Carsten Sachse

European Molecular Biology Laboratory, Heidelburg, Baden-Württemberg, Germany

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Publications (31)362.19 Total impact

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    ABSTRACT: Transcription of genes encoding small structured RNAs such as transfer RNAs, spliceosomal U6 small nuclear RNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. Here we present the cryo-electron microscopy structures of the Saccharomyces cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, which allow the building of a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82-C34-C31 heterotrimer in close proximity to the stalk. The C53-C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets.
    Nature 11/2015; DOI:10.1038/nature16143 · 41.46 Impact Factor
  • Terje Johansen · Carsten Sachse ·

    Oncotarget 06/2015; 6(19). DOI:10.18632/oncotarget.4590 · 6.36 Impact Factor
  • Tanmay A M Bharat · Garib N Murshudov · Carsten Sachse · Jan Löwe ·
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    ABSTRACT: Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.
    Nature 04/2015; 523(7558):1-5. DOI:10.1038/nature14356 · 41.46 Impact Factor
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    ABSTRACT: The scaffold protein p62/SQSTM1 is involved in protein turnover and signaling and is commonly found in dense protein bodies in eukaryotic cells. In autophagy, p62 acts as a selective autophagy receptor that recognizes and shuttles ubiquitinated proteins to the autophagosome for degradation. The structural organization of p62 in cellular bodies and the interplay of these assemblies with ubiquitin and the autophagic marker LC3 remain to be elucidated. Here, we present a cryo-EM structural analysis of p62. Together with structures of assemblies from the PB1 domain, we show that p62 is organized in flexible polymers with the PB1 domain constituting a helical scaffold. Filamentous p62 is capable of binding LC3 and addition of long ubiquitin chains induces disassembly and shortening of filaments. These studies explain how p62 assemblies provide a large molecular scaffold for the nascent autophagosome and reveal how they can bind ubiquitinated cargo. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 04/2015; 11(5). DOI:10.1016/j.celrep.2015.03.062 · 8.36 Impact Factor
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    ABSTRACT: Clathrin-mediated endocytosis, the main trafficking route from the plasma membrane to the cytoplasm, is critical to many fundamental cellular processes. Clathrin, coupled to the membrane by adaptor proteins, is thought to play a major structural role in endocytosis by self-assembling into a cage-like lattice around the forming vesicle. Although clathrin adaptors are essential for endocytosis, little is known about their structural role in this process. Here we show that the membrane-binding domains of two conserved clathrin adaptors, Sla2 and Ent1, co-assemble in a PI(4,5)P2-dependent manner to form organized lattices on membranes. We determined the structure of the co-assembled lattice by electron cryo-microscopy and designed mutations that specifically impair the lattice formation in vitro. We show that these mutations block endocytosis in vivo. We suggest that clathrin adaptors not only link the polymerized clathrin to the membrane but also form an oligomeric structure, which is essential for membrane remodeling during endocytosis. Copyright © 2015 Elsevier Inc. All rights reserved.
    Developmental Cell 04/2015; 33(2):150-162. DOI:10.1016/j.devcel.2015.02.023 · 9.71 Impact Factor
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    ABSTRACT: Measles is a highly contagious human disease. We used cryo-electron microscopy and single particle-based helical image analysis to determine the 4.3 Å resolution structure of the helical nucleocapsid formed by the folded domain of the Measles Virus nucleoprotein encapsidating an RNA. The resulting pseudoatomic model of the Measles Virus nucleocapsid offers important insights into the mechanism of the helical polymerisation of nucleocapsids of negative-strand RNA viruses, in particular via the exchange subdomains of the nucleoprotein.The structure reveals the mode of the nucleoprotein-RNA interaction and explains why each nucleoprotein of Measles Virus binds six nucleotides whereas the Respiratory Syncytial Virus nucleoprotein binds seven. It provides a rational basis for further analysis of Measles Virus replication and transcription, and reveals potential targets for drug design. Copyright © 2015, American Association for the Advancement of Science.
    Science 04/2015; 348(6235). DOI:10.1126/science.aaa5137 · 33.61 Impact Factor
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    ABSTRACT: Biotin-mediated carboxylation of short-chain fatty acid coenzyme A esters is a key step in lipid biosynthesis that is carried out by multienzyme complexes to extend fatty acids by one methylene group. Pathogenic mycobacteria have an unusually high redundancy of carboxyltransferase genes and biotin carboxylase genes, creating multiple combinations of protein/protein complexes of unknown overall composition and functional readout. By combining pull-down assays with mass spectrometry, we identified nine binary protein/protein interactions and four validated holo acyl-coenzyme A carboxylase complexes. We investigated one of these - the AccD1-AccA1 complex from Mycobacterium tuberculosis with hitherto unknown physiological function. Using genetics, metabolomics and biochemistry we found that this complex is involved in branched amino-acid catabolism with methylcrotonyl coenzyme A as the substrate. We then determined its overall architecture by electron microscopy and found it to be a four-layered dodecameric arrangement that matches the overall dimensions of a distantly related methylcrotonyl coenzyme A holo complex. Our data argue in favor of distinct structural requirements for biotin-mediated γ-carboxylation of α-β unsaturated acid esters and will advance the categorization of acyl-coenzyme A carboxylase complexes. Knowledge about the underlying structural/functional relationships will be crucial to make the target category amenable for future biomedical applications.
    PLoS Pathogens 02/2015; 11(2):e1004623. DOI:10.1371/journal.ppat.1004623 · 7.56 Impact Factor
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    ABSTRACT: With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require comparative characterization, we have used tobacco mosaic virus (TMV) as a test specimen to study the quality of 3D image reconstructions from data recorded on the two direct electron detector cameras, K2 Summit and Falcon II. Using DED movie frames, we explored related image-processing aspects and compared the performance of micrograph-based and segment-based motion correction approaches. In addition, we investigated the effect of dose deposition on the atomic-resolution structure of TMV and show that radiation damage affects negative carboxyl chains first in a side-chain specific manner. Finally, using 450,000 asymmetric units and limiting the effects of radiation damage, we determined a high-resolution cryo-EM map at 3.35 Å resolution. Here, we provide a comparative case study of highly ordered TMV recorded on different direct electron detectors to establish recording and processing conditions that enable structure determination up to 3.2 Å in resolution using cryo-EM.
    Journal of Structural Biology 12/2014; 189(2). DOI:10.1016/j.jsb.2014.12.002 · 3.23 Impact Factor
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    ABSTRACT: The structure and function of the γ-secretase proteases are of vast interest because of their critical roles in cellular and disease processes. We established a novel purification protocol for γ-secretase complex that involves a conformation and complex-specific nanobody, yielding highly pure and active enzyme. Using single particle electron microscopy, we analyzed the γ-secretase structure and its conformational variability. Under steady state conditions the complex adopts three major conformations, which are different in overall compactness and relative position of the nicastrin ectodomain. Occupancy of the active or substrate binding sites by inhibitors differentially stabilize sub-populations of particles with compact conformations, whereas a Familial Alzheimer Disease-linked mutation results in enrichment of extended-conformation complexes with increased flexibility. Our study presents the γ-secretase complex as a dynamic population of inter-converting conformations, involving rearrangements at the nanometer scale and high level of structural interdependence between subunits. The fact that protease inhibition or clinical mutations, which affect Aβ generation, enrich for particular subpopulations of conformers indicates the functional relevance of the observed dynamic changes, which are likely instrumental for highly allosteric behavior of the enzyme.
    Journal of Cell Science 12/2014; 128(3). DOI:10.1242/jcs.164384 · 5.43 Impact Factor
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    ABSTRACT: Mycobacteria have an unusual redundancy of six putative carboxyltransferase genes that form high-molecular weight holo acyl coenzyme A carboxylase complexes with a complementary set of three biotin carboxylase genes. Most of these enzyme complexes use small fatty acid coenzyme A esters as substrate, to allow their extension by one methylene group via a carboxybiotin-mediated α-carboxylation reaction. Redundant occurrence of these complexes was assumed to be related to highly complex enzymatic requirements in lipid biosynthesis, as the mycobacterial thick cell wall comprises unusual very long chain fatty acids, including mycolic acid. We have solved two high-resolution crystal structures of the 350 kDa hexameric assemblies of two different acyl coenzyme A carboxylase hexameric assemblies, AccD5 and AccD6 [1; Anandhakrishnan et al., unpublished], and characterized these enzyme complexes functionally. In a second step we investigated the acyl coenzyme A carboxylase complex AccD1-AccA1 from Mycobacteria tuberculosis with hitherto unknown function. By using a metabolomics approach we found that AccD1-AccA1 is involved in branched amino acid catabolism, which was not investigated in mycobacteria before [Ehebauer et al, unpublished. Using an in vitro assay, we show that the enzyme complex uses methylcrotonyl coenzyme A as substrate]. We determined the overall architecture of the 700 kDa AccD1-AccA1 complex to be formed from three layers of a central AccD1 hexameric ring, flanked by two distal tiers composed of three AccA1 subunits each. Our electron microscopy data match the overall dimensions of a methylcrotonyl coenzyme A holo complex with known structure and thus support our functional findings. Our data suggest a unique functional role of the AccD1-AccA1 complex within the Mycobacterium tuberculosis acyl coenzyme A carboxylase interactome. Ultimately, it is our goal to solve this and related structures of ACCase holo complexes by high-resolution crystallography as well. The abstract is dedicated to Louis Delbaere with whom I shared time during my PhD at the University of Basel, Switzerland.
    Acta Crystallographica Section A: Foundations and Advances 08/2014; 70(a1):C429-C429. DOI:10.1107/S2053273314095709
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    ABSTRACT: Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties.
    PLoS Pathogens 04/2014; 10(4):e1004091. DOI:10.1371/journal.ppat.1004091 · 7.56 Impact Factor
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    Ambroise Desfosses · Rodolfo Ciuffa · Irina Gutsche · Carsten Sachse ·
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    ABSTRACT: Helical reconstruction from electron cryomicrographs has become a routine technique for macromolecular structure determination of helical assemblies since the first days of Fourier-based three-dimensional image reconstruction. In the past decade, the single-particle technique has had an important impact on the advancement of helical reconstruction. Here, we present the software package SPRING that combines Fourier based symmetry analysis and real-space helical processing into a single workflow. One of the most time-consuming steps in helical reconstruction is the determination of the initial symmetry parameters. First, we propose a class-based helical reconstruction approach that enables the simultaneous exploration and evaluation of many symmetry combinations at low resolution. Second, multiple symmetry solutions can be further assessed and refined by single-particle based helical reconstruction using the correlation of simulated and experimental power spectra. Finally, the 3D structure can be determined to high resolution. In order to validate the procedure, we use the reference specimen Tobacco Mosaic Virus (TMV). After refinement of the helical symmetry, a total of 50,000 asymmetric units from two micrographs are sufficient to reconstruct a subnanometer 3D structure of TMV at 6.4 Åresolution. Furthermore, we introduce the individual programs of the software and discuss enhancements of the helical reconstruction workflow. Thanks to its user-friendly interface and documentation, SPRING can be utilized by the novice as well as the expert user. In addition to the study of well-ordered helical structures, the development of a streamlined workflow for single-particle based helical reconstruction opens new possibilities to analyze specimens that are heterogeneous in symmetries.
    Journal of Structural Biology 11/2013; 185(1). DOI:10.1016/j.jsb.2013.11.003 · 3.23 Impact Factor
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    ABSTRACT: Both subunits of αβ-tubulin that comprise the core components of microtubules bind GTP. GTP binding to α-tubulin has a structural role, whereas β-tubulin binds and hydrolyses GTP to regulate microtubule dynamics. γ-tubulin, another member of the tubulin superfamily that seeds microtubule nucleation at microtubule-organizing centres, also binds GTP; however, the importance of this association remains elusive. To address the role of GTP binding to γ-tubulin, we systematically mutagenized the GTP contact residues in the yeast γ-tubulin Tub4. Tub4(GTP)-mutant proteins that exhibited greatly reduced GTP affinity still assembled into the small γ-tubulin complex. However, tub4(GTP) mutants were no longer viable, and had defects in interaction between γ-tubulin and αβ-tubulin, decreased microtubule nucleation and defects in microtubule organization. In vitro and in vivo data show that only γ-tubulin loaded with GTP nucleates microtubules. Our results suggest that GTP recruitment to γ-tubulin enhances its interaction with αβ-tubulin similarly to GTP recruitment to β-tubulin.
    Nature Cell Biology 10/2013; 15(11). DOI:10.1038/ncb2863 · 19.68 Impact Factor
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    Harry H Low · Carsten Sachse · Linda A Amos · Jan Löwe ·

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    ABSTRACT: Centrioles and basal bodies are essential for the formation of cilia, flagella, and centrosomes. They exhibit a characteristic ninefold symmetry imparted by a cartwheel thought to contain rings of SAS-6 proteins. We used cryoelectron tomography to investigate the architecture of the exceptionally long cartwheel of the flagellate Trichonympha. We found that the cartwheel is a stack of central rings that exhibit a vertical periodicity of 8.5 nanometers and is able to accommodate nine SAS-6 homodimers. The spokes that emanate from two such rings associate into a layer, with a vertical periodicity of 17 nanometers on the cartwheel margin. Thus, by using the power of biodiversity, we unveiled the architecture of the cartwheel at the root of the ninefold symmetry of centrioles and basal bodies.
    Science 07/2012; 337(6094):553. DOI:10.1126/science.1222789 · 33.61 Impact Factor
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    ABSTRACT: The assembly of retroviruses such as HIV-1 is driven by oligomerization of their major structural protein, Gag. Gag is a multidomain polyprotein including three conserved folded domains: MA (matrix), CA (capsid) and NC (nucleocapsid). Assembly of an infectious virion proceeds in two stages. In the first stage, Gag oligomerization into a hexameric protein lattice leads to the formation of an incomplete, roughly spherical protein shell that buds through the plasma membrane of the infected cell to release an enveloped immature virus particle. In the second stage, cleavage of Gag by the viral protease leads to rearrangement of the particle interior, converting the non-infectious immature virus particle into a mature infectious virion. The immature Gag shell acts as the pivotal intermediate in assembly and is a potential target for anti-retroviral drugs both in inhibiting virus assembly and in disrupting virus maturation. However, detailed structural information on the immature Gag shell has not previously been available. For this reason it is unclear what protein conformations and interfaces mediate the interactions between domains and therefore the assembly of retrovirus particles, and what structural transitions are associated with retrovirus maturation. Here we solve the structure of the immature retroviral Gag shell from Mason-Pfizer monkey virus by combining cryo-electron microscopy and tomography. The 8-Å resolution structure permits the derivation of a pseudo-atomic model of CA in the immature retrovirus, which defines the protein interfaces mediating retrovirus assembly. We show that transition of an immature retrovirus into its mature infectious form involves marked rotations and translations of CA domains, that the roles of the amino-terminal and carboxy-terminal domains of CA in assembling the immature and mature hexameric lattices are exchanged, and that the CA interactions that stabilize the immature and mature viruses are almost completely distinct.
    Nature 06/2012; 487(7407):385-9. DOI:10.1038/nature11169 · 41.46 Impact Factor
  • A. Rohou · M. Schmidt · C. Sachse · M. Fändrich · N. Grigorieff ·

    Microscopy and Microanalysis 07/2010; 16:1018-1019. DOI:10.1017/S1431927610061532 · 1.88 Impact Factor
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    ABSTRACT: The 18 kDa TSPO protein is a polytopic mitochondrial outer membrane protein involved in a wide range of physiological functions and pathologies, including neurodegeneration and cancer. The pharmacology of TSPO has been extensively studied, but little is known about its biochemistry, oligomeric state, and structure. We have expressed, purified, and characterized a homologous protein, TspO from Rhodobacter sphaeroides, and reconstituted it as helical crystals. Using electron cryomicroscopy and single-particle helical reconstruction, we have determined a three-dimensional structure of TspO at 10 A resolution. The structure suggests that monomeric TspO comprises five transmembrane alpha helices that form a homodimer, which is consistent with the dimeric state observed in detergent solution. Furthermore, the arrangement of transmembrane domains of individual TspO subunits indicates a possibility of two substrate translocation pathways per dimer. The structure provides the first insight into the molecular architecture of TSPO/PBR protein family that will serve as a framework for future studies.
    Structure 06/2010; 18(6):677-87. DOI:10.1016/j.str.2010.03.001 · 5.62 Impact Factor
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    Carsten Sachse · Nikolaus Grigorieff · Marcus Fändrich ·
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    ABSTRACT: (Chemical Equation Presented) Versatile nanomaterial: Unusually high nanoscale flexibility was displayed by amyloid fibils in electron microscopy studies (see picture). This finding is relevant for understanding amyloid pathogenicity and for potential biotechnological applications.
    Angewandte Chemie International Edition 02/2010; 49(7):1321-3. DOI:10.1002/anie.200904781 · 11.26 Impact Factor
  • Carsten Sachse · Nikolaus Grigorieff · Marcus Fändrich ·

    Angewandte Chemie 02/2010; 122(7):1343 - 1345. DOI:10.1002/ange.200904781

Publication Stats

993 Citations
362.19 Total Impact Points


  • 2010-2015
    • European Molecular Biology Laboratory
      • Structural and Computational Biology Unit (Heidelberg)
      Heidelburg, Baden-Württemberg, Germany
  • 2008
    • Brandeis University
      Волтам, Massachusetts, United States
    • Martin Luther University Halle-Wittenberg
      Halle-on-the-Saale, Saxony-Anhalt, Germany
  • 2006-2008
    • Leibniz Institute for Age Research - Fritz Lipmann Institute
      Jena, Thuringia, Germany
  • 2007
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States