Cryo-EM Structures of Eastern Equine Encephalitis Virus Reveal Mechanisms of Virus Disassembly and Antibody Neutralization

Alphaviruses are enveloped pathogens that cause arthritis and encephalitis. Here, we report a 4.4-Å cryoelectron microscopy (cryo-EM) structure of eastern equine encephalitis virus (EEEV), an alphavirus that causes fatal encephalitis in humans. Our analysis provides insights into viral entry into host cells. The envelope protein E2 showed a binding site for the cellular attachment factor heparan sulfate. The presence of a cryptic E2 glycan suggests how EEEV escapes surveillance by lectin-expressing myeloid lineage cells, which are sentinels of the immune system. A mechanism for nucleocapsid core release and disassembly upon viral entry was inferred based on pH changes and capsid dissociation from envelope proteins. The EEEV capsid structure showed a viral RNA genome binding site adjacent to a ribosome binding site for viral genome translation following genome release. Using five Fab-EEEV complexes derived from neutralizing antibodies, our investigation provides insights into EEEV host cell interactions and protective epitopes relevant to vaccine design

Characterisation of the Morphological and Surface Properties of Organic Micro-Crystalline Particles

The surface properties of single and agglomerated micro-crystals are characterised using the micro-focus X-ray beams available at a third generation synchrotron light source together with other laboratory facilities. The influence of the crystallisation environment, on the resultant product crystals is studied by both varying the cooling rates during crystallisation and through the addition of impurities and cross-correlated with the morphology and size changes. Unmodified urea crystallised in 99% ethanol produce needle-like crystals whilst addition of 4% of biuret in crystallisation of urea produce a more prismatic crystal shape. At faster cooling rates smaller sized crystals are produced and vice versa. Dispersive surface energy analysis using inverse gas chromatography (IGC) shows that unmodified urea has lower dispersive surface energy than urea modified by biuret. The dispersive surface energy also increases as the cooling rates increased. Both the morphology changes and surface energy measurements are validated using molecular modelling. The morphological prediction intermolecular force calculations of unmodified urea and urea modified by biuret are used to calculate a weighted value for the whole crystals’ dispersive surface energies. The results are in good agreement with experimental results from IGC. The sorption of urea crystals on water moisture showed that unmodified urea samples adsorbed water higher than urea modified by biuret by the observation of the percentage of mass change with respect to the relative humidity. The study of variability within powdered samples was found that no significant different in the unit cell parameters values of each single crystals. The orientation relationship between agglomerated micro-crystalline particles of aspirin showed the agglomerates tend to interact at the faces that have ability to form bonding. In urea samples, most of the agglomerates are mostly aligned due to epitaxy growth of the crystals. The XMT experiment also was carried out on agglomerated α-LGA and the 3-dimensional (3D) shape the samples were obtained

Structural insights into the basis and evolution of interactions in multi-subunit protein assemblies. tryptophan synthase and titin FNIII-repeats

Cellular processes benefit from evolutionary shaping when optimized protein-protein interactions result in enhanced functionality. In fact, most cellular proteins are tightly embedded into biological networks that function following a modularity principle. Modularity, whether based on components as parts of stable protein complexes or as dynamic units that interact only transiently (as in signalling and metabolic cascades), facilitates the combinatorial generation of complexity in protein networks through the re-wiring of modules in addition to the diversification of individual proteins – thereby increasing the “evolvability” of the system. The mechanisms that drive the emergence and evolution of molecular recognition in protein networks remain unclear. It is difficult to justify such evolution on the basis of organismic advantage, since the latter might only be noticeable once full pathways and cascades have evolved. It is then likely that the evolution of protein-protein interactions is in the first instance driven by a molecular principle of local advantage to the protein system itself - for example, molecular stability. Unfortunately, it is difficult to gain insights into the evolution of protein-protein interactions since the pathways of evolutionary shaping normally let intermediates of evolution disappear. Subsequently, conclusions are more usually drawn from the comparison of proteins between different species and by mutagenesis probing. In the current study, we aim at gaining an insight into the evolutionary shaping of proteins surfaces for hetero-complex formation by studying two systems at an early stage of development: Tryptophan Synthase B2b (TrpB2b) from S. solfataricus and the modular interfaces of the poly-FNIII tandems in the muscle filament titin. In the case of TrpB2b, the evolution of inter-subunit communication is addressed in addition. Both structures have been elucidated using X-ray crystallography and a comparative analysis of their surfaces has been carried out. The architectural elements subjected to evolutionary pressure have been identified and conclusions on their relation to function and evolution have been drawn

Transparentni nanokompozitni filmovi za primenu u plastičnoj elektronici

As solar cell technology gains more and more attraction every year, research keeps up with trends by an exponential number of published papers every year. These papers cover a wide range of topics regarding solar cells and the individual components of which they are comprised, which is the subject of this thesis. Different topics of research have been addressed in order to better understand and optimize individual solar cell components (layers) such as silver nanowires (AgNWs), AgNW based nanocomposite and Ag dendrites for the application as transparent electrodes, polymer nanocomposites (PNC) for the possible application as protective layers (encapsulants) as well as organic tandem solar cells as a whole device, utilizing AgNWs as the transparent electrode. The first part of the thesis concentrates on AgNWs and an AgNW based nanocomposite. These AgNWs were synthesized by a simple polyol reduction process. The main focus of this research was elucidating the solid-state wetting and subsequently welding mechanisms that occur during annealing of AgNWs before a layer of aluminum doped zinc oxide (AZO) is deposited on them, for the enhancement of properties essential for an electrode in a solar cell. Microstructural characterization using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that solid-state wetting and subsequent welding occurred only between nanowires whose contact geometry is characterized by an enormous difference in radii of curvature. Results also indicated that, for two AgNWs in contact, during annealing, the AgNW in contact through a smaller radius of curvature dissolves, Ag atoms diffuse and are incorporated in a welded zone between the AgNWs whose crystallographic orientation is inherent from the AgNW in contact through a large radius of curvature. Wetting angle between two welded AgNWs was measured to be below 4.8°, indicating almost complete wetting. Direct atomic column measurements were performed in order to elucidate crystal lattice distortion. Tomography was employed to better understand the morphology of the welded zone and confirmed the welding mechanism based on cross sectional transmission electron microscopy imaging. Electron diffraction orientation and strain mapping were performed in order to elucidate possible strain fluctuation across the AgNWs as well as confirm crystallographic orientation of the welded zone. Crystal lattice distortion, directly measured by atomic column displacements in drift corrected cross-sectional atomic resolution scanning transmission electron microscopy images of an AgNW, prepared by focused ion beam (FIB), demonstrated non-uniform distribution of strain in five twin segments of the nanowire...S obzirom na porast interesovanja za solarnim tehnologijama, istraživanja idu u korak sa takvim trendovima što se manifestuje eksponencijalnim rastom broja objavljenih naučnih radova svake godine. Ovi radovi pokrivaju širok spektar tema vezanih za solarne ćelije i individualne slojeve od kojih su one sačinjene, što je i predmet proučavanja ove teze. Istraživanja su se odvijala u nekoliko pravaca kako bi se bolje razumeli i optimizovali individualni slojevi (komponente) solarnih ćelija kao što su nano-žice srebra, nanokompoziti na bazi nano-žica srebra i dendriti srebra, za primenu kao transparentne elektrode, polimerni nanokompoziti za primenu kao zaštitni slojevi (enkapsulanati) kao i kompletne organske tandem solarne ćelije koje koriste nano-žice srebra kao transparentne elektrode. Prvi deo teze fokusiran je na nano-žice srebra i nanokompozite na bazi nanožica srebra. Nano-žice su sintetisane jednostavnom metodom poliol redukcije. Osnova ovog istraživanja bila je razumevanje mehanizama kvašenja u čvrstom stanju i zavarivanja do kojih dolazi prilikom zagrevanja nano-žica, pre nego što je sloj cink oksida dopiran aluminijumom nanešen radi poboljšanja svojstava, od suštinskog značaja za elektrodu u solarnoj ćeliji. Mikrostrukturna karakterizacija korišćenjem skenirajuće elektronske mikroskopije (SEM) i transmisione elektronske mikroskopije (TEM) otkrila je da se kvašenje i zavarivanje odvijaju samo između nano-žica čiji su poluprečnici zakrivljenja u tački dodira veoma različiti. Rezultati su takođe pokazali da, kod dve nano-žice u kontaktu, prilikom zagrevanja, atomi srebra iz područja nano-žice u kontaktu malog poluprečnika zakrivljenja difunduju i ugrađuju se u zonu zavarenog spoja, koja povezuje dve nano-žice. Kristalografska orijentacija zavarenog spoja nasleđena je od nano-žice u velikog poluprečnika zakrivljenja u tački dodira. Ugao kvašenja između dve nano-žice manji je od 4.8°, što je indikator potpunog kvašenja. Tomografija je korišćena kako bi se bolje razumela morfologija zavarenog spoja i potvrdio mehanizam zavarivanja, ustanovljen na osnovu rezultata transmisione elektronske mikroskopije poprečnih preseka nano-žica. Mapiranje orientacije i naprezanja elektroskom difrakcijom urađeno je kako bi se utvrdila raspodela naprezanja u nano-žicama i potvrdila kristalografska orijentacija zavarene zone. Distorzija kristalne rešetke direktno je merena mapiranjem pomeranja atomskih kolona na slikama skenirajuće transmisione elektronske mikroskopije poprečnih preseka nano-žica, pripremljenih fokusiranim jonskim snopom (FIB metodom). Ustanovljena je neravnomerna raspodela naprezanja u dvojnikovanim segmentima nano-žica..

MC 2019 Berlin Microscopy Conference - Abstracts

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2019", die vom 01. bis 05.09.2019, in Berlin stattfand

Structural Studies of Non-Enveloped Viruses Associated with Human Diseases

Non-enveloped viruses encompass many human pathogens which are responsible for a broad range of diseases that have very significant impacts on human health. By studying the structures of such viruses, insights can be gained into aspects of their lifecycle, including receptor attachment, genome uncoating and capsid assembly. This information can then serve as a structural platform for the design of targeted antivirals and vaccines. This thesis aimed to use cryo-electron microscopy (cryo-EM) to structurally characterise the structures, and receptor binding, of two important human pathogens, BK polyomavirus (BKV) and Coxsackievirus A24v (CV-A24v). BKV causes polyomavirus-associated nephropathy and haemorrhagic cystitis in immunosuppressed patients. These are diseases for which we currently have limited treatment options. Initially, a modest resolution structure (~7 Å) of BKV is used to investigate the organisation of the viral genome and minor capsid proteins. Subsequently, high-resolution structures of BKV alone (3.8 Å) and in complex with the receptor fragment of GT1b ganglioside (3.4 Å) and heparin (3.6 Å) were determined. Collectively, these structures provide insights into capsid assembly, rationalise how GT1b enhances infection over smaller gangliosides studied previously and provide the first structural clues for glycosaminoglycan binding to BKV. CV-A24v is responsible for large outbreaks of acute haemorrhagic conjunctivitis (AHC), a painful, contagious eye disease. Here, ICAM-1 is identified as an essential receptor for CV-A24v and the high-resolution cryo-EM structure (3.9 Å) of the virus–ICAM-1 complex is presented, which reveals the critical ICAM-1–binding residues within the capsid. These data could help identify a possible conserved mode of receptor engagement among ICAM-1–binding enteroviruses. In addition, structures of the uncoating intermediates of CV-A24v are presented which describe the molecular basis of capsid expansion. Furthermore, the molecular details of a branched pocket factor binding site in CV-A24v are described which is unique amongst currently structurally characterised human enteroviruses

The Transmission Electron Microscope

The book "The Transmission Electron Microscope" contains a collection of research articles submitted by engineers and scientists to present an overview of different aspects of TEM from the basic mechanisms and diagnosis to the latest advancements in the field. The book presents descriptions of electron microscopy, models for improved sample sizing and handling, new methods of image projection, and experimental methodologies for nanomaterials studies. The selection of chapters focuses on transmission electron microscopy used in material characterization, with special emphasis on both the theoretical and experimental aspect of modern electron microscopy techniques. I believe that a broad range of readers, such as students, scientists and engineers will benefit from this book

A combined EMR and DFT study of radiation-induced defects in sucrose and glucose 1-phosphate

Ionizing radiation induces radicals in organic matter, i.e. molecules with one or more unpaired electrons. This dissertation deals with a detailed study of radicals induced by X-ray radiation in solid sucrose (also known as common household sugar) and in the dipotassium salt of glucose 1-phosphate (K2G1P). The radiation chemistry of these materials is relevant in the context of radiation damage to DNA and (emergency) dosimetry. Next to the actual research results, which will be discussed below, the dissertation contains several, quite extensive chapters of a more general nature. The research strategy employed mainly comprises electron-magnetic-resonance (EMR) measurements (EPR, ENDOR, HYSCORE, …) and theoretical ab-initio calculations based on density functional theory (DFT). Essentially, radical structures are identified and scrutinized by comparing DFT-calculated and experimentally determined EMR parameters. These are the main results with respect to radiation-induced radicals in sucrose: (i) all major stable radiation-induced radicals are characterized and identified. Each of them requires formation of a carbonyl group and scission of the glycosidic bond. (ii) The transformation of the EPR spectrum during the first four hours after RT irradiation is shown to be due to the decay of several semistable species to diamagnetic products. (iii) Six (more) primary radicals (studied by in-situ irradiation at 10 K and subsequent EMR measurements at 10 K) were characterized and three of the four dominant radicals were identified as H-abstracted species. In K2G1P, the radicals present at 77 K after in-situ X-ray irradiation at 77 K were studied. Four radical species were characterized and identified. The dominant radical is chemically identical to one of the major radicals in sucrose and has a broken sugar-phosphate junction. Finally, it was shown, via first-order perturbation theory, that an ambiguity can arise in the determination of hyperfine tensors for low-symmetry paramagnetic centers with S=1/2 and I=1/2 – which can lead to erroneous radical identification. A firm theoretical basis is thus provided for a problem that was since long known to exist, but not always recognized or adequately dealt with in the literature

Tenth European Powder Diffraction Conference – Geneva, September 1-4, 2006

Zeitschrift für Kristallographie. Supplement Volume 26 presents the complete Proceedings of all contributions to the X European Powder Diffraction Conference in Geneva 2006: Method Development and Application, Instrumental Software Development, Materials Supplement Series of Zeitschrift für Kristallographie publishes Proceedings and Abstracts of international conferences on the interdisciplinary field of crystallography