The structural analysis serves as a bridge to link the structure of materials to their properties. Revealing the structure details allows a better understanding on the growth mechanisms and properties of materials, and a further designed synthesis of functional materials. The widely used methods based on X-ray diffraction have certain limitations for the structural analysis when crystals are small, poorly crystallized or contain many defects. As electrons interact strongly with matter and can be focused by electromagnetic lenses to form an image, electron crystallography (EC) approaches become prime candidates for the structural analysis of a wide range of materials that cannot be done using X-rays, particularly nanomaterials with poor crystallinity. Three-dimensional electron diffraction tomography (3D EDT) is a recently developed method to automatically collect 3D electron diffraction data. By combining mechanical specimen tilt and electronic e-beam tilt, a large volume of reciprocal space can be swept at a fine step size to ensure the completeness and accuracy of the diffraction data with respect to both position and intensity. Effects of the dynamical scattering are enormously reduced as most of the patterns are collected at conditions off the zone axes. In this thesis, 3D EDT has been used for unit cell determination (COF-505), phase identifications and structure solutions (ZnO, Ba-Ta3N5, Zn-Sc, and V4O9), and the study of layer stacking faults (ETS-10 and SAPO-34 nanosheets). High-resolution transmission electron microscope (HRTEM) imaging shows its particular advantages over diffraction by allowing observations of crystal structure projections and the 3D potential map reconstruction. HRTEM imaging has been used to visualize fine structures of different materials (hierarchical zeolites, ETS-10, and SAPO-34). Reconstructed 3D potential maps have been used to locate the positions of metal ions in a woven framework (COF-505) and elucidate the pore shape and connectivity in a silica mesoporous crystal. The last part of this thesis explores the combination with X-ray crystallography to obtain more structure details.

Electron crystallography is an important technique for studying micro- and nano-sized materials. It has two important advantages over X-ray crystallography for structural studies: 1) crystals millions of times smaller than those needed for X-ray diffraction can be studied; 2) it is possible to; focus the electrons to form an image. The local atomic arrangement can be seen directly by high-resolution transmission electron microscopy (HRTEM). The crystallographic structure factor phases, which are lost in recording diffraction patterns, are present in HRTEM images and can be determined experimentally. The main disadvantages of electron crystallography compared to X-ray diffraction are that the data are difficult to collect, often incomplete and suffer from dynamic scattering. New methods need to be developed to overcome these problems. In this work, structure determination of several unique and complex porous materials including zeolites and mesoporous silica is demonstrated. None of the structures of these materials could be solved by X-ray crystallography. New techniques are also developed in order to overcome the disadvantages of electron crystallography. The new techniques include a digital sampling method for collecting precession electron diffraction data and a rotation method for automatic collection of complete 3D electron diffraction data. A number of practical issues concerning data collection and data processing are described and the data quality is analysed. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Submitted.

Electron crystallography has evolved as a powerful method for structural characterization of a wide range of materials. It has two significant advantages over other methods for structure determination, e.g. X-ray diffraction. Electrons interact much more strongly with matter compared to X-rays and they can be focused by electromagnetic lenses to form images with atomic resolution. These advantages make electron crystallography a unique tool for characterization of crystalline materials suffering from small crystal size and complex or disordered structures.      Zeolites are a class of microporous materials with significance in several applications. They often possess complex and disordered structures, which demand large efforts in the structure determination.      Over the last years, two new electron crystallographic methods have been developed; the rotation electron diffraction (RED) and the structure projection reconstruction from a through-focus series of high resolution transmission electron microscopy (HRTEM) images. In this thesis, they will be applied for structure determination of four new zeolite structures, including EMM-25 and EMM-23 with two ordered structures, and ITQ-39 and ITQ-38 with disordered structures. Each of the structure solutions have different challenges to overcome. The high silica borosilicate EMM-25 was solved by the RED method. The aluminosilicate EMM-23 was solved by a combination of HRTEM and RED. The structure solution of two materials with disordered structures, ITQ-39 and ITQ-38, will be described. For materials containing disorders, structure projection images are of utmost importance.      Furthermore, the mesoporosity inside hierarchically porous ZSM-5 crystals was studied by a combination of focused ion beam (FIB) and HRTEM imaging. The last part of this thesis explores STEM imaging for use in structure determination from 3D reconstruction. / <p>At the time of the doctoral defence the following papers were unpublished and had a status as follows: Papers 4 and 5: Manuscipts; Paper 10: Manuscript</p>

Lipopolysaccharide (LPS), one of the main components of Gram-negative bacterial cell walls, is a potent endotoxin. Structures of the unique protective monoclonal antibody (mAb) WN1 222-5 in complex with Escherichia coli R2 and R4 LPS core regions show that recognition occurs in a manner similar to the innate immune receptor Toll-like receptor 4 (TLR4). Inner core LPS is shown to exist in a conserved epitope with multiple intramolecular interactions that allows the conserved epitope to bind strongly to mAb WN1 222-5. The structure of mAb FDP4, directed against truncated E. coli J-5 LPS, shows a deep pocket combining site specific for a terminal epitope that does not allow room for wild type (wt) LPS. Research into these anti-LPS binding mAbs opens up new avenues for potential septic shock therapy. The explosion of new techniques and bright x-ray sources in the 80’s and 90’s led to rapid advancement of protein x-ray crystallography; however, structure determination on some of the most important problems is now stalled due to the general inability to grow crystals of sufficient size. Recent advances in electron microscopy (EM) technology has led to improved beam characteristics, which has allowed the initiation of research to develop EM as a viable alternative to x-ray crystallography. In this research, method development using standard equipment to explore potential avenues for analysing three-dimensional protein crystals via EM has been explored. / Graduate / 0982 / 0487 / 0537 / kgomery@uvic.ca

Membrane proteins are involved in a number of disease pathologies and thus comprise a large number of drug targets. Determination of the high-resolution three-dimensional structure is essential for rational drug design, but several hurdles need to be overcome, primarily the over-expression and purification of said membrane proteins. Human leukotriene C4 synthase (hLTC4S), an 18 kDa integral membrane protein localized in the outer nuclear membrane of eosinophils and basophils, catalyzes the conjugation of LTA4 and reduced glutathione to produce LTC4. LTC4 and its metabolites LTD4 and LTE4 are the cysteinyl leukotrienes implicated in bronchoconstriction and inflammation pathways. The focus of my project involves optimizing the over-expression and purification of hLTC4S, which was heterologously expressed in Schizosaccharomyces pombe, purified by immobilized affinity chromatography, and finally "polished" with a buffer exchange step to remove excess co-purified lipids. The optimized protocol yielded ~1 mg of ~90% homogenous, pure protein per liter of cell culture. The finalized purified protein can then be used for further investigation of two-dimensional crystals by electron crystallography with the overall goal of structure determination.

Electron crystallography is complementary to X-ray crystallography. Single crystal X-ray diffraction requires the size of a crystal to be larger than about 5 × 5 × 5 μm3 while a TEM allows a million times smaller crystals being studied. This advantage of electron crystallography has been used to solve new structures of small crystals. One method which has been used to collect electron diffraction data is rotation electron diffraction (RED) developed at Stockholm University. The RED method combines the goniometer tilt and beam tilt in a TEM to achieve 3D electron diffraction data. Using a high angle tilt sample holder, RED data can be collected to cover a tilt range of up to 140o.  Here the crystal structures of several different compounds have been determined using RED. The structure of needle-like crystals on the surface of NiMH particles was solved as La(OH)2. A structure model of metal-organic layers has been built based on RED data. A 3D MOF structure was solved from RED data. Two halide perovskite structures and two newly synthesized aluminophosphate structures were solved. For those beam sensitive crystals characterized here, sample cooling down to -170oC was used to reduce the beam damage. The low temperature not only reduces electron beam damage, but also keeps the structure more stable in the high vacuum in a TEM and improves the quality of the diffraction data. It is shown that cooling can improve the resolution of diffraction data for MOFs and zeolites, for samples undergoing phase changes at low temperature, the data quality could be worse by cooling. In summary, cooling can improve the ED data quality as long as the low temperature does not trigger structural changes. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 3: Submitted.</p>

The membrane proteins signal peptide peptidase, signal peptide peptidase like and presenilin are intramembrane aspartyl proteases located in the endoplasmic reticulum, plasma membrane and organelle. These membrane proteins are able to catalyze a hydrolytic reaction in a hydrophobic space. The downstream consequences of these reactions impact a variety of cellular functions such as cytokine production, inflammatory responses, embryogenesis, and immune system regulation. Additionally, the aspartyl proteases such as signal peptide peptidase and presenilin, a part of the γ-secretase complex, hydrolyze peptides leading to pathogen maturation and Alzheimer’s disease, respectively. Electron crystallography offers the unique aspect of studying membrane proteins in a near native state. Determining the structures of Haloarcula morismortui and Methanoculleus marisnigri JR1 signal peptide peptidases by electron crystallography may provide insight into how a hydrolysis reaction occurs in a hydrophobic environment and how the protein determines which transmembrane signal peptides to cleave. Additionally, structure determination may help answer questions regarding why human presenilin, part of the γ-secretase complex, incorrectly processes amyloid precursor protein into amyloid-beta peptides leading to Alzheimer’s disease. Such structural data may not only shed light on how amyloid precursor protein is processed but how other proteins are processed by signal peptide peptidase leading to immune responses, cell signaling, and pathogen maturation. In addition, structure-function data may have an impact on pharmaceutical drug designs that targets signal peptide peptidase, signal peptide peptidase like, and/or presenilin. To determine the structure of aspartyl proteases, two archaeal signal peptide peptidases were used for two-dimensional crystallization trials to be able to study their structure by electron crystallography. Haloarcula morismortui and Methanoculleus marisnigri JR1 signal peptide peptidases, both human signal peptide peptidase homologues, were recombinantly over-expressed and purified. During dialysis trials, various lipid-to-protein ratios, sodium chloride concentrations, temperatures, detergents and a variety of other variables were tested. Methanoculleus marisnigri JR1 signal peptide peptidase showed the most promising results in terms of crystallinity. Optimizing dialysis conditions, specifically narrowing the lipid to protein ratio, resulted in two-dimensional crystals. Ordered arrays measuring up to 200 nm x 200 nm were observed. These ordered arrays have been shown to be reproducible amongst multiple batches of purified Methanoculleus marisnigri JR1 signal peptide peptidase. Preliminary projection maps of negatively stained ordered arrays show unit cell dimensions of a = 178 Å, b = 160 Å, γ = 92.0 Å and a = 175 Å, b = 167 Å, γ = 92.0 Å. The monomer measurements are approximately 70 Å by 80 Å. This is the first time a signal peptide peptidase homologue has been crystallized by two-dimensional crystallization.

No description available.

Electron crystallography of two-dimensional crystals is a structure-determination method well suited to the study of membrane protein structure-function. Two-dimensional crystals consist of ordered arrays of protein within reconstituted lipid bilayers, an arrangement that mimics the natural membrane environment. In this work we describe our recent progress in the use of this method with three different proteins, each providing a window into a separate paradigm in the electron crystallographic pipeline. Specific crystallization conditions for human leukotriene C₄ synthase (LTC₄S) have previously been determined, but our continued refinement of purification and crystallization has identified a number of additional parameters that greatly affect crystal size and quality, and we have developed a protocol to rapidly and reproducibly grow large, non-mosaic crystals of LTC₄S. The human gamma-glutamyl carboxylase (GGCX) has also been crystallized, but is sensitive to cryo-EM sample preparation conditions and we present here the successful reproduction of crystallization and refinement of cryo-EM sample preparation conditions. Lastly, we describe our crystallization screens with the Vibrio cholerae sodium-pumping NADH:ubiquinone reductase complex (Na⁺-NQR), and identify the factors critical to membrane reconstitution of the complex, a necessary first step towards crystallization. We also describe a semi-quantitative crystal screening protocol we have developed that provides quick and accurate method to assess two- dimensional crystallization trials, and discuss some general observations in optimization of membrane protein purification and two-dimensional crystallization for electron crystallography.

[ES] Los materiales microporosos como las zeolitas han sido destacados por su amplio impacto en procesos catalíticos, medio ambiente e industria. El conocimiento de la estructura cristalina de la materia y el estudio de sus propiedades y aplicaciones están intrínsecamente relacionadas. Por lo tanto, las técnicas convencionales de difracción como la difracción de rayos X en monocristal y la difracción de rayos X en polvo (SCXRD y PXRD, respectivamente) han sido los métodos habituales para obtener información sobre la disposición estructural de los átomos. Sin embargo, la limitación para obtener cristales lo suficientemente grandes para ser medidos por SCXRD y la fuerte superposición de picos en los patrones de PXRD dificultan con frecuencia la determinación estructural de estos materiales con estructuras complejas y unidades de parámetros de celda grandes. Es por ello por lo que el desarrollo de nuevas técnicas de difracción que permitan la determinación estructural es de suma importancia. Por lo tanto, es fundamental estudiar técnicas de difracción de última generación para la caracterización estructural en nuevos materiales microporosos, como las zeolitas. El objetivo de este trabajo es investigar la Tomografía de Difracción de Electrones (EDT), también conocida como Difracción de Electrones Tridimensional (3DED). Estos métodos pueden ser una técnica excepcional para el análisis estructural de materiales microporosos utilizando Microscopios Electrónicos de Transmisión (TEM) convencionales y ya disponibles. Estas técnicas generan patrones de difracción de monocristales de tamaño nanométrico con intensidades de dispersión mejoradas utilizando haces de electrones. Estos patrones se adquieren mientras el nanocristal gira dentro del goniómetro del TEM, resultando en patrones de difracción de electrones (ED) de pequeños cristales individuales de dimensiones nanométricas. Además, estos métodos requieren el desarrollo y la optimización de todos los parámetros experimentales que a menudo exigen experiencia en TEM. Además, muchos materiales sufren daños estructurales por la interacción con el haz de electrones. Para abordar este problema, la combinación de la técnica de tomografía por difracción de electrones con precesión (PEDT), que aplica una precesión del haz, junto con un manual de 3DED fácil de usar, minimizará estos efectos, permitiendo una adquisición de datos, procesamiento y determinación de estructuras exitosos. Esta metodología se ha aplicado para resolver la estructura de nuevos materiales microporosos denominados ITQ-70 e ITQ-35, en forma sin calcinar, así como una zeolita ya conocida en forma sin calcinar con dos moléculas orgánicas diferentes en su interior como la ITQ-52. Todas estas estructuras se presentan en forma sin calcinar conteniendo agentes directores de estructura en su interior. Este enfoque conducirá a la localización atómica completa de materiales de zeolita híbridos inorgánicos y orgánicos mediante la accesibilidad de técnicas de difracción de rayos X y 3DED. Este avance ofrecerá un protocolo general prometedor para desarrollar una nueva herramienta de caracterización para una determinación precisa de la estructura de materiales cristalinos pequeños. / [CA] Els materials microporosos com les zeolites han sigut destacats pel seu ampli impacte en processos catalítics, medi ambient i indústria. El coneixement de l'estructura cristal·lina de la matèria i l'estudi de les seues propietats i aplicacions estan intrínsecament relacionats. Per tant, les tècniques convencionals de difracció com la difracció de raigs X en monocristall i la difracció de raigs X en pols (SCXRD i PXRD, respectivament) han sigut els mètodes habituals per a obtindre informació sobre la disposició estructural dels àtoms. No obstant això, la limitació per a obtindre cristalls prou grans per a ser mesurats per SCXRD i la forta superposició de pics en els patrons de PXRD dificulten freqüentment la determinació estructural d'aquests materials amb estructures complexes i unitats de paràmetres de cel·la grans. És per això pel que el desenvolupament de noves tècniques de difracció que permeten la determinació estructural és de summa importància. Per tant, és fonamental estudiar tècniques de difracció d'última generació per a la caracterització estructural en nous materials microporosos, com les zeolites. L'objectiu d'aquest treball és investigar la Tomografia de Difracció d'Electrons (EDT), també coneguda com a Difracció d'Electrons Tridimensional (3DED). Aquests mètodes poden ser una tècnica excepcional per a l'anàlisi estructural de materials microporosos utilitzant Microscopis Electrònics de Transmissió (TEM) convencionals i ja disponibles. Aquestes tècniques generen patrons de difracció de monocristalls de grandària nanomètrica amb intensitats de dispersió millorades utilitzant feixos d'electrons. Aquests patrons s'adquireixen mentre el nanocristall gira dins del goniòmetre del TEM, resultant en patrons de difracció d'electrons (ED) de xicotets cristalls individuals de dimensions nanomètriques. A més, aquests mètodes requereixen el desenvolupament i l'optimització de tots els paràmetres experimentals que sovint exigeixen experiència en TEM. A més, els forts efectes de difracció múltiple o efectes dinàmics poden dificultar l'obtenció precisa d'intensitats cinemàtiques a més que molts materials pateixen danys estructurals causats per la interacció amb el feix d'electrons. Per a abordar aquest problema, la combinació amb la tècnica de tomografia de difracció d'electrons amb precessió (PEDT), que aplica una precessió del feix juntament amb un manual de 3DED fàcil d'utilitzar, minimitzarà aquests efectes; portant a una recol·lecció de dades, processament i determinació d'estructures exitosos. Aquesta metodologia s'ha aplicat per a resoldre l'estructura de nous materials microporosos denominats ITQ-70 i ITQ-35, en forma sense calcinar, així com una zeolita ja coneguda en forma sense calcinar amb dues molècules orgàniques diferents en el seu interior com la ITQ-52. Totes aquestes estructures es presenten en forma sense calcinar contenint agents directors d'estructura en el seu interior. Aquest enfocament conduirà a la localització atòmica completa de materials de zeolita híbrids inorgànics i orgànics mitjançant l'accessibilitat de tècniques de difracció de raigs X i 3DED. Aquest avanç oferirà un protocol general prometedor per a desenvolupar una nova ferramenta de caracterització per a una determinació precisa de l'estructura de materials cristal·lins xicotets. / [EN] Microporous materials such as zeolites have been highlighted due to their broad impact on catalytic processes and industry. The knowledge of their crystalline structure and the study of their properties and applications are intrinsically related. Therefore, conventional diffraction techniques such as single-crystal and powder X-ray diffraction (SCXRD and PXRD) have been the usual methods to retrieve structural information. However, the limitation in growing crystals large enough for SCXRD and the strong peak overlapping in PXRD patterns frequently hamper the structure determination of these materials, with complex structures and large unit cell parameters. Therefore, studying state-of-the-art diffraction techniques for structural characterization in new microporous materials, such as zeolites, is paramount. The incentive of this work is to investigate Electron Diffraction Tomography (EDT), also known as Three-Dimensional Electron Diffraction (3DED). These methods can be an exceptional technique for structural analysis of microporous materials using conventional and already available Transmission Electron Microscopes (TEM). These techniques generate single-nanocrystal diffraction patterns with enhanced scattering intensities using electron beams. These patterns are acquired as the nanocrystal rotates within the TEM's goniometer, resulting in electron diffraction (ED) patterns from single small crystals of nanometer-scale dimensions. These methods require developing and optimizing all experimental parameters, often demanding TEM expertise operators. Additionally, strong multiple diffractions ("dynamical effects") can hinder the collection of accurate intensities. Moreover, many materials suffer structural damage by the interaction with the electron beam. To address this issue, the combination of the precession electron diffraction tomography (PEDT) technique, which applies a precession of the beam, together with a fast 3DED procedure will minimize these effects, leading to a successful data collection, processing, and structure determination. This methodology has been applied to solve the structure new microporous materials, named ITQ-70 and ITQ-35, in as-made form containing organic guest molecules, and one already known as-made zeolites structure with two different guest organic molecules, ITQ-52. This approach will lead to the complete atomic localization of the hybrid inorganic and organic zeolitic materials through the approachability of 3DED techniques combined with X-ray diffraction refinements. This will offer a promising general protocol for developing a novel characterization tool for an accurate structure determination from small crystal materials. / Este trabajo ha sido posible gracias al Instituto de Tecnología Química (ITQ), que me ofreció la posibilidad de realizar esta tesis doctoral, y al Ministerio de Ciencia y Educación que me otorgó un contrato predoctoral (PRE2018-083623), permitiéndome llevar a cabo esta tesis doctoral dentro del programa Severo Ochoa (SEV-2016-0683-18-3). Asimismo, expreso mi gratitud a los Servicios de Microscopia de la Universitat Politécnica de València por facilitarme los recursos de microscopia necesarios. Agradezco también a la Unión Europea y al Gobierno de España por la financiación de este trabajo a través de los proyectos CEX2021–001230-S, PID2022-136934OB- 100 y TED2021–130191B-C41. Y a la Generalitat Valenciana (Prometeo 2021/077). Además, parte de este estudio forma parte del programa Advanced Materials y apoyado por el MCIN con financiación parcial de los fondos Next Generation EU (PRTR-C17. I1) y por la Generalitat Valenciana (MFA/2022/012 and MFA/2022/047). / Tirado Castaño, JI. (2025). Advanced Electron Diffraction Techniques for Structural Elucidation of Microporous Materials [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/214460