Hierarchical self-assembly of nucleotide-appended oligopyrenotides into defined supramolecular objects

Supramolecular DNA assembly blends DNA building blocks with synthetic organic molecules giving structural and functional advantages. Incorporating extended aromatic molecules as connectors of DNA strands allows folding of these strands through π-π stacking (DNA 'foldamers'). In previous work it was shown that short oligopyrenotides behave as staircase-like foldamers, which cooperatively self-assemble into 2D supramolecular polymers in aqueous medium. Herein, we demonstrate that 10-mer DNA-sequence conjugated with seven pyrene unites forms dimensionally-defined supramolecular polymers under thermodynamic conditions in water. We present the self-assembly behavior, morphologycal studies (AFM and TEM), and the spectroscopic properties (UV/vis, CD) of the investigated pyrene - conjugated DNA-sequence.

Switching on the fluorescence of 2-aminopurine by site-selective microhydration

​2-Aminopurine (​2AP) is a fluorescent isomer of ​adenine and has a fluorescence lifetime of ~11 ns in water. It is widely used in biochemical settings as a site-specific fluorescent probe of DNA and RNA structure and base-flipping and -folding. These assays assume that ​2AP is intrinsically strongly fluorescent. Here, we show this not to be the case, observing that gas-phase, jet-cooled ​2-aminopurine and ​9-methyl-2-aminopurine have very short fluorescence lifetimes (156 ps and 210 ps, respectively); they are, to all intents and purposes, non-fluorescent. We find that the lifetime of ​2-aminopurine increases dramatically when it is part of a hydrate cluster, 2AP·(H2O)n, where n = 1–3. Not only does it depend on the presence of water molecules, it also depends on the specific hydrogen-bonding site to which they attach and on the number of H2O molecules at that site. We selectively microhydrate ​2-aminopurine at its sugar-edge, cis-amino or trans-amino sites and see that its fluorescence lifetime increases by 4, 50 and 95 times (to 14.5 ns), respectively.

Backscattered energetic neutral atoms from the Moon in the Earth's plasma sheet observed by Chandarayaan-1/Sub-keV Atom Reflecting Analyzer instrument

We present the observations of energetic neutral atoms (ENAs) produced at the lunar surface in the Earth's magnetotail. When the Moon was located in the terrestrial plasma sheet, Chandrayaan-1 Energetic Neutrals Analyzer (CENA) detected hydrogen ENAs from the Moon. Analysis of the data from CENA together with the Solar Wind Monitor (SWIM) onboard Chandrayaan-1 reveals the characteristic energy of the observed ENA energy spectrum (the e-folding energy of the distribution function) ∼100 eV and the ENA backscattering ratio (defined as the ratio of upward ENA flux to downward proton flux) <∼0.1. These characteristics are similar to those of the backscattered ENAs in the solar wind, suggesting that CENA detected plasma sheet particles backscattered as ENAs from the lunar surface. The observed ENA backscattering ratio in the plasma sheet exhibits no significant difference in the Southern Hemisphere, where a large and strong magnetized region exists, compared with that in the Northern Hemisphere. This is contrary to the CENA observations in the solar wind, when the backscattering ratio drops by ∼50% in the Southern Hemisphere. Our analysis and test particle simulations suggest that magnetic shielding of the lunar surface in the plasma sheet is less effective than in the solar wind due to the broad velocity distributions of the plasma sheet protons.

Relating orogen width to shortening, erosion, and exhumation during Alpine collision

We investigate along-strike width changes of the thickened, accreted lower plate (TALP) in the Central and in the Eastern Alps. We set the width of the TALP in relation to the inferred amount of collisional shortening and exhumation along six orogen-scale cross sections. Taking the present-day, along-strike gradients in the amount of collisional shortening to represent the temporal evolution of the collisional wedge, it may be concluded that the cross-sectional area of the TALP diminishes during ongoing shortening, indicating that the erosional flux outpaced the accretionary flux. Higher amounts of collisional shortening systematically coincide with smaller widths of the TALP and dramatic increases of the reconstructed eroded rock column. Higher amounts of shortening also coincide with larger amplitudes of orogen-scale, upright folds, with higher exhumation and with higher exhumation rates. Hence, erosion did play a major role in reducing by >30 km the vertical crustal thickness in order to accommodate and allow shortening by folding. Long-term climate differences cannot explain alternating changes of width by a factor of almost 2 along straight segments of the orogen on length scales less than 200 km, as observed from the western Central Alps to the easternmost Eastern Alps. Sedimentary or paleontological evidences supporting such paleo-climatic differences are lacking, suggesting that erosional processes did not directly control the width of the orogen.

Molecular Consequences of the SERPINH1/HSP47 Mutation in the Dachshund Natural Model of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a heritable connective tissue disease characterized by bone fragility and increased risk of fractures. Up to now, mutations in at least 18 genes have been associated with dominant and recessive forms of OI that affect the production or post-translational processing of procollagen or alter bone homeostasis. Among those, SERPINH1 encoding heat shock protein 47 (HSP47), a chaperone exclusive for collagen folding in the ER, was identified to cause a severe form of OI in dachshunds (L326P) as well as in humans (one single case with a L78P mutation). To elucidate the disease mechanism underlying OI in the dog model, we applied a range of biochemical assays to mutant and control skin fibroblasts as well as on bone samples. These experiments revealed that type I collagen synthesized by mutant cells had decreased electrophoretic mobility. Procollagen was retained intracellularly with concomitant dilation of ER cisternae and activation of the ER stress response markers GRP78 and phospho-eIF2α, thus suggesting a defect in procollagen processing. In line with the migration shift detected on SDS-PAGE of cell culture collagen, extracts of bone collagen from the OI dog showed a similar mobility shift, and on tandem mass spectrometry, the chains were post-translationally overmodified. The bone collagen had a higher content of pyridinoline than control dog bone. We conclude that the SERPINH1 mutation in this naturally occurring model of OI impairs how HSP47 acts as a chaperone in the ER. This results in abnormal post-translational modification and cross-linking of the bone collagen.

Insights into post-translational processing of beta-galactosidase in an animal model resembling late infantile human G-gangliosidosis

G(M1)-gangliosidosis is a lysosomal storage disorder caused by a deficiency of ss-galactosidase activity. Human GM1-gangliosidosis has been classified into three forms according to the age of clinical onset and specific biochemical parameters. In the present study, a canine model for type II late infantile human GM1-gangliosidosis was investigated 'in vitro' in detail. For a better understanding of the molecular pathogenesis underlying G(M1)-gangliosidosis the study focused on the analysis of the molecular events and subsequent intracellular protein trafficking of beta-galactosidase. In the canine model the genetic defect results in exclusion or inclusion of exon 15 in the mRNA transcripts and to translation of two mutant precursor proteins. Intracellular localization, processing and enzymatic activity of these mutant proteins were investigated. The obtained results suggested that the beta-galactosidase C-terminus encoded by exons 15 and 16 is necessary for correct C-terminal proteolytic processing and enzyme activity but does not affect the correct routing to the lysosomes. Both mutant protein precursors are enzymatically inactive, but are transported to the lysosomes clearly indicating that the amino acid sequences encoded by exons 15 and 16 are necessary for correct folding and association with protective protein/cathepsin A, whereas the routing to the lysosomes is not influenced. Thus, the investigated canine model is an appropriate animal model for the human late infantile form and represents a versatile system to test gene therapeutic approaches for human and canine G(M1)-gangliosidosis.

The tectonometamorphic evolution of the Sesia–Dent Blanche nappes (internal Western Alps): review and synthesis

This study reviews and synthesizes the present knowledge on the Sesia–Dent Blanche nappes, the highest tectonic elements in the Western Alps (Switzerland and Italy), which comprise pieces of pre-Alpine basement and Mesozoic cover. All of the available data are integrated in a crustal-scale kinematic model with the aim to reconstruct the Alpine tectono-metamorphic evolution of the Sesia–Dent Blanche nappes. Although major uncertainties remain in the pre-Alpine geometry, the basement and cover sequences of the Sesia–Dent Blanche nappes are seen as part of a thinned continental crust derived from the Adriatic margin. The earliest stages of the Alpine evolution are interpreted as recording late Cretaceous subduction of the Adria-derived Sesia–Dent Blanche nappes below the South-Alpine domain. During this subduction, several sheets of crustal material were stacked and separated by shear zones that rework remnants of their Mesozoic cover. The recently described Roisan-Cignana Shear Zone of the Dent Blanche Tectonic System represents such a shear zone, indicating that the Sesia–Dent Blanche nappes represent a stack of several individual nappes. During the subsequent subduction of the Piemonte–Liguria Ocean large-scale folding of the nappe stack (including the Roisan-Cignana Shear Zone) took place under greenschist facies conditions, which indicates partial exhumation of the Dent Blanche Tectonic System. The entrance of the Briançonnais micro-continent within the subduction zone led to a drastic change in the deformation pattern of the Alpine belt, with rapid exhumation of the eclogite-facies ophiolite bearing units and thrust propagation towards the foreland. Slab breakoff probably was responsible for allowing partial melting in the mantle and Oligocene intrusions into the most internal parts of the Sesia–Dent Blanche nappes. Finally, indentation of the Adriatic plate into the orogenic wedge resulted in the formation of the Vanzone back-fold, which marks the end of the pervasive ductile deformation within the Sesia–Dent Blanche nappes during the earliest Miocene.

Geometry and kinematics of the Roisan-Cignana Shear Zone, and the orogenic evolution of the Dent Blanche Tectonic System (Western Alps)

The Dent Blanche Tectonic System (DBTS) is a composite thrust sheet derived from the previously thinned passive Adriatic continental margin. A kilometric high-strain zone, the Roisan-Cignana Shear Zone (RCSZ) defines the major tectonic boundary within the DBTS and separates it into two subunits, the Dent Blanche s.s. nappe to the northwest and the Mont Mary nappe to the southeast. Within this shear zone, tectonic slices of Mesozoic and pre-Alpine meta-sediments became amalgamated with continental basement rocks of the Adriatic margin. The occurrence of high pressure assemblages along the contact between these tectonic slices indicates that the amalgamation occurred prior to or during the subduction process, at an early stage of the Alpine orogenic cycle. Detailed mapping, petrographic and structural analysis show that the Roisan-Cignana Shear Zone results from several superimposed Alpine structural and metamorphic stages. Subduction of the continental fragments is recorded by blueschist-facies deformation, whereas the Alpine collision is reflected by a greenschist facies overprint associated with the development of large-scale open folds. The postnappe evolution comprises the development of low-angle brittle faults, followed by large-scale folding (Vanzone phase) and finally brittle extensional faults. The RCSZ shows that fragments of continental crust had been torn off the passive continental margin prior to continental collision, thus recording the entire history of the orogenic cycle. The role of preceding Permo-Triassic lithospheric thinning, Jurassic rifting, and ablative subduction processes in controlling the removal of crustal fragments from the reactivated passive continental margin is discussed. Results of this study constrain the temporal sequence of the tectono-metamorphic processes involved in the assembly of the DBTS, but they also show limits on the interpretation. In particular it remains difficult to judge to what extent precollisional rifting at the Adriatic continental margin preconditioned the efficiency of convergent processes, i.e. accretion, subduction, and orogenic exhumation.

Modeling the Histidine–Phenylalanine Interaction: The NH···π Hydrogen Bond of Imidazole·Benzene

NH···π hydrogen bonds occur frequently between the amino acid side groups in proteins and peptides. Data-mining studies of protein crystals find that ~80% of the T-shaped histidine···aromatic contacts are CH···π, and only ~20% are NH···π interactions. We investigated the infrared (IR) and ultraviolet (UV) spectra of the supersonic-jet-cooled imidazole·benzene (Im·Bz) complex as a model for the NH···π interaction between histidine and phenylalanine. Ground- and excited-state dispersion-corrected density functional calculations and correlated methods (SCS-MP2 and SCS-CC2) predict that Im·Bz has a Cs-symmetric T-shaped minimum-energy structure with an NH···π hydrogen bond to the Bz ring; the NH bond is tilted 12° away from the Bz C₆ axis. IR depletion spectra support the T-shaped geometry: The NH stretch vibrational fundamental is red shifted by −73 cm⁻¹ relative to that of bare imidazole at 3518 cm⁻¹, indicating a moderately strong NH···π interaction. While the Sₒ(A1g) → S₁(B₂u) origin of benzene at 38 086 cm⁻¹ is forbidden in the gas phase, Im·Bz exhibits a moderately intense Sₒ → S₁ origin, which appears via the D₆h → Cs symmetry lowering of Bz by its interaction with imidazole. The NH···π ground-state hydrogen bond is strong, De=22.7 kJ/mol (1899 cm⁻¹). The combination of gas-phase UV and IR spectra confirms the theoretical predictions that the optimum Im·Bz geometry is T shaped and NH···π hydrogen bonded. We find no experimental evidence for a CH···π hydrogen-bonded ground-state isomer of Im·Bz. The optimum NH···π geometry of the Im·Bz complex is very different from the majority of the histidine·aromatic contact geometries found in protein database analyses, implying that the CH···π contacts observed in these searches do not arise from favorable binding interactions but merely from protein side-chain folding and crystal-packing constraints. The UV and IR spectra of the imidazole·(benzene)₂ cluster are observed via fragmentation into the Im·Bz+ mass channel. The spectra of Im·Bz and Im·Bz₂ are cleanly separable by IR hole burning. The UV spectrum of Im·Bz₂ exhibits two 000 bands corresponding to the Sₒ → S₁ excitations of the two inequivalent benzenes, which are symmetrically shifted by −86/+88 cm⁻¹ relative to the 000 band of benzene.

Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines

Self-amplifying replicon RNA (RepRNA) are large molecules (12-14kb); their self-replication amplifies mRNA template numbers, affording several rounds of antigen production, effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) - absolute requirements for vaccine design - were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA molecules cannot be transferred to RepRNA due to its greater size and complexity; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines.

Single-molecule force spectroscopy of membrane proteins from membranes freely spanning across nanoscopic pores.

Single-molecule force spectroscopy (SMFS) provides detailed insight into the mechanical (un)folding pathways and structural stability of membrane proteins. So far, SMFS could only be applied to membrane proteins embedded in native or synthetic membranes adsorbed to solid supports. This adsorption causes experimental limitations and raises the question to what extent the support influences the results obtained by SMFS. Therefore, we introduce here SMFS from native purple membrane freely spanning across nanopores. We show that correct analysis of the SMFS data requires extending the worm-like chain model, which describes the mechanical stretching of a polypeptide, by the cubic extension model, which describes the bending of a purple membrane exposed to mechanical stress. This new experimental and theoretical approach allows to characterize the stepwise (un)folding of the membrane protein bacteriorhodopsin and to assign the stability of single and grouped secondary structures. The (un)folding and stability of bacteriorhodopsin shows no significant difference between freely spanning and directly supported purple membranes. Importantly, the novel experimental SMFS setup opens an avenue to characterize any protein from freely spanning cellular or synthetic membranes.

Functional interplay of SP family members and nuclear factor Y is essential for transcriptional activation of the human Calreticulin gene

Calreticulin (CALR) is a highly conserved, multifunctional protein involved in a variety of cellular processes including the maintenance of intracellular calcium homeostasis, proper protein folding, differentiation and immunogenic cell death. More recently, a crucial role for CALR in the pathogenesis of certain hematologic malignancies was discovered: in clinical subgroups of acute myeloid leukemia, CALR overexpression mediates a block in differentiation, while somatic mutations have been found in the majority of patients with myeloproliferative neoplasms with nonmutated Janus kinase 2 gene (JAK2) or thrombopoietin receptor gene (MPL). However, the mechanisms underlying CALR promoter activation have insufficiently been investigated so far. By dissecting the core promoter region, we could identify a functional TATA-box relevant for transcriptional activation. In addition, we characterized two evolutionary highly conserved cis-regulatory modules (CRMs) within the proximal promoter each composed of one binding site for the transcription factors SP1 and SP3 as well as for the nuclear transcription factor Y (NFY) and we verified binding of these factors to their cognate sites in vitro and in vivo.

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.

Modeling Techniques for the High-Resolution Interpretation of Cryo-Electron Microscopy Reconstructions

Essential biological processes are governed by organized, dynamic interactions between multiple biomolecular systems. Complexes are thus formed to enable the biological function and get dissembled as the process is completed. Examples of such processes include the translation of the messenger RNA into protein by the ribosome, the folding of proteins by chaperonins or the entry of viruses in host cells. Understanding these fundamental processes by characterizing the molecular mechanisms that enable then, would allow the (better) design of therapies and drugs. Such molecular mechanisms may be revealed trough the structural elucidation of the biomolecular assemblies at the core of these processes. Various experimental techniques may be applied to investigate the molecular architecture of biomolecular assemblies. High-resolution techniques, such as X-ray crystallography, may solve the atomic structure of the system, but are typically constrained to biomolecules of reduced flexibility and dimensions. In particular, X-ray crystallography requires the sample to form a three dimensional (3D) crystal lattice which is technically di‑cult, if not impossible, to obtain, especially for large, dynamic systems. Often these techniques solve the structure of the different constituent components within the assembly, but encounter difficulties when investigating the entire system. On the other hand, imaging techniques, such as cryo-electron microscopy (cryo-EM), are able to depict large systems in near-native environment, without requiring the formation of crystals. The structures solved by cryo-EM cover a wide range of resolutions, from very low level of detail where only the overall shape of the system is visible, to high-resolution that approach, but not yet reach, atomic level of detail. In this dissertation, several modeling methods are introduced to either integrate cryo-EM datasets with structural data from X-ray crystallography, or to directly interpret the cryo-EM reconstruction. Such computational techniques were developed with the goal of creating an atomic model for the cryo-EM data. The low-resolution reconstructions lack the level of detail to permit a direct atomic interpretation, i.e. one cannot reliably locate the atoms or amino-acid residues within the structure obtained by cryo-EM. Thereby one needs to consider additional information, for example, structural data from other sources such as X-ray crystallography, in order to enable such a high-resolution interpretation. Modeling techniques are thus developed to integrate the structural data from the different biophysical sources, examples including the work described in the manuscript I and II of this dissertation. At intermediate and high-resolution, cryo-EM reconstructions depict consistent 3D folds such as tubular features which in general correspond to alpha-helices. Such features can be annotated and later on used to build the atomic model of the system, see manuscript III as alternative. Three manuscripts are presented as part of the PhD dissertation, each introducing a computational technique that facilitates the interpretation of cryo-EM reconstructions. The first manuscript is an application paper that describes a heuristics to generate the atomic model for the protein envelope of the Rift Valley fever virus. The second manuscript introduces the evolutionary tabu search strategies to enable the integration of multiple component atomic structures with the cryo-EM map of their assembly. Finally, the third manuscript develops further the latter technique and apply it to annotate consistent 3D patterns in intermediate-resolution cryo-EM reconstructions. The first manuscript, titled An assembly model for Rift Valley fever virus, was submitted for publication in the Journal of Molecular Biology. The cryo-EM structure of the Rift Valley fever virus was previously solved at 27Å-resolution by Dr. Freiberg and collaborators. Such reconstruction shows the overall shape of the virus envelope, yet the reduced level of detail prevents the direct atomic interpretation. High-resolution structures are not yet available for the entire virus nor for the two different component glycoproteins that form its envelope. However, homology models may be generated for these glycoproteins based on similar structures that are available at atomic resolutions. The manuscript presents the steps required to identify an atomic model of the entire virus envelope, based on the low-resolution cryo-EM map of the envelope and the homology models of the two glycoproteins. Starting with the results of the exhaustive search to place the two glycoproteins, the model is built iterative by running multiple multi-body refinements to hierarchically generate models for the different regions of the envelope. The generated atomic model is supported by prior knowledge regarding virus biology and contains valuable information about the molecular architecture of the system. It provides the basis for further investigations seeking to reveal different processes in which the virus is involved such as assembly or fusion. The second manuscript was recently published in the of Journal of Structural Biology (doi:10.1016/j.jsb.2009.12.028) under the title Evolutionary tabu search strategies for the simultaneous registration of multiple atomic structures in cryo-EM reconstructions. This manuscript introduces the evolutionary tabu search strategies applied to enable a multi-body registration. This technique is a hybrid approach that combines a genetic algorithm with a tabu search strategy to promote the proper exploration of the high-dimensional search space. Similar to the Rift Valley fever virus, it is common that the structure of a large multi-component assembly is available at low-resolution from cryo-EM, while high-resolution structures are solved for the different components but lack for the entire system. Evolutionary tabu search strategies enable the building of an atomic model for the entire system by considering simultaneously the different components. Such registration indirectly introduces spatial constrains as all components need to be placed within the assembly, enabling the proper docked in the low-resolution map of the entire assembly. Along with the method description, the manuscript covers the validation, presenting the benefit of the technique in both synthetic and experimental test cases. Such approach successfully docked multiple components up to resolutions of 40Å. The third manuscript is entitled Evolutionary Bidirectional Expansion for the Annotation of Alpha Helices in Electron Cryo-Microscopy Reconstructions and was submitted for publication in the Journal of Structural Biology. The modeling approach described in this manuscript applies the evolutionary tabu search strategies in combination with the bidirectional expansion to annotate secondary structure elements in intermediate resolution cryo-EM reconstructions. In particular, secondary structure elements such as alpha helices show consistent patterns in cryo-EM data, and are visible as rod-like patterns of high density. The evolutionary tabu search strategy is applied to identify the placement of the different alpha helices, while the bidirectional expansion characterizes their length and curvature. The manuscript presents the validation of the approach at resolutions ranging between 6 and 14Å, a level of detail where alpha helices are visible. Up to resolution of 12 Å, the method measures sensitivities between 70-100% as estimated in experimental test cases, i.e. 70-100% of the alpha-helices were correctly predicted in an automatic manner in the experimental data. The three manuscripts presented in this PhD dissertation cover different computation methods for the integration and interpretation of cryo-EM reconstructions. The methods were developed in the molecular modeling software Sculptor (http://sculptor.biomachina.org) and are available for the scientific community interested in the multi-resolution modeling of cryo-EM data. The work spans a wide range of resolution covering multi-body refinement and registration at low-resolution along with annotation of consistent patterns at high-resolution. Such methods are essential for the modeling of cryo-EM data, and may be applied in other fields where similar spatial problems are encountered, such as medical imaging.

Gene clustering of the small leucine -rich repeat gene family

The small leucine-rich repeat proteoglycans (or SLRPs) are a group of extracellular proteins (ECM) that belong to the leucine-rich repeat (LRR) superfamily of proteins. The LRR is a protein folding motif composed of 20–30 amino acids with leucines in conserved positions. LRR-containing proteins are present in a broad spectrum of organisms and possess diverse cellular functions and localization. In mammals, the SLRPs are abundant in connective tissues, such as bones, cartilage, tendons, skin, and blood vessels. We have discovered a new member of the class I small leucine rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N-terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression of asporin partially overlaps with the expression of decorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22-9g21.3 where asporin is part of a SLRP gene cluster that includes ECM2, osteoadherin, and osteoglycin. This gene cluster of four LRR-encoding genes is embedded in a 238 kilobase intron of another novel gene named Tes9orf that is expressed primarily in the testes of the adult mouse. The SLRP genes are not present in Drosophila or C. elegans , but reside in three separate gene clusters in the puffer fish, mice and humans. Targeted disruption of individual mouse SLRP genes display minor connective tissue defects such as skin fragility, tendon laxity, minor growth plate defects, and mild osteoporosis. However, double and triple knockouts of SLRP genes exacerbate these phenotypes. Both the double epiphycan/biglycan and the triple PRELP/fibromodulin/biglycan knockout mice exhibit premature osteoarthritis. ^