TRIM21 is a cytosolic ubiquitin ligase and an antibody receptor that providesa last line of defense against invading pathogens. By utilizing the diversity ofantibody repertoire to identify pathogens, TRIM21 serves as a link betweenintrinsic cellular defense and adaptive immunity. A variety of diseases havebeen linked to mutations of the TRIM family, including cancer, inflammatorydiseases, and autoimmune diseases. In this project, TRIM21 was producedand purified from Escherichia coli, (E.coli). Protein characterization wasperformed with SDS-PAGE, size exclusion chromatography and cryo-electronmicroscopy (cryo-EM). Previously TRIM21 has been shown to form a dimerwhen produced in SF9. Results from size exclusion chromatography show thatTRIM21 form a larger complex when expressed in E.coli. Cryo-EM resultsshow that the complex structure is more globular than previously thought.Purified TRIM21 was bound to the antibody IC100. SDS-PAGE and sizeexclusion chromatography results show much lower affinity to antibodies thanexpected. / TRIM21 är en cytosolisk ubiquitinligas- och antikroppsreceptor som ger ensista försvarslinje mot invaderande virus. Genom att använda mångfalden avantikroppsrepertoar för att identifiera patogener, fungerar TRIM21 som enlänk mellan inre cellulärt försvar och adaptiv immunitet. En mängd olikasjukdomar har kopplats till mutationer i TRIM-familjen, inklusive cancer,inflammatoriska sjukdomar och autoimmuna sjukdomar. I detta projekt produceradesoch renades TRIM21 från Escherichia coli, (E.coli). Proteinkarakteriseringutfördes med SDS-PAGE, gelfiltreringskromatografi och kryo-elektronmikroskopi(cryo-EM). Tidigare har TRIM21 visat sig bilda en dimer när den producerasi SF9. Resultat från gelfiltrering visar att TRIM21 bildar ett större komplexnär det uttrycks i E.coli. Cryo-EM-resultat visar att den komplexa strukturenär mer klotformig än man tidigare trott. Renad TRIM21 bands till antikroppenIC100. SDS-PAGE och gel-filtrerings resultat visar mycket lägre affinitet tillantikroppar än förväntat.

Nanomedicine is one of the most promising fields in nanotechnology nowadays. The use of nanoparticles as carriers aims to improve efficiency of drugs that possess low solubility in aqueous environment (very hydrophobic molecules) or that have a lot of undesired side effects. In this way, nanoparticles offer both a protection for the molecules and a carrying vehicle. On this ground, cubosomes are nanoparticles capable of storing both hydrophilic and hydrophobic molecules within its structure, in addition, cubosomes have approximately 50% hydrophilic and hydrophobic areas. Therefore, they can carry much more molecules than liposomes for instance. In particular, cubosomes are quite easy to produce due to its base product, lipids (like monoolein (GMO) or phytantriol (PHY)) that self-assembly in water media. In this project, both lipids were chosen to produce the cubosomes from well-established protocols in literature. A model drug, miletofsine (MILT), was chosen to study the interaction of such nanosystem with a guest molecule. GMO cubosomes revealed to have Im3m crystallographic symmetry and lattice parameter 15.3(7) nm, particles presented sizes 300(8) nm and moderate polydispersion 0.160(20). TEM revealed squared particles with sizes ~350 nm, cryo-EM presented particles with internal structure and varied size (from 200 to > 500 nm). From FFT analysis, the calculated lattice parameter remained in the order of ~10 nm compatible with SAXS measurements. MILT loading into cubosomes was possible up to 4% w/w without loss of cubosomes structure. For 5% w/w MILT, the nanoparticles were already loosing their crystalline structure, as evidenced by cryo-EM. TEM analysis reveals that as more MILT is loaded into the cubosomes, their sizes increased. For sample 1.5% w/w MILT cryo-EM presents nanoparticles with organized internal structure and an envelope (hypothesized to be a polymer coating) in its surface. Calculated lattice parameters are in the order of ~10 nm. Myverol (Myv) is a commercial mixture that contains ~60% GMO, in this project it was proposed a bottom up protocol for Myv-based cubosomes. The production of these nanoparticles also revealed, by SAXS, Im3m symmetry and lattice parameter 12.30(12) nm. DLS revealed particle size 280(5) nm and moderate polydispersion 0.115(52). TEM shows square and cubic nanoparticles with sizes ~500 nm. MILT loading into Myv-cubosomes revealed that the drug interacts with the nanoparticle by enlarging their lattice parameter as more MILT is loaded (up to 4% w/w). Curiously, for some MILT concentrations the presence of other unknown cubic structures was evidenced by SAXS. TEM revealed nanoparticles with huge polydispersion, with sizes raging from 200 nm to 2 µm. PHY based cubosomes were successfully reproduced by the chosen protocol, in both water, PBS buffer and 2.25% glycerol medium. SAXS revealed crystallographic structure Pn3m and lattice parameter 6.74(04) nm. DLS measured sizes ~450 nm and moderate polydispersion 0.161(10). NTA measurements were consistent with DLS, revealing a broad size distribution and total particle concentration of ~1016 particles/mL for each sample. TEM revealed square and rounder particles in varied size. Cryo-EM micrographs presented particles with internal structure and varied size confirming moderate polydispersion. The FFT analysis revealed calculated lattice parameters ~6.5 nm, compatible with SAXS data. Samples were submitted to lyophilization and found that after re-hydration they still hold the same characteristics (morphology, size) as the original sample. Extrusion was also performed in order to improve polydispersion and control particle size, again cubosomes held their internal structure after the process, diminishing their sizes and improving monodispersion. MILT was loaded into cubosomes via co-solubilization and addition after the nanoparticles were formed. Up to 5% w/w the cubosomes incorporated MILT without loss of crystallographic structure, but at 10%, 15% and 20% w/w, the drug provoked phase change for Im3m symmetry. At the lower concentrations, MILT enlarged the lattice parameter of cubosomes and it was hypothesized that MILT inserted itself into the bilayer of the nanoparticles. DLS reveales that the drug does not change particle size or polydispersion. TEM revealed square and rounder particles in sizes slightly bigger than DLS. For sample 4% w/w, Cryo-EM presented particles with internal structure and calculated lattice parameter ~7 nm compatible with SAXS measurements for this sample. Co-solubilization and addition after nanoparticle preparation proved out to have the same effect on cubosomes loaded with MILT. All samples were submitted to higher temperatures to investigate phase change, based on phase diagram of the lipid. It was found that for the blank samples at 65 °C the cubosomes suffer phase change for isotropic phase L2, when MILT is loaded into the nanoparticles this phase change does not happen. DLS revealed also that at higher temperatures, particle size does not change, neither polydispersion. Finally, cubosomes proved to be remarkable nanoparticles that hold their physico-chemical characteristics even when submitted to extreme environments (lyophylization, extrusion and higher temperatures.) / Nanomedicina é o campo de estudo mais promissor dentro da nanotecnologia atualmente. O uso de nanopartículas visa melhorar a eficiência de fármacos que possuem baixa solubilidade em meios aquosos (moléculas muito hidrofóbicas) ou que possuem muitos efeitos colaterais indesejados. Neste contexto, as nanopartículas oferecem proteção e veículo para tais moléculas. Para isso, cubossomos são nanopartículas capazes de encapsular tanto as moléculas hidrofóbicas como as hidrofílicas em sua estrutura. Cubossomos também apresentam aproximadamente 50% de áreas hidrofílica e hidrofóbica, sendo capaz de encapsular grandes quantidades de moléculas teóricamente. Particularmente, cubossomos são nanopartículas de fácil produção devido à sua matéria prima serem lipídios (por exemplo, monoleína (GMO) ou fitantriol (PHY)) que se auto associam em meio aquoso. Neste projeto, os dois lipídios citados foram escolhidos para a produção dos cubossomos empregando-se protocolos bem estabelecidos da literatura. Uma fármaco modelo, miltefosina (MILT), foi escolhida para o estudo da interação com as nanopartículas. Cubossomos de monoleína (GMO) revelaram simetria cristalográfica Im3m e parâmetro de rede de 15.3(7) nm, as nanopartículas apresentaram tamanhos em torno de 300(8) nm e PDI 0.160(20). MET revelou partículas quadradas com tamanhos ~350 nm e a crio microscopia mostrou partículas com estrutura interna bem definida e tamanhos variados (200 a 500 nm), os parâmetros de rede calculados se mostraram da ordem de ~10 nm, compatíveis com as medidas de SAXS. O encapsulamento da MILT nos cubossomos foi possível até 4% w/w sem perda de morfologia. Para 5% w/w MILT as nanopartículas já apresentavam perda de cristalinidade na sua estrutura, evidenciado por crio microscopia. Análise por MET revelou que quanto mais MILT era encapsulada nos cubossomos, maiores ficaram as nanopartículas. Com a amostra de 1.5% w/w foi feita a crio microscopia, que revelou cubossomos com estrutura interna bem definida e um envelope (possivelmente formado pelo polímero) na sua superfície. Os parâmetros de rede calculados foram da ordem de ~10 nm também. O myverol (Myv) é uma mistura comercial que contém aproximadamente 60% de GMO, e neste projeto foi proposto um protocolo bottom up para cubossomos feitos de Myv. A produção dessas nanopartículas também revelou, por SAXS, estrutura cristalográfica Im3m e um parâmetro de rede de 12.30(12) nm. DLS apresentou partículas de tamanho 280(5) nm e polidispersão moderada 0.115(52). MET mostrou partículas quadradas e cúbicas com tamanhos de ~500 nm. O encapsulamento da MILT revelou que o fármaco interage com os cubossomos aumentando seu parâmetro de rede, até uma concentração de 4% w/w. Curiosamente, para algumas concentrações de MILT havia presença de outras estruturas evidenciadas por SAXS. MET revelou nanopartículas com muita polidispersão, com tamanhos variando entre 200 nm e 2 µm. Cubossomos de PHY foram reproduzidos com sucesso a partir do protocolo escolhido, em meios aquoso, tampão PBS e 2.25% glicerol. SAXS revelou nanopartículas com simetria cristalográfica Pn3m e parâmetro de rede 6.74(04) nm. Por DLS, o tamanho das partículas foi de ~450 nm e polidispersão moderada 0.161(10). Medidas de NTA foram consistentes com DLS, mostrando uma larga distribuição de tamanhos e concentração de partículas ~1016 partículas/mL. MET revelou cubossomos quadrados e mais arredondados de tamanhos variados. Criomicroscopia apresentou partículas com estrutura interna bem definida, tamanhos variados (confirmando a polidispersão) e parâmetro de rede calculado em ~6.5 nm, compatível com medidas de SAXS. Essas amostras também foram submetidas a liofilização e descobriu-se que mesmo depois da re-hidratação, as partículas ainda mantiveram as mesmas características da amostra original. A extrusão também foi feita com o objetivo de melhorar a polidispersão e controlar o tamanho das partículas, novamente, os cubossomos demonstraram manter sua estrutura interna depois desse processo, diminuindo seus tamanhos e diminuindo a polidispersão dos sistema. MILT foi encapsulada de duas formas: passiva (co-solubilização) e ativa (adição depois que as nanopartículas foram formadas). Com até 5% w/w de MILT incorporada, os cubossomos mantiveram sua estrutura cristalográfica, porém em concentrações de 10%, 15% e 20% w/w, o fármaco provocou transição de fase para simetria Im3m. Em baixas concentrações, MILT aumentou os parâmetros de rede dos cubossomos e a hipótese levantada foi que a droga se insere na bicamada lipídica das nanopartículas. DLS revelou que MILT não altera o tamanho das partículas nem sua polidispersão. MET revelou partículas quadradas e arredondadas com tamanhos maiores que os medidos por DLS. Para a amostra 4% w/w, a crio microscopia foi realizada e as partículas encontradas apresentaram estrutura interna e parâmetro de rede calculado ~7 nm, compatível com medidas de SAXS. Co-solubilização e adição depois do preparo se mostraram equivalentes para o encapsulamento da MILT. Todas as amostras também foram submetidas a um estudo de temperaturas para investigar transições de fase, baseando-se nos diagramas de fase dos lipídios. Foi descoberto que os cubossomos, sem a droga, a 65 °C sofrem transição para a fase isotrópica L2 e quando MILT está incorporada essa transição não acontece. DLS também revelou que as partículas não têm seus tamanhos alterados com o aumento de temperatura. Por fim, cubossomos mostraram ser excepcionais conseguindo manter suas características físico-químicas mesmo quando submetidos a ambientes extremos, como a liofilização, a extrusão e a altas temperaturas.

Nanomedicine is one of the most promising fields in nanotechnology nowadays. The use of nanoparticles as carriers aims to improve efficiency of drugs that possess low solubility in aqueous environment (very hydrophobic molecules) or that have a lot of undesired side effects. In this way, nanoparticles offer both a protection for the molecules and a carrying vehicle. On this ground, cubosomes are nanoparticles capable of storing both hydrophilic and hydrophobic molecules within its structure, in addition, cubosomes have approximately 50% hydrophilic and hydrophobic areas. Therefore, they can carry much more molecules than liposomes for instance. In particular, cubosomes are quite easy to produce due to its base product, lipids (like monoolein (GMO) or phytantriol (PHY)) that self-assembly in water media. In this project, both lipids were chosen to produce the cubosomes from well-established protocols in literature. A model drug, miletofsine (MILT), was chosen to study the interaction of such nanosystem with a guest molecule. GMO cubosomes revealed to have Im3m crystallographic symmetry and lattice parameter 15.3(7) nm, particles presented sizes 300(8) nm and moderate polydispersion 0.160(20). TEM revealed squared particles with sizes ~350 nm, cryo-EM presented particles with internal structure and varied size (from 200 to > 500 nm). From FFT analysis, the calculated lattice parameter remained in the order of ~10 nm compatible with SAXS measurements. MILT loading into cubosomes was possible up to 4% w/w without loss of cubosomes structure. For 5% w/w MILT, the nanoparticles were already loosing their crystalline structure, as evidenced by cryo-EM. TEM analysis reveals that as more MILT is loaded into the cubosomes, their sizes increased. For sample 1.5% w/w MILT cryo-EM presents nanoparticles with organized internal structure and an envelope (hypothesized to be a polymer coating) in its surface. Calculated lattice parameters are in the order of ~10 nm. Myverol (Myv) is a commercial mixture that contains ~60% GMO, in this project it was proposed a bottom up protocol for Myv-based cubosomes. The production of these nanoparticles also revealed, by SAXS, Im3m symmetry and lattice parameter 12.30(12) nm. DLS revealed particle size 280(5) nm and moderate polydispersion 0.115(52). TEM shows square and cubic nanoparticles with sizes ~500 nm. MILT loading into Myv-cubosomes revealed that the drug interacts with the nanoparticle by enlarging their lattice parameter as more MILT is loaded (up to 4% w/w). Curiously, for some MILT concentrations the presence of other unknown cubic structures was evidenced by SAXS. TEM revealed nanoparticles with huge polydispersion, with sizes raging from 200 nm to 2 µm. PHY based cubosomes were successfully reproduced by the chosen protocol, in both water, PBS buffer and 2.25% glycerol medium. SAXS revealed crystallographic structure Pn3m and lattice parameter 6.74(04) nm. DLS measured sizes ~450 nm and moderate polydispersion 0.161(10). NTA measurements were consistent with DLS, revealing a broad size distribution and total particle concentration of ~1016 particles/mL for each sample. TEM revealed square and rounder particles in varied size. Cryo-EM micrographs presented particles with internal structure and varied size confirming moderate polydispersion. The FFT analysis revealed calculated lattice parameters ~6.5 nm, compatible with SAXS data. Samples were submitted to lyophilization and found that after re-hydration they still hold the same characteristics (morphology, size) as the original sample. Extrusion was also performed in order to improve polydispersion and control particle size, again cubosomes held their internal structure after the process, diminishing their sizes and improving monodispersion. MILT was loaded into cubosomes via co-solubilization and addition after the nanoparticles were formed. Up to 5% w/w the cubosomes incorporated MILT without loss of crystallographic structure, but at 10%, 15% and 20% w/w, the drug provoked phase change for Im3m symmetry. At the lower concentrations, MILT enlarged the lattice parameter of cubosomes and it was hypothesized that MILT inserted itself into the bilayer of the nanoparticles. DLS reveales that the drug does not change particle size or polydispersion. TEM revealed square and rounder particles in sizes slightly bigger than DLS. For sample 4% w/w, Cryo-EM presented particles with internal structure and calculated lattice parameter ~7 nm compatible with SAXS measurements for this sample. Co-solubilization and addition after nanoparticle preparation proved out to have the same effect on cubosomes loaded with MILT. All samples were submitted to higher temperatures to investigate phase change, based on phase diagram of the lipid. It was found that for the blank samples at 65 °C the cubosomes suffer phase change for isotropic phase L2, when MILT is loaded into the nanoparticles this phase change does not happen. DLS revealed also that at higher temperatures, particle size does not change, neither polydispersion. Finally, cubosomes proved to be remarkable nanoparticles that hold their physico-chemical characteristics even when submitted to extreme environments (lyophylization, extrusion and higher temperatures.) / Nanomedicina é o campo de estudo mais promissor dentro da nanotecnologia atualmente. O uso de nanopartículas visa melhorar a eficiência de fármacos que possuem baixa solubilidade em meios aquosos (moléculas muito hidrofóbicas) ou que possuem muitos efeitos colaterais indesejados. Neste contexto, as nanopartículas oferecem proteção e veículo para tais moléculas. Para isso, cubossomos são nanopartículas capazes de encapsular tanto as moléculas hidrofóbicas como as hidrofílicas em sua estrutura. Cubossomos também apresentam aproximadamente 50% de áreas hidrofílica e hidrofóbica, sendo capaz de encapsular grandes quantidades de moléculas teóricamente. Particularmente, cubossomos são nanopartículas de fácil produção devido à sua matéria prima serem lipídios (por exemplo, monoleína (GMO) ou fitantriol (PHY)) que se auto associam em meio aquoso. Neste projeto, os dois lipídios citados foram escolhidos para a produção dos cubossomos empregando-se protocolos bem estabelecidos da literatura. Uma fármaco modelo, miltefosina (MILT), foi escolhida para o estudo da interação com as nanopartículas. Cubossomos de monoleína (GMO) revelaram simetria cristalográfica Im3m e parâmetro de rede de 15.3(7) nm, as nanopartículas apresentaram tamanhos em torno de 300(8) nm e PDI 0.160(20). MET revelou partículas quadradas com tamanhos ~350 nm e a crio microscopia mostrou partículas com estrutura interna bem definida e tamanhos variados (200 a 500 nm), os parâmetros de rede calculados se mostraram da ordem de ~10 nm, compatíveis com as medidas de SAXS. O encapsulamento da MILT nos cubossomos foi possível até 4% w/w sem perda de morfologia. Para 5% w/w MILT as nanopartículas já apresentavam perda de cristalinidade na sua estrutura, evidenciado por crio microscopia. Análise por MET revelou que quanto mais MILT era encapsulada nos cubossomos, maiores ficaram as nanopartículas. Com a amostra de 1.5% w/w foi feita a crio microscopia, que revelou cubossomos com estrutura interna bem definida e um envelope (possivelmente formado pelo polímero) na sua superfície. Os parâmetros de rede calculados foram da ordem de ~10 nm também. O myverol (Myv) é uma mistura comercial que contém aproximadamente 60% de GMO, e neste projeto foi proposto um protocolo bottom up para cubossomos feitos de Myv. A produção dessas nanopartículas também revelou, por SAXS, estrutura cristalográfica Im3m e um parâmetro de rede de 12.30(12) nm. DLS apresentou partículas de tamanho 280(5) nm e polidispersão moderada 0.115(52). MET mostrou partículas quadradas e cúbicas com tamanhos de ~500 nm. O encapsulamento da MILT revelou que o fármaco interage com os cubossomos aumentando seu parâmetro de rede, até uma concentração de 4% w/w. Curiosamente, para algumas concentrações de MILT havia presença de outras estruturas evidenciadas por SAXS. MET revelou nanopartículas com muita polidispersão, com tamanhos variando entre 200 nm e 2 µm. Cubossomos de PHY foram reproduzidos com sucesso a partir do protocolo escolhido, em meios aquoso, tampão PBS e 2.25% glicerol. SAXS revelou nanopartículas com simetria cristalográfica Pn3m e parâmetro de rede 6.74(04) nm. Por DLS, o tamanho das partículas foi de ~450 nm e polidispersão moderada 0.161(10). Medidas de NTA foram consistentes com DLS, mostrando uma larga distribuição de tamanhos e concentração de partículas ~1016 partículas/mL. MET revelou cubossomos quadrados e mais arredondados de tamanhos variados. Criomicroscopia apresentou partículas com estrutura interna bem definida, tamanhos variados (confirmando a polidispersão) e parâmetro de rede calculado em ~6.5 nm, compatível com medidas de SAXS. Essas amostras também foram submetidas a liofilização e descobriu-se que mesmo depois da re-hidratação, as partículas ainda mantiveram as mesmas características da amostra original. A extrusão também foi feita com o objetivo de melhorar a polidispersão e controlar o tamanho das partículas, novamente, os cubossomos demonstraram manter sua estrutura interna depois desse processo, diminuindo seus tamanhos e diminuindo a polidispersão dos sistema. MILT foi encapsulada de duas formas: passiva (co-solubilização) e ativa (adição depois que as nanopartículas foram formadas). Com até 5% w/w de MILT incorporada, os cubossomos mantiveram sua estrutura cristalográfica, porém em concentrações de 10%, 15% e 20% w/w, o fármaco provocou transição de fase para simetria Im3m. Em baixas concentrações, MILT aumentou os parâmetros de rede dos cubossomos e a hipótese levantada foi que a droga se insere na bicamada lipídica das nanopartículas. DLS revelou que MILT não altera o tamanho das partículas nem sua polidispersão. MET revelou partículas quadradas e arredondadas com tamanhos maiores que os medidos por DLS. Para a amostra 4% w/w, a crio microscopia foi realizada e as partículas encontradas apresentaram estrutura interna e parâmetro de rede calculado ~7 nm, compatível com medidas de SAXS. Co-solubilização e adição depois do preparo se mostraram equivalentes para o encapsulamento da MILT. Todas as amostras também foram submetidas a um estudo de temperaturas para investigar transições de fase, baseando-se nos diagramas de fase dos lipídios. Foi descoberto que os cubossomos, sem a droga, a 65 °C sofrem transição para a fase isotrópica L2 e quando MILT está incorporada essa transição não acontece. DLS também revelou que as partículas não têm seus tamanhos alterados com o aumento de temperatura. Por fim, cubossomos mostraram ser excepcionais conseguindo manter suas características físico-químicas mesmo quando submetidos a ambientes extremos, como a liofilização, a extrusão e a altas temperaturas.

No description available.

<div>Viruses are a group of contagious microbes that have compact structures, containing a nucleic acid core and a protein shell. The replication of viruses requires assistance from hosts which can be almost any cellular organism. Viral infections are often associated with diseases and have been a major threat to the human race. To cope with viral diseases, we need to understand viruses, including their structures, life cycle, pathogenesis and interactions with their hosts. The first structure of a human virus was determined by the Rossmann lab in 1985 using X-ray crystallography.</div><div>Thanks to the recent advances in both hardware and software, cryo-electron microscopy (cryo-EM) has emerged as a powerful tool to study virus structures. Cryo-EM allows structural determination for a wide range of specimens to high resolution comparable to what can be achieved by X-ray crystallography. Currently two techniques of cryo-EM are commonly used in structural virology: single particles analysis (SPA) and electron tomography (ET). </div><div>Single particle analysis has been used to determine the structures of viruses complexed with host factors in three studies that are to be discussed with more details in chapters 2-4. </div><div>The structure of B19 parvovirus complexed with Fabs of a neutralizing human antibody was determined to 3.2 Å resolution. This structure showed that amino acids from three neighboring VP2 proteins form a quaternary structure epitope. In addition, the structure of human rhinovirus-C (RV-C) complexed with its cellular receptor, CDHR3, was determined to 3.9 Å resolution. Despite the low occupancy of the receptors, a “powerful” localized 3D classification procedure helped to select viral particles that had more bound receptors. Furthermore, structures were determined to 10 Å resolution of bacteriophage ΦX174 bound to lipopolysaccharide (LPS) bilayers, before and after genome ejection. These structures showed a series of conformational changes that occurred when a phage penetrated the bacterial membranes. These studies are good examples of applying cryo-EM to investigate virus-host interactions.</div><div>However, single particle analysis requires samples to be isolated, homogenous and monodispersed. On the contrary, tomography allows in situ studies and is applicable to samples with more flexibility and more heterogeneity. In the case of ΦX174, the structural changes that are involved in the assembly of the H-tube during infection remains a huge mystery. To provide an environment that is more similar to the surface of a bacterial cell, LPS-containing liposomes were mixed with ΦX174 viruses. It was then observed that the ΦX174 particles bound to these liposomes in a very compact manner which was impossible interpret with single particle analysis. Using cryo-ET, 3D volumes of liposome-ΦX174 complexes were reconstructed and structural details were visualized by sub-tomogram classification and averaging.</div><div>The emergence of cryo-EM has not only made high-resolution structural studies possible but also broadened the scope of samples with which virologists could work. Moreover, studies on flexible and heterogeneous complexes between viruses and host factors are now possible using either single particle analysis or electron tomography. These techniques will help us to understand virus-host relationships and finally, to develop effective anti-viral therapies.</div>

Adeno-associated Virus (AAV) Rep proteins are multifunctional proteins that carry out various DNA transactions required for the life cycle of AAV. The Rep proteins have been found to be important for genome replication, gene regulation, site-specific integration and play an essential role in genome packaging. There are two main groups of Rep proteins: large and small Reps; both groups are SF3 helicase family members. During DNA packaging, studies have shown that the small Rep proteins are critical to produce fully packed particles. Using stopped-flow kinetic analysis, we show a significant difference in helicase activity between the small and large Rep proteins that support the notion that the small Rep proteins are the primary motor to package DNA due to more efficient motor activity. That leaves the large Rep proteins to serve a different role during packaging. In previous studies, we have shown that the large Rep proteins have the ability to change their oligomeric state depending on the nature of the DNA substrate. We can observe double octameric rings with single-stranded DNA (ssDNA) and heptameric complex with double-stranded DNA (dsDNA). To understand Rep protein structural plasticity, we solved a 6.96 Å cryo-EM structure of Rep68*/ssDNA complex illustrating that the formation of Rep octamer rings is dominated by interactions between their N-terminal origin-binding domain (OBD) using the same interface utilized to recognize dsDNA specifically. Our analysis of the structural data suggests that the double octameric ring structure is stabilized by ssDNA that bridges octameric rings together. The structure shows that the helicase domains are highly flexible and that ssDNA is present at the center of the ring. In addition, we have solved a preliminary 12 Å model of Rep68*/dsDNA complex showing a heptameric ring encircling a DNA molecule. Our structural and functional data offer insights to the various Rep-DNA scaffolds that can perform diverse functions during the AAV life cycle.

The gram-negative bacteria Escherichia coli, a neutralophile, is remarkable in its defenses against acid stress. Of interest to our laboratory is the inducible lysine decarboxylase (LdcI) system, an acid resistance system which renders acid resistance to E. coli in mild acid stress (~pH 5). It was found that this enzyme forms an extremely large (~3.3 MDa) and tight complex (Kd ~ 0.56 μM) with a MoxR AAA+ ATPase named Regulatory ATPase Variant A (RavA). The cryo-EM structure at 14 Å was determined. Through size-exclusion chromatography (SEC) experiments, the binding sites on both LdcI and RavA have been determined. It is proposed that the complex can form through both charged and hydrophobic interactions. In the course of these studies, unexpected observations led to the characterization of the LARA domain of RavA as an amyloid protein under in vitro conditions. The physiological significance of this observation is still under investigation.

The gram-negative bacteria Escherichia coli, a neutralophile, is remarkable in its defenses against acid stress. Of interest to our laboratory is the inducible lysine decarboxylase (LdcI) system, an acid resistance system which renders acid resistance to E. coli in mild acid stress (~pH 5). It was found that this enzyme forms an extremely large (~3.3 MDa) and tight complex (Kd ~ 0.56 μM) with a MoxR AAA+ ATPase named Regulatory ATPase Variant A (RavA). The cryo-EM structure at 14 Å was determined. Through size-exclusion chromatography (SEC) experiments, the binding sites on both LdcI and RavA have been determined. It is proposed that the complex can form through both charged and hydrophobic interactions. In the course of these studies, unexpected observations led to the characterization of the LARA domain of RavA as an amyloid protein under in vitro conditions. The physiological significance of this observation is still under investigation.

X-Linked Retinoschisis (XLRS) is a currently incurable, progressive retinal degeneration that affects approximately 1:20,000 males. Sufferers have a loss of retinal structure and visual acuity, leading to blindness. The condition is caused by mutation of the RS1 gene encoding the retinal-specific protein retinoschisin. Retinoschisin is critical in maintaining the normal, ordered retinal architecture, with deletion in mice models leading to loss of both structure and visual processing, analogous to XLRS sufferers. However, re-introduction of retinoschisin using adeno-associated viral vectors leads to complete rescue in these models. Despite the importance of retinoschisin in maintaining retinal architecture, the mechanism by which it maintains this structure remains unknown. As a result, this study aimed to structurally characterise retinoschisin and XLRS-associated point mutants R141H and H207Q to gain insight into the mechanism of retinoschisin action. To this end, retinoschisin was expressed and purified from HEK 293-EBNA cells and the structure of both monomeric and octameric retinoschisin was investigated using Small-Angle X-Ray Scattering (SAXS) and Cryo-electron microscopy (Cryo-EM). Monomeric retinoschisin was found to adopt an elongated structure that allowed for the tight association of the subunits into a planer propeller structure. However, in solution conditions the octamer also stably self-assembled into a dimer of octamers, for which the structure was solved using cryo-EM. This allowed for construction of a quasi-atomic model, enabling mapping of XLRS-associated point mutations on the complex. Two major classes of mutation were identified, in the intra-octamer and inter-octamer interfaces, suggesting a mechanism of pathology for these mutants. Observation of clustered conservative mutations at the inter-octamer interface suggested the dimer of octamers may be physiologically relevant. Furthermore, comparison of the R141H mutant to the wild-type revealed an additional mutated site in the propeller tips. Here, R141H was suggested to induce a small conformational change and alter an interaction site. Another mutant, H207Q, however, induced a destabilization of the assembled retinoschisin molecule. In conclusion, we purified and structurally characterised human retinoschisin, identifying a new hexadecameric oligomer. The structure of this allowed for identification of distinct classes of mutations on the assembled molecule and a hypothesis of the mechanism of retinoschisin action in the retina.

Les kinétoplastides sont un groupe de protozoaires, et qui menace plus de 400 millions de personnes dans le monde entier. Ils possèdent des segments d'expansion d'ARNr (SE) inhabituellement plus larges dans les sous-unités 40S. Ici, nous avons purifié à partir de lysats de cellules de T. cruzi des complexes d'initiation natifs (48S IC) et des sous-unités de 40S natives que nous avons ensuite analysées par cryo-ME. La structure des 48S IC révèle certains des aspects spécifiques de la traduction aux kinétoplastides, tels qu'un réseau d’interaction complexe entre eIF3 et SEs. En outre, notre structure met en évidence le rôle de DDX60 dans l'initiation de la traduction chez les kinétoplastides. La structure d'une sous-unité 40S native révèle l'existence d'un facteur non caractérisé (appelé ηF). Le site de liaison de ηF suggère un rôle dans le contrôle de la traduction. De plus, nous avons rapporté́ la structure d’une nouvelle protéine ribosomale (-r) spécifique des kinétoplastides (KSRP). Notre travail pose les premières bases structurales des aspects spécifiques de l'initiation de la traduction chez les kinétoplastides. / Kinetoplastid is a group of flagellated protozoans, which threatens more than 400 million people world-wide. They possess unusual large rRNA expansion segments (ES) in the 40S, such as ES6S, ES7S and ES9S and their location suggests an involvement in the initiation process. Furthermore, all mature mRNAs possess a conserved 5’ spliced-leader. Here, we purified from T. cruzi cell lysates native initiation complexes and native 40S subunits that we then analysed by cryo-EM. The structure of native initiation complexes reveals several kinetoplastid-specific aspects of translation, such as an intricate interaction network between eIF3 and ES6S and ES7S. Furthermore, it reveals the role of DDX60 in translation initiation in kinetoplastids. The structure of native 40S subunits reveals the existence of an uncharacterized factor (termed ηF) bound at platform of the 40S. The binding site of ηF suggests a role in translational control. Moreover, we reported a novel kinetoplastid-specific ribosomal (r-) protein (KSRP) bound to the 40S subunit. Our work represents the first structural characterization of kinetoplastids-specific aspects of translation initiation.