Wolfgang Baumeister

..We have successfully tested the experimental setup and the whole procedure with two types of adherent fluorescently labelled cells, a neuronal cell line and keratinocytes, both grown directly on EM grids...

..Cryo-electron tomography has proven to be a powerful technique for helping us understand the relationships between flagellar structure and function and the design of macromolecular machines in situ...

..In silico simulations of EM data collection and tomographic reconstruction identify the limits in revealing the filaments due to their length, concentration and orientation.PACS Codes: 87.64.Ee...

..This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete...

..Tomograms with molecular resolution are essentially images of the cellular proteome and, in conjunction with advanced pattern recognition techniques, they can be used to map the molecular landscape inside organelles and cells...

....

..This opens up exciting perspectives for visualizing the molecular organization of the cytoplasm...

..Moreover, cryo-EM can be combined in several ways with X-ray diffraction to enhance the resolution of cryo-EM and the applicability of crystallography. Electron tomography holds promise for visualizing machines at work inside cells...

..Analyses of the resulting hybrid structure of the HsTPPII holocomplex provide new insights into the mechanism of maturation and activation...

..We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram...

..The strands inherently heterogeneous in length are thus locked into a discrete oligomeric state. Our data indicate that the unique spindle form of the holo-complex represents an assembly motif stabilizing a highly active state...

..Tricorn protease appeared to act as the core of a proteolytic system; when it interacted with several smaller proteins, it displayed multicatalytic activities...

..The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP...

..Together with the C-terminal helices of the six PCI-domain subunits they form a very large coiled-coil bundle, which appears to serve as a flexible anchoring device for all the lid subunits...

..In addition, a variable mass near the mouth of the ATPase ring has been identified as Rpn10, a multiubiquitin receptor, by correlating the electron microscopy data with quantitative mass spectrometry...

..The binding of a peptide aldehyde inhibitor marks the active site in the central cavity at the amino termini of the beta subunits and suggests a novel proteolytic mechanism...

..On the basis of the mutual positions of Rpn10 and Rpn13, we propose a model for polyubiquitin binding to the 26S proteasome...

..In situ studies with intact E. coli cells allowed us to demonstrate that 100S ribosomes do exist in vivo and represent an easily reversible state of quiescence; they readily vanish when the growth medium is replenished...

..The application of this procedure to the 26S proteasome from Drosophila has allowed us to determine the three-dimensional structure of the complex to a resolution of 2.9 nm and the prospects for further improvements are good...

..On the basis of our structural data, we could model the motility modes of these cells and explain how helical Mollicutes can propel themselves by means of coordinated length changes of their cytoskeletal ribbons...

..This study represents a first step towards generating a more comprehensive cellular atlas of macromolecular complexes...

..In contrast, two neighboring residues in the D2 section of the pore had to be exchanged simultaneously, to achieve a drastic inhibition of GFP unfolding...

..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...

..Two-dimensional crystals grown on lipid monolayers yielded images of the ATPase molecules in "end-on" orientation at 1.9 nm resolution...

..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...

....

..The correlation method presented here can be expected to provide new insights into the structure-function relationship of supramolecular organization in neurons...

..At a resolution of 5 to 6 nanometers, single macromolecules with distinct shapes, such as the 26S proteasome, can be identified in an unperturbed cellular environment...

..An integrated model is presented which sheds light on the early steps of protein degradation by the 26S complex...

..However, the lacking alignment of the substrate by the double-Glu motif causes the endopeptidolytic K(cat)/K(M) of TPPII to be very low...

..We have performed a tomographic reconstruction, followed by averaging over approx. 150 individual reconstructions, of Drosophila 26S proteasomes suspended in a thin layer of amorphous ice...

..Therefore, it is quite likely that Tricorn also acts in vivo downstream of the proteasome and, in cooperation with its interacting factors, completes protein catabolic pathways...

..Sequence variations in the protrusion regions that are found in the eukaryotic TRiC/CCT subunits may provide different structural propensities and hence serve different roles in substrate recognition...

..The recombinant alpha-complex as well as the native thermosome could be reconstituted in vitro from their dissociated subunits in the presence of Mg-ATP...

..6 MDa. We have used electron cryomicroscopy to determine the structure of the Tricorn capsids to a resolution of 1.3 nm...

..The distinct neighbor orientations found in situ resemble configurations of bacterial polysomes in vitro, indicating a conserved supramolecular organization with implications for nascent polypeptide folding...

..Here, we summarize our current knowledge about TPPII with a focus on structural aspects. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome...

....

..Its performance is demonstrated with a single particle analysis case study and with a batch tomography application...

..Here, we will review these findings and discuss future directions towards the elucidation of the molecular landscape of the neuron...

..These data allowed us to compute an atomic model for the CP-AAA-ATPase subcomplex. In addition to this atomic model, further subunits can be mapped approximately, which lets us hypothesize on the substrate path during its degradation...

..Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners...

....

..This study thus provides 3D structural information regarding the prefusion conformation of an intact unstained retrovirus surface protein...

..Changes in the position of the CP/T were accompanied by structural rearrangements in the NPC scaffold...

..Microtubules from extracted sporozoites and Toxoplasma gondii tachyzoites showed a similar density distribution, suggesting that the putative protein is conserved among Apicomplexa and serves to stabilize microtubules...

..TOM represents a new way of working with the electron microscope and can serve as the basis for future high-throughput applications...

..These forces can be attributed to the translocation of substrate en route to the active sites that are harbored deep inside the proteasome...

..In some cases, ATP synthases could be identified without ambiguity. These findings confirm the feasibility of investigating the structural biology of mammalian cells in three dimensions and at a resolution of 6-8 nm...

..At the current resolution of approximately 4 nm, macromolecules in the size range of 0.5-1 MDa can be identified with good fidelity...

..Thus, this method appears to be well suited to study the conformational changes that occur during the functional cycle of the protein...

..The significant sequence similarity to the various subunits of eukaryotic proteasomes make it likely that proteasomal proteins are encoded by one gene family of ancient origin...

....

..We suggest that the tricorn capsid, in addition to its intrinsic proteolytic activity, serves as the organizing center of a multienzyme complex...

..Our data reveal an analogy between the ATPase cycles of the two groups of chaperonins and enable us to put forward a model of thermosome action...

..Although these proteins exhibit homology to known sequences, their structures, subunit compositions, and biological functions are not yet known...

..The application of cryo-ET to intact cells provides novel insights into the structure and the spatial organization of the cytoskeleton in prokaryotic and eukaryotic cells...

..The systematic and comprehensive interpretation of such tomograms will provide unprecedented insight into the molecular organization of cellular landscapes...

..We also introduce a novel cryo-planing procedure as a method that could facilitate thinning of large areas of vitreous ice prior to cryo-fluorescence, FIB thinning, and cryo-electron tomography...

..Application of our approach to cryo-electron tomographic data of ice-embedded thermosomes revealed distinct conformations that are in good agreement with results obtained by previous single particle studies...

..Reciprocal interactions of the N-terminal part of subunits from neighboring strands are probably involved in the formation of the native quaternary structure, lending the TPPII spindle a stability higher than that of single strands...

..Modelling of the intact TLP monomer suggests that this protease has an additional side entrance to its active site as in the DPP-IV or tricorn protease complexes...

..These striking structural similarities confirm the proposition that all these molecules belong to a single protein family which is structurally and functionally related to the GroEL class of molecular chaperones...

..We were able to demonstrate that structural information up to a resolution of 16.8Å can be recovered using our CTF correction approach, whereas the uncorrected 3D map had a resolution of only 26.2Å...

..Visual proteomics' aims to complement and extend mass-spectrometry-based inventories, and to provide a quantitative description of the macromolecular interactions that underlie cellular functions...

..An example of the latter-a 5-fold symmetric particle is-given. Second, electron cryotomography offers an incisive probe to examine crowding in different cellular compartments...

....

..Interestingly, immunoprecipitation analysis with TAA43 specific antibodies found a fraction of native TAA43 associated with Thermoplasma ribosomal proteins...

....

..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...

..Generally, this is the first study that employs whole cell cryo-ET to address structural details of pathogen-host cell interaction...

....

..This opens up exciting prospects for mapping the territorial distribution of macromolecules and for analyzing molecular interactions in situ...

..Experiments with inactive mutant PIPs indicate that the activities elicited by interacting with TRI are contributed by PIP...

..Actin filaments located on the outside usually associate with the vesicle membrane...

..The experiments further indicate that the assembly pathways of the Rhodococcus and of the Thermoplasma proteasome differ in some important details...

..Significant progress has been made during the past two years in elucidating the structural principles and the enzymatic mechanism of the 20S proteasome...

..These data suggest that other proteolytic systems may exist that can compensate for the loss of proteasome function in T. acidophilum...

....

..5 nm) three-dimensional maps of both the whole 500 kDa complex and the N-terminally truncated valosine-containing protein-like ATPase from T. acidophilum complex lacking the putative substrate binding domain...

..The same closure can be triggered by the crystallization buffer. Thus, the allosteric regulation of group II chaperonins appears different from that of their group I counterparts...

..We report also for the first time the in vitro assembly of chimeric proteasomes composed of Frankia and Rhodococcus erythropolis subunits, which are correctly assembled and proteolytically active...

..Close relationships to a number of other amino-terminal hydrolases have emerged, making the proteasomal subunits the prototype of this newly discovered structural superfamily...

..Interestingly, the putative NLS sequence found in T. acidophilum was also functional as a nuclear targeting sequence...

..The effect of Mg(2+) and K(+) nucleotide cycling is documented. We conclude that archaeal chaperonins have unique allosteric properties and discuss them in the light of the mechanism established for the group I chaperonins...

..Here, we show that the conformation of the native alphabeta-thermosome of Thermoplasma acidophilum in vitrified ice is strictly regulated by adenine nucleotides...

..Electron microscopy shows the purified protein to form single and double homo-hexameric rings. Although the symmetry is different, the appearance of the complexes formed of two rings resembles the 20S proteasome and Hsp60/GroEL...

....

..59. The RPL15 amino acid sequence shows significant similarity (> 35% identity) to the L15/YL10 proteins of various eukaryotes...

....

..Even at large ice thicknesses zero-loss filtering always gives better image contrast. Most probable loss imaging can only help where there is no intensity in the zero-loss image, at very large thicknesses (lambda > 8)...

..Its homomultimeric organisation differs from the typically dimeric structure of its eubacterial and eukaryotic counterparts...

..The method should facilitate higher-throughput single-particle analysis by substantially reducing the time needed for protein purification, as demonstrated for three complexes from Thermoplasma acidophilum...

..These data support the proposed assembly-dependent activation model in which the S2-S3 loop acts as an activation switch...

....

..Spacers were found distributed throughout the discs, including regions that are distant from the rim region of discs...

..This suggests an important role of the inter domain for the ordered assembly of the ARC ATPase...

..A low-resolution structure of the 26S proteasome has been obtained by electron microscopy, but the precise arrangement of subunits in the 19S complex is unclear...

..An accelerating voltage of 300 kV is advantageous, mainly for specimens thicker than 100 nm. Experimental investigations so far have resulted in a resolution that is worse by a factor of 2-5 as compared to theory...