Nanoscale mosaicity revealed in peptide microcrystals by scanning electron nanodiffraction.

Changes in lattice structure across sub-regions of protein crystals are challenging to assess when relying on whole crystal measurements. Because of this difficulty, macromolecular structure determination from protein micro and nanocrystals requires assumptions of bulk crystallinity and domain block substructure. Here we map lattice structure across micron size areas of cryogenically preserved three-dimensional peptide crystals using a nano-focused electron beam. This approach produces diffraction from as few as 1500 molecules in a crystal, is sensitive to crystal thickness and three-dimensional lattice orientation. Real-space maps reconstructed from unsupervised classification of diffraction patterns across a crystal reveal regions of crystal order/disorder and three-dimensional lattice tilts on the sub-100nm scale. The nanoscale lattice reorientation observed in the micron-sized peptide crystal lattices studied here provides a direct view of their plasticity. Knowledge of these features facilitates an improved understanding of peptide assemblies that could aid in the determination of structures from nano- and microcrystals by single or serial crystal electron diffraction

Structures at the Core of Mammalian Prions

Prion diseases, also known as Transmissible Spongiform Encephalopathies, are neurodegenerative diseases that pose a major threat to both humans and animals. They fall into a category of misfolding diseases known as amyloidoses where accumulation of fibrous protein aggregates correlates with disease symptoms. Unlike other amyloid diseases, prion diseases are infectious and not strictly linked with age. Victims of prion diseases experience dementia, hallucinations, and an inability to take care of themselves among other symptoms before inevitably succumbing to disease. While the time between presentation of symptoms and death is often less than one year, symptoms can take decades to appear. This makes the immediate cause of sporadic disease difficult to determine. Prion diseases currently lack any form of treatment or means of prevention outside of selective breeding in sheep, which takes advantage of a disease-preventing sequence polymorphism. Infectious prions share β-sheet rich cores that produce a cross-β diffraction pattern not observed in natively folded PrPC. Knowledge of the atomic structures adopted by prions will aid not only in structure-based drug design and prion disease prevention, but also provide answers to the centuries old question of what makes a protein infectious.The aim of this dissertation is to uncover the structural characteristics of prions that distinguish them from other amyloids. The dissertation is also aimed at uncovering molecular explanations for species barriers, whereby PrPSc from one species converts PrPC of another species to a misfolded form with an efficiency dependent on both the original and newly infected host prions. These aims will be achieved through a combination of technological advancements made in atomic-level amyloid structure determination, with a focus on micro-crystal electron diffraction (MicroED) of the building blocks that make up prion cores (chapters 1 and 2) and single particle cryo-electron microscopy (cryo-EM) of prion filaments (chapters 3 and 4)

Structures at the Core of Mammalian Prions

Prion diseases, also known as Transmissible Spongiform Encephalopathies, are neurodegenerative diseases that pose a major threat to both humans and animals. They fall into a category of misfolding diseases known as amyloidoses where accumulation of fibrous protein aggregates correlates with disease symptoms. Unlike other amyloid diseases, prion diseases are infectious and not strictly linked with age. Victims of prion diseases experience dementia, hallucinations, and an inability to take care of themselves among other symptoms before inevitably succumbing to disease. While the time between presentation of symptoms and death is often less than one year, symptoms can take decades to appear. This makes the immediate cause of sporadic disease difficult to determine. Prion diseases currently lack any form of treatment or means of prevention outside of selective breeding in sheep, which takes advantage of a disease-preventing sequence polymorphism. Infectious prions share β-sheet rich cores that produce a cross-β diffraction pattern not observed in natively folded PrPC. Knowledge of the atomic structures adopted by prions will aid not only in structure-based drug design and prion disease prevention, but also provide answers to the centuries old question of what makes a protein infectious.The aim of this dissertation is to uncover the structural characteristics of prions that distinguish them from other amyloids. The dissertation is also aimed at uncovering molecular explanations for species barriers, whereby PrPSc from one species converts PrPC of another species to a misfolded form with an efficiency dependent on both the original and newly infected host prions. These aims will be achieved through a combination of technological advancements made in atomic-level amyloid structure determination, with a focus on micro-crystal electron diffraction (MicroED) of the building blocks that make up prion cores (chapters 1 and 2) and single particle cryo-electron microscopy (cryo-EM) of prion filaments (chapters 3 and 4)

Data-driven challenges and opportunities in crystallography.

Structural biology is in the midst of a revolution fueled by faster and more powerful instruments capable of delivering orders of magnitude more data than their predecessors. This increased pace in data gathering introduces new experimental and computational challenges, frustrating real-time processing and interpretation of data and requiring long-term solutions for data archival and retrieval. This combination of challenges and opportunities is driving the exploration of new areas of structural biology, including studies of macromolecular dynamics and the investigation of molecular ensembles in search of a better understanding of conformational landscapes. The next generation of instruments promises to yield even greater data rates, requiring a concerted effort by institutions, centers and individuals to extract meaning from every bit and make data accessible to the community at large, facilitating data mining efforts by individuals or groups as analysis tools improve

Data-driven challenges and opportunities in crystallography.

Structural biology is in the midst of a revolution fueled by faster and more powerful instruments capable of delivering orders of magnitude more data than their predecessors. This increased pace in data gathering introduces new experimental and computational challenges, frustrating real-time processing and interpretation of data and requiring long-term solutions for data archival and retrieval. This combination of challenges and opportunities is driving the exploration of new areas of structural biology, including studies of macromolecular dynamics and the investigation of molecular ensembles in search of a better understanding of conformational landscapes. The next generation of instruments promises to yield even greater data rates, requiring a concerted effort by institutions, centers and individuals to extract meaning from every bit and make data accessible to the community at large, facilitating data mining efforts by individuals or groups as analysis tools improve

Analysis of Global and Site-Specific Radiation Damage in Cryo-EM

Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data

Homochiral and racemic MicroED structures of a peptide repeat from the ice-nucleation protein InaZ

The ice-nucleation protein InaZ from Pseudomonas syringae contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. Ab initio structures of this repeat segment, resolved to 1.1 Å by microfocus X-ray crystallography and to 0.9 Å by the cryo-EM method MicroED, were determined from both racemic and homochiral crystals. The benefits of racemic protein crystals for structure determination by MicroED were evaluated and it was confirmed that the phase restriction introduced by crystal centrosymmetry increases the number of successful trials during the ab initio phasing of the electron diffraction data. Both homochiral and racemic GSTSTA form amyloid-like protofibrils with labile, corrugated antiparallel β-sheets that mate face to back. The racemic GSTSTA protofibril represents a new class of amyloid assembly in which all-left-handed sheets mate with their all-right-handed counterparts. This determination of racemic amyloid assemblies by MicroED reveals complex amyloid architectures and illustrates the racemic advantage in macromolecular crystallography, now with submicrometre-sized crystals

Cryo-EM structure of a human prion fibril with a hydrophobic, protease-resistant core.

Self-templating assemblies of the human prion protein are clinically associated with transmissible spongiform encephalopathies. Here we present the cryo-EM structure of a denaturant- and protease-resistant fibril formed in vitro spontaneously by a 9.7-kDa unglycosylated fragment of the human prion protein. This human prion fibril contains two protofilaments intertwined with screw symmetry and linked by a tightly packed hydrophobic interface. Each protofilament consists of an extended beta arch formed by residues 106 to 145 of the prion protein, a hydrophobic and highly fibrillogenic disease-associated segment. Such structures of prion polymorphs serve as blueprints on which to evaluate the potential impact of sequence variants on prion disease