Machine Learning on Neutron and X-Ray Scattering

Neutron and X-ray scattering represent two state-of-the-art materials characterization techniques that measure materials' structural and dynamical properties with high precision. These techniques play critical roles in understanding a wide variety of materials systems, from catalysis to polymers, nanomaterials to macromolecules, and energy materials to quantum materials. In recent years, neutron and X-ray scattering have received a significant boost due to the development and increased application of machine learning to materials problems. This article reviews the recent progress in applying machine learning techniques to augment various neutron and X-ray scattering techniques. We highlight the integration of machine learning methods into the typical workflow of scattering experiments. We focus on scattering problems that faced challenge with traditional methods but addressable using machine learning, such as leveraging the knowledge of simple materials to model more complicated systems, learning with limited data or incomplete labels, identifying meaningful spectra and materials' representations for learning tasks, mitigating spectral noise, and many others. We present an outlook on a few emerging roles machine learning may play in broad types of scattering and spectroscopic problems in the foreseeable future.Comment: 56 pages, 12 figures. Feedback most welcom

Biological Systems Workbook: Data modelling and simulations at molecular level

Nowadays, there are huge quantities of data surrounding the different fields of biology derived from experiments and theoretical simulations, where results are often stored in biological databases that are growing at a vertiginous rate every year. Therefore, there is an increasing research interest in the application of mathematical and physical models able to produce reliable predictions and explanations to understand and rationalize that information. All these investigations are helping to overcome biological questions pushing forward in the solution of problems faced by our society. In this Biological Systems Workbook, we aim to introduce the basic pieces allowing life to take place, from the 3D structural point of view. We will start learning how to look at the 3D structure of molecules from studying small organic molecules used as drugs. Meanwhile, we will learn some methods that help us to generate models of these structures. Then we will move to more complex natural organic molecules as lipid or carbohydrates, learning how to estimate and reproduce their dynamics. Later, we will revise the structure of more complex macromolecules as proteins or DNA. Along this process, we will refer to different computational tools and databases that will help us to search, analyze and model the different molecular systems studied in this course

Innovative ferritin nanocages for drug-delivery and biotechnological applications

The work presented in this thesis aimed to set out the basis for the rational design of innovative protein-based carriers for drug-delivery and biotechnological applications. In this context, ferritin stood out as promising protein system due to its remarkable characteristics. Ferritins are versatile biocompatible proteins scaffolds that display a cage-like structure that provides shielding of the cavity’s content from harsh external conditions, and are amenable to modifications in a relatively straightforward manner. Previously published information noted that the proteic cage of ferritins presents an unexpected degree of plasticity in that it can undergo significant structural rearrangements and thus trap small molecules within the internal cavity, a process referred to as “encapsulation”. However the assembled state of ferritin’s cage has always been seen as an impermeable structure, in which the communication between the internal cavity and the exterior is uniquely governed by the gating effect of the channels located at the threefold symmetry axes. In order to clarify the process of molecule confinement within ferritin the internal cavity, a critical analysis of the process of ligand entry/release through the protein matrices of ferritins of different origin was carried. Thus the kinetics of the disulfide bond formation between DTNB and engineered cysteines at selected positions were followed. Kinetics were found to be in the order of tens of seconds, a time frame likely to reflect the slow crossing of the protein matrix.The obtained data indicates that the protein matrix does not provide a significant barrier against bulky ligands such as DTNB, which, due to its dimensions (8-10 Å) and its net negative charge was thought unlinke to cross the protein shell. Within this vision, the technological effort of protein engineering for payload delivery may be most conveniently addressed to modification of the properties of the surface of the internal cavity rather than to possible rearrangements of the threefold channels. In an effort to develop versatile ferritin-based drug-delivery systems, archaeal ferritins appeared particularly promising as scaffolds. These proteins displays unique assembly properties and extreme thermodynamic stability, however lack the cell recognition properties of human ferritin. Thus a chimeric protein was designed in which the relevant recognition sequence of human ferritin was grafted into the corresponding sequence into archaeal ferritin surface exposed regions in order to confer specific recognition of human epitopes while keeping the unique salt-dependent assembly reaction. The construct structure was determined by X-ray crystallography and successfully shown to be actively uptaken via the Transferrin Receptor 1, a receptor known to be overexpressed in cancer cells. With the aim of improving current encapsulation methods the assembly properties of the novel archaeal-human chimeric ferritin nanocarrier were investigated and the effect of divalent cation investigated. Data demonstrated that physiological Mg2+ concentrations are sufficient to promote full assembly and that assembly takes place in a highly cooperative and fast manner, driven mostly by hydrophobic forces. However, at present, the effect of divalent cations has not been translated into a model of assembly mechanism, though, taken together, data indicates that subunit oligomerization may possibly follow an analogous mechanism as identified for cation-induced assembly of viral capsids. The understanding of the major forces governing the assembly provides key elements for the development of strategies for efficient encapsulation/ release of probes in a controlled way. On a different approach, the project aimed to exploit the versatility of ferritins for bioimaging applications by inserting an extra functional segment per monomer for binding of luminescent lanthanides, within mouse H ferritin’s cavity. This extra segment possesses one high affinity Terbium binding site provided by six coordinating aminoacid side chains and a tryptophan residue in its close proximity for FRET sensitization. Accordingly, the construct demonstrated lanthanide fluorescence detectable in the pM concentration range and demonstrated to be actively uptaken by selected tumor cell lines by confocal microscopy and FACS analysis of their FITC derivatives. Crystallographic data shown that introduction segment did not disrupt the cage assembly, and the presence of a total of 56 Tb(III) atoms per 24mer. These systems could be used for advanced cell imaging applications, merging the recognition capabilities of ferritins with the notable properties of lanthanide-based fluorescence

Protein Shape Description and its Application to Shape Comparison

ProSHADE Repository: http://fg.oisin.rc-harwell.ac.uk/projects/proshade/There are currently over 138, 000 known macromolecular structures deposited in the wwPDB (Worldwide Protein Data Bank) database. While all the macromolecular structure files contain information about a particular structure, the collection of these files also allows combining the macromolecular structures to obtain statistical information about macromolecules in general. This fact has been the basis for many structural biology methods including the molecular replacement method used in X-ray crystallography or homologous structure restraints in the refinement methods. With the success of methods based on prior information, it is feasible that novel methods could be developed and current methods improved using further prior information; more specifically, by using the structure density-map shape similarity instead of sequence or model similarity. Therefore, this project introduces a mathematical framework for computing three different measures of macromolecular three-dimensional shape similarity and demonstrates how these descriptors can be applied in symmetry detection and protein-domain clustering. The ability to detect cyclic (C), dihedral (D), tetrahedral (T), octahedral (O) and icosahedral (I) symmetry groups as well as computing all associated symmetry elements has direct applications in map averaging and reducing the storage requirements by storing only the asymmetric information. Moreover, by having the capacity to find structures with similar shape, it was possible to reduce the size of the BALBES protein domain database by more than 18.7% and thus achieve proportional speed-up in the searching parts of its applications. Finally, the development of the method described in this project has many possible applications throughout structural biology. The method could, for example, facilitate matching and fitting of protein domains into the density maps produced by the electron-microscopy techniques, or it could allow for molecular-replacement candidate search using shape instead of sequence similarity. To allow for the development of any further applications, software for applying the methods described here is also presented and released for the community.Medical Research Council ( MC_US_A025_1012

A General, Symmetry-Based Approach for the Assembly of Proteins into Nanoscale Polyhedra.

The assembly of individual protein subunits into large-scale symmetrical structures is widespread in Nature and confers unique biological properties which have potential applications in nano-technology and medicine. While efforts to functionalize and repurpose existing protein complexes have been mainly successful, designing well-defined de novo protein complexes remains an unsolved problem. A major challenge in engineering de novo symmetrical assemblies has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Prior de novo protein cages have been developed with moderate success, but suffer from a lack of generalizability and require significant computational effort and screening of mutant fusion proteins. The design and optimization of a simple, generalizable approach to designing novel fusion proteins which assemble into cage-like structures will be the subject of this dissertation. We show that by genetically fusing a C4-symmetric coiled-coil to the C-terminus of a C3-symmetric trimeric protein via a short, flexible linker, we can assemble a well-defined 24-subunit protein cage with octahedral symmetry. The flexible nature of these assemblies alleviates the need for rigorous interface modeling, requiring only minimal computation to determine the length of the linker sequence. This is the first de novo designed symmetrical protein complex to incorporate a C4 symmetry element, and we anticipate this method can be applied to a wider variety of proteins and symmetries, which may open up a new avenue of research into designer protein cages with unique, built-in functionalities.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/120840/1/sciore_1.pd

Symmetry-Based Design of Protein Nano-Cages

The self-assembly of protein subunits into large-scale oligomeric structures is a powerful and ubiquitous feature of biology, with viral capsids providing an excellent example. These assemblies perform a diverse set of functional and structural roles in living systems. Additionally, proteins can be modified both genetically and chemically to introduce new properties. Because of these attractive properties, natural and de novo designed protein assemblies have already been evaluated for various applications in medicine and materials science. This thesis explores a recently developed, generalizable coiled coil based strategy for de novo designing protein cages to utilize for applications in these fields. The design strategy relies on the combination of 2 rotational symmetry elements, one provided by the natural, building block protein (BBP) and the other provided by the coiled coil, to specify a protein cage of the desired geometry. The oligomerization of the coiled coil brings the copies of the BBP together, leading to the assembly of protein cages. By employing BBPs and coiled coils of different rotational symmetries, cages of various sizes and geometries have been designed, including both tetrahedral and octahedral protein cages. This thesis extends these studies to more ambitious design targets and explores the generalizability of this approach. Because the design strategy requires well-specified homo-oligomeric parallel-coiled coils, the utility of several selected de novo designed coiled coils was first evaluated as off-the-shelf components for protein assembly, using green fluorescent protein as a model system. This study revealed context-dependent oligomerization state changes for some of these coiled coils. Next, the potential of elaborating previously designed protein cages by attaching additional protein domains to free end of the coiled coil was investigated. As a proof-of-concept, an octahedral cage was elaborated by fusing a large monomeric protein to the free end of the coiled coil assembly domain. This design successfully self-assembled into a homogeneous octahedral protein cage of ~ 1.8 MDa, significantly the addition of the extra protein domain dramatically improved the yield and efficiency of protein assembly. The design strategy was extended to the de novo design of an icosahedral protein cage by fusing a pentameric coiled coil to the trimeric BBP previously utilized for octahedral and tetrahedral cage designs. After optimization, a construct with an 8-residue oligo-glycine spacer successfully assembled into a hyperstable 60-subunit protein cage with icosahedral geometry and molecular weight of ~ 2.1 MDa. Surprisingly, these cages captured short DNA strands during purification which were important to maintain the homogeneity of the cages. The cages could be transiently disassembled by treating with Dnase; the re-assembled cages were significantly more heterogeneous. The hyperstability and ability to capture DNA are new emergent properties of this design that arise from assembly and were not evident in previously designed cages. Finally, the potential of extending this symmetry-based strategy to design protein cages that assemble in response to environmental stimuli was investigated. This study was conducted by fusing a de novo designed metal-dependent coiled coil to the trimeric BBP. The construct successfully assembled into discrete particles in the presence of divalent transition metal ions; adding metal chelators or decreasing pH led to disassembly of these particles into their trimeric form.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/149900/1/scristie_1.pd

Image Processing Algorithms for Electron Microscopy

The development of the electron microscopy (EM) has led to new algorithms devoted to the analysis of the images obtained by the EM know as micrographs. These images are characterized for being noisy, making the extraction of information a difficult task. To that end, several reconstruction packages are being developed that provide with powerful tools to denoise, extract and analyze the micrographs to obtain the 3D model (volume) of the sample. Since there is no a gold standard that can be followed to process all micrographs in a single way, researches rely on the combination of algorithms of several packages to get the optimal result. However, the lack of consensus between the packages makes difficult to combine different programs. Also, the algorithms should be made as simple and automatic as possible to facilitate the task of the researchers without degrading the quality of the results. The purpose of this bachelor thesis is to introduce required algorithms that can be used to reconstruct the 3D model of a sample out of the micrographs acquired by the electron microscope. The analysis will be focused on two individual and complementary tasks: the development of a mathematical basis aimed at the automatization and simplification of the deformation calculations carried out to optimize the 3D templates used to reconstruct new 3D models, and the protocolization of a package that uses ab initio methods to avoid the requirements of a 3D template to reconstruct a model. The algorithms will be tested in order to validate and analyze their performance, so they can be used in real problems and applications.Ingeniería Biomédic