslinking mass spectrometry allowed the mapping of key protein interfaces within the Y-shaped complex. Thus, the thermophilic Nup84 complex can serve as a structural model for higher eukaryotic Nup107-Nup160 assemblies to gain insight into its possible configuration within the NPC scaffold.
Fourier ring correlation as a resolution criterion for super-resolution microscopy.
Banterle, N., Bui, K.H., Lemke, E.A. & Beck, M.
J Struct Biol. 2013 Sep;183(3):363-7. doi: 10.1016/j.jsb.2013.05.004. Epub 2013May 16.
Optical nanoscopy techniques using localization based image reconstruction, also termed super-resolution microscopy (SRM), have become a standard tool to bypass the diffraction limit in fluorescence light microscopy. The localization precision measured for the detected fluorophores is commonly used to describe the maximal attainable resolution. However, this measure takes not all experimental factors, which impact onto the finally achieved resolution, into account. Several other methods to measure the resolution of super-resolved images were previously suggested, typically relying on intrinsic standards, such as molecular rulers, or on a priori knowledge about the specimen, e.g. its spatial frequency content. Here we show that Fourier ring correlation provides an easy-to-use, laboratory consistent standard for measuring the resolution of SRM images. We provide a freely available software tool that combines resolution measurement with image reconstruction.
Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines.
Ori, A., Banterle, N., Iskar, M., Andres-Pons, A., Escher, C., Khanh Bui, H., Sparks, L., Solis-Mezarino, V., Rinner, O., Bork, P., Lemke, E.A. & Beck, M.
Mol Syst Biol. 2013 Mar 19;9:648. doi: 10.1038/msb.2013.4.
To understand the structure and function of large molecular machines, accurate knowledge of their stoichiometry is essential. In this study, we developed an integrated targeted proteomics and super-resolution microscopy approach to determine the absolute stoichiometry of the human nuclear pore complex (NPC), possibly the largest eukaryotic protein complex. We show that the human NPC has a previously unanticipated stoichiometry that varies across cancer cell types, tissues and in disease. Using large-scale proteomics, we provide evidence that more than one third of the known, well-defined nuclear protein complexes display a similar cell type-specific variation of their subunit stoichiometry. Our data point to compositional rearrangement as a widespread mechanism for adapting the functions of molecular machines toward cell type-specific constraints and context-dependent needs, and highlight the need of deeper investigation of such structural variants.
Facilitated aggregation of FG nucleoporins under molecular crowding conditions.
Milles, S., Huy Bui, K., Koehler, C., Eltsov, M., Beck, M. & Lemke, E.A.
EMBO Rep. 2013 Feb;14(2):178-83. doi: 10.1038/embor.2012.204. Epub 2012 Dec 14.
Intrinsically disordered and phenylalanine-glycine-rich nucleoporins (FG Nups) form a crowded and selective transport conduit inside the NPC that can only be transited with the help of nuclear transport receptors (NTRs). It has been shown in vitro that FG Nups can assemble into two distinct appearances, amyloids and hydrogels. If and how these phenomena are linked and if they have a physiological role still remains unclear. Using a variety of high-resolution fluorescence and electron microscopic (EM) tools, we reveal that crowding conditions mimicking the NPC environment can accelerate the aggregation and amyloid formation speed of yeast and human FG Nups by orders of magnitude. Aggregation can be inhibited by NTRs, providing a rationale on how the cell might control amyloid formation of FG Nups. The superb spatial resolving power of EM also reveals that hydrogels are enlaced amyloid fibres, and these findings have implications for existing transport models and for NPC assembly.
False discovery rate estimation for cross-linked peptides identified by mass spectrometry.
Walzthoeni, T., Claassen, M., Leitner, A., Herzog, F., Bohn, S., Forster, F., Beck, M. & Aebersold, R.
Nat Methods. 2012 Sep;9(9):901-3. doi: 10.1038/nmeth.2103. Epub 2012 Jul 8.
The mass spectrometric identification of chemically cross-linked peptides (CXMS) specifies spatial restraints of protein complexes; these values complement data obtained from common structure-determination techniques. Generic methods for determining false discovery rates of cross-linked peptide assignments are currently lacking, thus making data sets from CXMS studies inherently incomparable. Here we describe an automated target-decoy strategy and the software tool xProphet, which solve this problem for large multicomponent protein complexes.
Structural probing of a protein phosphatase 2A network by chemical cross-linking and mass spectrometry.
Herzog, F., Kahraman, A., Boehringer, D., Mak, R., Bracher, A., Walzthoeni, T., Leitner, A., Beck, M., Hartl, F.U., Ban, N., Malmstrom, L. & Aebersold, R.
Science. 2012 Sep 14;337(6100):1348-52.
The identification of proximate amino acids by chemical cross-linking and mass spectrometry (XL-MS) facilitates the structural analysis of homogeneous protein complexes. We gained distance restraints on a modular interaction network of protein complexes affinity-purified from human cells by applying an adapted XL-MS protocol. Systematic analysis of human protein phosphatase 2A (PP2A) complexes identified 176 interprotein and 570 intraprotein cross-links that link specific trimeric PP2A complexes to a multitude of adaptor proteins that control their cellular functions. Spatial restraints guided molecular modeling of the binding interface between immunoglobulin binding protein 1 (IGBP1) and PP2A and revealed the topology of TCP1 ring complex (TRiC) chaperonin interacting with the PP2A regulatory subunit 2ABG. This study establishes XL-MS as an integral part of hybrid structural biology approaches for the analysis of endogenous protein complexes.
High-throughput subtomogram alignment and classification by Fourier space constrained fast volumetric matching.
Xu, M., Beck, M. & Alber, F.
J Struct Biol. 2012 May;178(2):152-64. doi: 10.1016/j.jsb.2012.02.014. Epub 2012Mar 7.
Cryo-electron tomography allows the visualization of macromolecular complexes in their cellular environments in close-to-live conditions. The nominal resolution of subtomograms can be significantly increased when individual subtomograms of the same kind are aligned and averaged. A vital step for such a procedure are algorithms that speedup subtomogram alignment and improve its accuracy to allow reference-free subtomogram classifications. Such methods will facilitate automation of tomography analysis and overall high throughput in the data processing. Building on previous work, here we propose a fast rotational alignment method that uses the Fourier equivalent form of a popular constrained correlation measure that considers missing wedge corrections and density variances in the subtomograms. The fast rotational search is based on 3D volumetric matching, which improves the rotational alignment accuracy in particular for highly distorted subtomograms with low SNR and tilt angle ranges in comparison to fast rotational matching of projected 2D spherical images. We further integrate our fast rotational alignment method in a reference-free iterative subtomogram classification scheme, and propose a local feature enhancement strategy in the classification process. As a proof of principle, we can demonstrate that the automatic method can successfully classify a large number of experimental subtomograms without the need of a reference structure.
The quantitative proteome of a human cell line.
Beck, M., Schmidt, A., Malmstroem, J., Claassen, M., Ori, A., Szymborska, A., Herzog, F., Rinner, O., Ellenberg, J. & Aebersold, R.
Mol Syst Biol. 2011 Nov 8;7:549. doi: 10.1038/msb.2011.82.
The generation of mathematical models of biological processes, the simulation of these processes under different conditions, and the comparison and integration of multiple data sets are explicit goals of systems biology that require the knowledge of the absolute quantity of the system's components. To date, systematic estimates of cellular protein concentrations have been exceptionally scarce. Here, we provide a quantitative description of the proteome of a commonly used human cell line in two functional states, interphase and mitosis. We show that these human cultured cells express at least approximately 10 000 proteins and that the quantified proteins span a concentration range of seven orders of magnitude up to 20 000 000 copies per cell. We discuss how protein abundance is linked to function and evolution.
Absolute quantification of microbial proteomes at different states by directed mass spectrometry.
Schmidt, A., Beck, M., Malmstrom, J., Lam, H., Claassen, M., Campbell, D. & Aebersold, R.
Mol Syst Biol. 2011 Jul 19;7:510. doi: 10.1038/msb.2011.37.
Over the past decade, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has evolved into the main proteome discovery technology. Up to several thousand proteins can now be reliably identified from a sample and the relative abundance of the identified proteins can be determined across samples. However, the remeasurement of substantially similar proteomes, for example those generated by perturbation experiments in systems biology, at high reproducibility and throughput remains challenging. Here, we apply a directed MS strategy to detect and quantify sets of pre-determined peptides in tryptic digests of cells of the human pathogen Leptospira interrogans at 25 different states. We show that in a single LC-MS/MS experiment around 5000 peptides, covering 1680 L. interrogans proteins, can be consistently detected and their absolute expression levels estimated, revealing new insights about the proteome changes involved in pathogenic progression and antibiotic defense of L. interrogans. This is the first study that describes the absolute quantitative behavior of any proteome over multiple states, and represents the most comprehensive proteome abundance pattern comparison for any organism to date.
Template-free detection of macromolecular complexes in cryo electron tomograms.
Xu, M., Beck, M. & Alber, F.
Bioinformatics. 2011 Jul 1;27(13):i69-76. doi: 10.1093/bioinformatics/btr207.
MOTIVATION: Cryo electron tomography (CryoET) produces 3D density maps of biological specimen in its near native states. Applied to small cells, cryoET produces 3D snapshots of the cellular distributions of large complexes. However, retrieving this information is non-trivial due to the low resolution and low signal-to-noise ratio in tomograms. Current pattern recognition methods identify complexes by matching known structures to the cryo electron tomogram. However, so far only a small fraction of all protein complexes have been structurally resolved. It is, therefore, of great importance to develop template-free methods for the discovery of previously unknown protein complexes in cryo electron tomograms. RESULTS: Here, we have developed an inference method for the template-free discovery of frequently occurring protein complexes in cryo electron tomograms. We provide a first proof-of-principle of the approach and assess its applicability using realistically simulated tomograms, allowing for the inclusion of noise and distortions due to missing wedge and electron optical factors. Our method is a step toward the template-free discovery of the shapes, abundance and spatial distributions of previously unknown macromolecular complexes in whole cell tomograms. CONTACT: email@example.com
mProphet: automated data processing and statistical validation for large-scale SRM experiments.
Reiter, L., Rinner, O., Picotti, P., Huttenhain, R., Beck, M., Brusniak, M.Y., Hengartner, M.O. & Aebersold, R.
Nat Methods. 2011 May;8(5):430-5. doi: 10.1038/nmeth.1584. Epub 2011 Mar 20.
Selected reaction monitoring (SRM) is a targeted mass spectrometric method that is increasingly used in proteomics for the detection and quantification of sets of preselected proteins at high sensitivity, reproducibility and accuracy. Currently, data from SRM measurements are mostly evaluated subjectively by manual inspection on the basis of ad hoc criteria, precluding the consistent analysis of different data sets and an objective assessment of their error rates. Here we present mProphet, a fully automated system that computes accurate error rates for the identification of targeted peptides in SRM data sets and maximizes specificity and sensitivity by combining relevant features in the data into a statistical model.
Exploring the spatial and temporal organization of a cell's proteome.
Beck, M., Topf, M., Frazier, Z., Tjong, H., Xu, M., Zhang, S. & Alber, F.
J Struct Biol. 2011 Mar;173(3):483-96. doi: 10.1016/j.jsb.2010.11.011. Epub 2010Nov 19.
To increase our current understanding of cellular processes, such as cell signaling and division, knowledge is needed about the spatial and temporal organization of the proteome at different organizational levels. These levels cover a wide range of length and time scales: from the atomic structures of macromolecules for inferring their molecular function, to the quantitative description of their abundance, and spatial distribution in the cell. Emerging new experimental technologies are greatly increasing the availability of such spatial information on the molecular organization in living cells. This review addresses three fields that have significantly contributed to our understanding of the proteome's spatial and temporal organization: first, methods for the structure determination of individual macromolecular assemblies, specifically the fitting of atomic structures into density maps generated from electron microscopy techniques; second, research that visualizes the spatial distributions of these complexes within the cellular context using cryo electron tomography techniques combined with computational image processing; and third, methods for the spatial modeling of the dynamic organization of the proteome, specifically those methods for simulating reaction and diffusion of proteins and complexes in crowded intracellular fluids. The long-term goal is to integrate the varied data about a proteome's organization into a spatially explicit, predictive model of cellular processes.
Beck, M., Claassen, M. & Aebersold, R.
Curr Opin Biotechnol. 2011 Feb;22(1):3-8. doi: 10.1016/j.copbio.2010.09.002. Epub2010 Oct 1.
Extensive proteome discovery projects using a variety of mass spectrometric techniques have identified proteins matching to 50-70% of the predicted gene models of various species. Comprehensive proteome coverage is desirable for the unbiased comparison of protein quantities between different biological states and for the meaningful comparison of data from multiple samples. Here we discuss the feasibility of this goal in the light of recent technological developments.
Structure of the 26S proteasome from Schizosaccharomyces pombe at subnanometer resolution.
Bohn, S., Beck, F., Sakata, E., Walzthoeni, T., Beck, M., Aebersold, R., Forster, F., Baumeister, W. & Nickell, S.
Proc Natl Acad Sci U S A. 2010 Dec 7;107(49):20992-7. doi:10.1073/pnas.1015530107. Epub 2010 Nov 22.
The structure of the 26S proteasome from Schizosaccharomyces pombe has been determined to a resolution of 9.1 A by cryoelectron microscopy and single particle analysis. In addition, chemical cross-linking in conjunction with mass spectrometry has been used to identify numerous residue pairs in close proximity to each other, providing an array of spatial restraints. Taken together these data clarify the topology of the AAA-ATPase module in the 19S regulatory particle and its spatial relationship to the alpha-ring of the 20S core particle. Image classification and variance analysis reveal a belt of high "activity" surrounding the AAA-ATPase module which is tentatively assigned to the reversible association of proteasome interacting proteins and the conformational heterogeneity among the particles. An integrated model is presented which sheds light on the early steps of protein degradation by the 26S complex.
Probing native protein structures by chemical cross-linking, mass spectrometry, and bioinformatics.
Leitner, A., Walzthoeni, T., Kahraman, A., Herzog, F., Rinner, O., Beck, M. & Aebersold, R.
Mol Cell Proteomics. 2010 Aug;9(8):1634-49. Epub 2010 Mar 31.
Chemical cross-linking of reactive groups in native proteins and protein complexes in combination with the identification of cross-linked sites by mass spectrometry has been in use for more than a decade. Recent advances in instrumentation, cross-linking protocols, and analysis software have led to a renewed interest in this technique, which promises to provide important information about native protein structure and the topology of protein complexes. In this article, we discuss the critical steps of chemical cross-linking and its implications for (structural) biology: reagent design and cross-linking protocols, separation and mass spectrometric analysis of cross-linked samples, dedicated software for data analysis, and the use of cross-linking data for computational modeling. Finally, the impact of protein cross-linking on various biological disciplines is highlighted.
Forster, F., Han, B.G. & Beck, M.
Methods Enzymol. 2010;483:215-43.
Visual proteomics attempts to generate molecular atlases by providing the position and angular orientation of protein complexes inside of cells. This is accomplished by template matching (pattern recognition), a cross-correlation-based process that matches the structure of a specific protein complex to the densities of the whole volume or subvolume of a cell, that is typically acquired by cryoelectron tomography. Thereby, a search is performed that scans the entire volume for structural templates contained in a database. In this chapter, we primarily describe the practical experiences gained with visual proteomics during the Leptospira interrogans proteome project [Beck et al. (2009). Visual proteomics of the human pathogen Leptospira interrogans. Nat. Methods 6, 817.]. We give a practical guide how to implement the method and review critical experimental and computational aspects in detail. Based on a survey that has been undertaken for protein complexes from Desulfovibrio vulgaris, we review the difficulty of generating reference structures in detail. Finally, we discuss the high yield targets for technical improvements.
Visual proteomics of the human pathogen Leptospira interrogans.
Beck, M., Malmstrom, J.A., Lange, V., Schmidt, A., Deutsch, E.W. & Aebersold, R.
Nat Methods. 2009 Nov;6(11):817-23. Epub 2009 Oct 18.
Systems biology conceptualizes biological systems as dynamic networks of interacting elements, whereby functionally important properties are thought to emerge from the structure of such networks. Owing to the ubiquitous role of complexes of interacting proteins in biological systems, their subunit composition and temporal and spatial arrangement within the cell are of particular interest. 'Visual proteomics' attempts to localize individual macromolecular complexes inside of intact cells by template matching reference structures into cryo-electron tomograms. Here we combined quantitative mass spectrometry and cryo-electron tomography to detect, count and localize specific protein complexes in the cytoplasm of the human pathogen Leptospira interrogans. We describe a scoring function for visual proteomics and assess its performance and accuracy under realistic conditions. We discuss current and general limitations of the approach, as well as expected improvements in the future.
Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans.
Malmstrom, J., Beck, M., Schmidt, A., Lange, V., Deutsch, E.W. & Aebersold, R.
Nature. 2009 Aug 6;460(7256):762-5. Epub 2009 Jul 15.
Mass-spectrometry-based methods for relative proteome quantification have broadly affected life science research. However, important research directions, particularly those involving mathematical modelling and simulation of biological processes, also critically depend on absolutely quantitative data--that is, knowledge of the concentration of the expressed proteins as a function of cellular state. Until now, absolute protein concentration measurements of a considerable fraction of the proteome (73%) have only been derived from genetically altered Saccharomyces cerevisiae cells, a technique that is not directly portable from yeast to other species. Here we present a mass-spectrometry-based strategy to determine the absolute quantity, that is, the average number of protein copies per cell in a cell population, for a large fraction of the proteome in genetically unperturbed cells. Applying the technology to the human pathogen Leptospira interrogans, a spirochete responsible for leptospirosis, we generated an absolute protein abundance scale for 83% of the mass-spectrometry-detectable proteome, from cells at different states. Taking advantage of the unique cellular dimensions of L. interrogans, we used cryo-electron tomography morphological measurements to verify, at the single-cell level, the average absolute abundance values of selected proteins determined by mass spectrometry on a population of cells. Because the strategy is relatively fast and applicable to any cell type, we expect that it will become a cornerstone of quantitative biology and systems biology.
Architecture and molecular mechanism of PAN, the archaeal proteasome regulatory ATPase.
Medalia, N., Beer, A., Zwickl, P., Mihalache, O., Beck, M., Medalia, O. & Navon, A.
J Biol Chem. 2009 Aug 21;284(34):22952-60. Epub 2009 Apr 10.
In Archaea, an hexameric ATPase complex termed PAN promotes proteins unfolding and translocation into the 20 S proteasome. PAN is highly homologous to the six ATPases of the eukaryotic 19 S proteasome regulatory complex. Thus, insight into the mechanism of PAN function may reveal a general mode of action mutual to the eukaryotic 19 S proteasome regulatory complex. In this study we generated a three-dimensional model of PAN from tomographic reconstruction of negatively stained particles. Surprisingly, this reconstruction indicated that the hexameric complex assumes a two-ring structure enclosing a large cavity. Assessment of distinct three-dimensional functional states of PAN in the presence of adenosine 5'-O-(thiotriphosphate) and ADP and in the absence of nucleotides outlined a possible mechanism linking nucleotide binding and hydrolysis to substrate recognition, unfolding, and translocation. A novel feature of the ATPase complex revealed in this study is a gate controlling the "exit port" of the regulatory complex and, presumably, translocation into the 20 S proteasome. Based on our structural and biochemical findings, we propose a possible model in which substrate binding and unfolding are linked to structural transitions driven by nucleotide binding and hydrolysis, whereas translocation into the proteasome only depends upon the presence of an unfolded substrate and binding but not hydrolysis of nucleotide.
Structural and functional insights into nucleocytoplasmic transport.
Beck, M. & Medalia, O.
Histol Histopathol. 2008 Aug;23(8):1025-33.
The cell nucleus is surrounded by a double membrane system, the nuclear envelope (NE), with the outer nuclear membrane being continuous with the endoplasmic reticulum. Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes, forming aqueous channels that allow free diffusion of small molecules but that also mediate the energy-dependent transport of large macromolecules. The NPC represents the largest known molecular complex and is composed of about 30 different proteins, termed nucleoporins (Nups). Here, we review recent studies that provide novel insight into the structural and functional organization of nucleocytoplasmic transport. In addition, prospects towards a high resolution model of the nuclear pore are discussed.
Identification of cross-linked peptides from large sequence databases.
Rinner, O., Seebacher, J., Walzthoeni, T., Mueller, L.N., Beck, M., Schmidt, A., Mueller, M. & Aebersold, R.
Nat Methods. 2008 Apr;5(4):315-8. Epub 2008 Mar 9.
We describe a method to identify cross-linked peptides from complex samples and large protein sequence databases by combining isotopically tagged cross-linkers, chromatographic enrichment, targeted proteomics and a new search engine called xQuest. This software reduces the search space by an upstream candidate-peptide search before the recombination step. We showed that xQuest can identify cross-linked peptides from a total Escherichia coli lysate with an unrestricted database search.
Snapshots of nuclear pore complexes in action captured by cryo-electron tomography.
Beck, M., Lucic, V., Forster, F., Baumeister, W. & Medalia, O.
Nature. 2007 Oct 4;449(7162):611-5. Epub 2007 Sep 12.
Nuclear pore complexes reside in the nuclear envelope of eukaryotic cells and mediate the nucleocytoplasmic exchange of macromolecules. Traffic is regulated by mobile transport receptors that target their cargo to the central translocation channel, where phenylalanine-glycine-rich repeats serve as binding sites. The structural analysis of the nuclear pore is a formidable challenge given its size, its location in a membranous environment and its dynamic nature. Here we have used cryo-electron tomography to study the structure of nuclear pore complexes in their functional environment, that is, in intact nuclei of Dictyostelium discoideum. A new image-processing strategy compensating for deviations of the asymmetric units (protomers) from a perfect eight-fold symmetry enabled us to refine the structure and to identify new features. Furthermore, the superposition of a large number of tomograms taken in the presence of cargo, which was rendered visible by gold nanoparticles, has yielded a map outlining the trajectories of import cargo. Finally, we have performed single-molecule Monte Carlo simulations of nuclear import to interpret the experimentally observed cargo distribution in the light of existing models for nuclear import.
Organization of actin networks in intact filopodia.
Medalia, O., Beck, M., Ecke, M., Weber, I., Neujahr, R., Baumeister, W. & Gerisch, G.
Curr Biol. 2007 Jan 9;17(1):79-84.
Filopodia are finger-like extensions of the cell surface that are involved in sensing the environment, in attachment of particles for phagocytosis, in anchorage of cells on a substratum, and in the response to chemoattractants or other guidance cues. Filopodia present an excellent model for actin-driven membrane protrusion. They grow at their tips by the assembly of actin and are stabilized along their length by a core of bundled actin filaments. To visualize actin networks in their native membrane-anchored state, filopodia of Dictyostelium cells were subjected to cryo-electron tomography. At the site of actin polymerization, a peculiar structure, the "terminal cone," is built of short filaments fixed with their distal end to the filopod's tip and with their proximal end to the flank of the filopod. The backbone of the filopodia consists of actin filaments that are shorter than the entire filopod and aligned in parallel or obliquely to the filopod's axis. We hypothesize that growth of the highly dynamic filopodia of Dictyostelium is accompanied by repetitive nucleation of actin polymerization at the filopod tip, followed by the rearrangement of filaments within the shaft.
Luminal particles within cellular microtubules.
Garvalov, B.K., Zuber, B., Bouchet-Marquis, C., Kudryashev, M., Gruska, M., Beck, M., Leis, A., Frischknecht, F., Bradke, F., Baumeister, W., Dubochet, J. & Cyrklaff, M.
J Cell Biol. 2006 Sep 11;174(6):759-65. Epub 2006 Sep 5.
The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells.
Cryo-electron tomography of biological specimens.
Leis, A. P., Beck, M., Gruska, M., Best, C., Hegerl, R., Baumeister, W., and Leis, J. W.
IEEE Signal Processing Magazine
Nuclear pore complex structure and dynamics revealed by cryoelectron tomography.
Beck, M., Forster, F., Ecke, M., Plitzko, J.M., Melchior, F., Gerisch, G., Baumeister, W. & Medalia, O.
Science. 2004 Nov 19;306(5700):1387-90. Epub 2004 Oct 28.
Nuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. To analyze their structure in a close-to-life state, we studied transport-active, intact nuclei from Dictyostelium discoideum by means of cryoelectron tomography. Subvolumes of the tomograms containing individual NPCs were extracted in silico and subjected to three-dimensional classification and averaging, whereby distinct structural states were observed. The central plug/transporter (CP/T) was variable in volume and could occupy different positions along the nucleocytoplasmic axis, which supports the notion that it essentially represents cargo in transit. Changes in the position of the CP/T were accompanied by structural rearrangements in the NPC scaffold.