Matching Items (7)
Description
Over the last century, X-ray crystallography has been established as the most successful technique for unravelling the structure-function relationship in molecules. For integral membrane proteins, growing well-ordered large crystals is a challenge and hence, there is room for improving current methods of macromolecular crystallography and for exploring complimentary techniques. Since protein function is deeply associated with its structural dynamics, static position of atoms in a macromolecule are insufficient to unlock the mechanism.
The availability of X-ray free electron lasers presents an opportunity to study micron-sized crystals that could be triggered (using light, small molecules or physical conditions) to capture macromolecules in action. This method of ‘Time-resolved serial crystallography’ answers key biological questions by capturing snapshots of conformational changes associated with multi-step reactions. This dissertation describes approaches for studying structures of large membrane protein complexes. Both macro and micro-seeding techniques have been implemented for improving crystal quality and obtaining high-resolution structures. Well-diffracting 15-20 micron crystals of active Photosystem II were used to perform time-resolved studies with fixed-target Roadrunner sample delivery system. By employing continuous diffraction obtained up to 2 A, significant progress can be made towards understanding the process of water oxidation.
Structure of Photosystem I was solved to 2.3 A by X-ray crystallography and to medium resolution of 4.8 A using Cryogenic electron microscopy. Using complimentary techniques to study macromolecules provides an insight into differences among methods in structural biology. This helps in overcoming limitations of one specific technique and contributes in greater knowledge of the molecule under study.
The availability of X-ray free electron lasers presents an opportunity to study micron-sized crystals that could be triggered (using light, small molecules or physical conditions) to capture macromolecules in action. This method of ‘Time-resolved serial crystallography’ answers key biological questions by capturing snapshots of conformational changes associated with multi-step reactions. This dissertation describes approaches for studying structures of large membrane protein complexes. Both macro and micro-seeding techniques have been implemented for improving crystal quality and obtaining high-resolution structures. Well-diffracting 15-20 micron crystals of active Photosystem II were used to perform time-resolved studies with fixed-target Roadrunner sample delivery system. By employing continuous diffraction obtained up to 2 A, significant progress can be made towards understanding the process of water oxidation.
Structure of Photosystem I was solved to 2.3 A by X-ray crystallography and to medium resolution of 4.8 A using Cryogenic electron microscopy. Using complimentary techniques to study macromolecules provides an insight into differences among methods in structural biology. This helps in overcoming limitations of one specific technique and contributes in greater knowledge of the molecule under study.
ContributorsRoy Chowdhury, Shatabdi (Author) / Fromme, Petra (Thesis advisor) / Ros, Alexandra (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2018
Description
Scientists have used X-rays to study biological molecules for nearly a century. Now with the X-ray free electron laser (XFEL), new methods have been developed to advance structural biology. These new methods include serial femtosecond crystallography, single particle imaging, solution scattering, and time resolved techniques.
The XFEL is characterized by high intensity pulses, which are only about 50 femtoseconds in duration. The intensity allows for scattering from microscopic particles, while the short pulses offer a way to outrun radiation damage. XFELs are powerful enough to obliterate most samples in a single pulse. While this allows for a “diffract and destroy” methodology, it also requires instrumentation that can position microscopic particles into the X-ray beam (which may also be microscopic), continuously renew the sample after each pulse, and maintain sample viability during data collection.
Typically these experiments have used liquid microjets to continuously renew sample. The high flow rate associated with liquid microjets requires large amounts of sample, most of which runs to waste between pulses. An injector designed to stream a viscous gel-like material called lipidic cubic phase (LCP) was developed to address this problem. LCP, commonly used as a growth medium for membrane protein crystals, lends itself to low flow rate jetting and so reduces the amount of sample wasted significantly.
This work discusses sample delivery and injection for XFEL experiments. It reviews the liquid microjet method extensively, and presents the LCP injector as a novel device for serial crystallography, including detailed protocols for the LCP injector and anti-settler operation.
The XFEL is characterized by high intensity pulses, which are only about 50 femtoseconds in duration. The intensity allows for scattering from microscopic particles, while the short pulses offer a way to outrun radiation damage. XFELs are powerful enough to obliterate most samples in a single pulse. While this allows for a “diffract and destroy” methodology, it also requires instrumentation that can position microscopic particles into the X-ray beam (which may also be microscopic), continuously renew the sample after each pulse, and maintain sample viability during data collection.
Typically these experiments have used liquid microjets to continuously renew sample. The high flow rate associated with liquid microjets requires large amounts of sample, most of which runs to waste between pulses. An injector designed to stream a viscous gel-like material called lipidic cubic phase (LCP) was developed to address this problem. LCP, commonly used as a growth medium for membrane protein crystals, lends itself to low flow rate jetting and so reduces the amount of sample wasted significantly.
This work discusses sample delivery and injection for XFEL experiments. It reviews the liquid microjet method extensively, and presents the LCP injector as a novel device for serial crystallography, including detailed protocols for the LCP injector and anti-settler operation.
ContributorsJames, Daniel (Author) / Spence, John (Thesis advisor) / Weierstall, Uwe (Committee member) / Kirian, Richard (Committee member) / Schmidt, Kevin (Committee member) / Arizona State University (Publisher)
Created2015
Description
Microfluidic devices represent a growing technology in the world of analytical chemistry. Serial femtosecond crystallography (SFX) utilizes microfluidic devices to generate droplets of an aqueous buffer containing protein crystals, which are then fired out as a jet in the beam of an X-ray free electron laser (XFEL). A crucial part of the device is its method of droplet detection. This project presents a design for a capacitive sensor that uses a unique electrode configuration to detect the difference in capacitance between the aqueous and oil phases. This design was developed using MATLAB and COMSOL Multiphysics simulations and printed using high-resolution 3D printing. Results show that this design can successfully distinguish between the two immiscible liquids, confirming it as a possible detection method in future SFX experiments.
ContributorsCorder, Cameron Dean (Author) / Ros, Alexandra (Thesis director) / Williams, Peter (Committee member) / Hayes, Mark (Committee member) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has enabled the determination of damage-free protein structures at ambient temperatures and of reaction intermediate species with time resolution on the order of hundreds of femtoseconds. However, currently available XFEL facility X-ray pulse structures waste the majority of continuously injected crystal sample, requiring a large quantity (up to grams) of crystal sample to solve a protein structure. Furthermore, mix-and-inject serial crystallography (MISC) at XFEL facilities requires fast mixing for short (millisecond) reaction time points (𝑡"), and current sample delivery methods have complex fabrication and assembly requirements.
To reduce sample consumption during SFX, a 3D printed T-junction for generating segmented aqueous-in-oil droplets was developed. The device surface properties were characterized both with and without a surface coating for improved droplet generation stability. Additionally, the droplet generation frequency was characterized. The 3D printed device interfaced with gas dynamic virtual nozzles (GDVNs) at the Linac Coherent Light Source (LCLS), and a relationship between the aqueous phase volume and the resulting crystal hit rate was developed. Furthermore, at the European XFEL (EuXFEL) a similar quantity and quality of diffraction data was collected for segmented sample delivery using ~60% less sample volume than continuous injection, and a structure of 3-deoxy-D-manno- octulosonate 8-phosphate synthase (KDO8PS) delivered by segmented injection was solved that revealed new structural details to a resolution of 2.8 Å.
For MISC, a 3D printed hydrodynamic focusing mixer for fast mixing by diffusion was developed to automate device fabrication and simplify device assembly. The mixer was characterized with numerical models and fluorescence microscopy. A variety of devices were developed to reach reaction intermediate time points, 𝑡", on the order of 100 – 103 ms. These devices include 3D printed mixers coupled to glass or 3D printed GDVNs and two designs of mixers with GDVNs integrated into the one device. A 3D printed mixer coupled to a glass GDVN was utilized at LCLS to study the oxidation of cytochrome c oxidase (CcO), and a structure of the CcO Pr intermediate was determined at 𝑡" = 8 s.
To reduce sample consumption during SFX, a 3D printed T-junction for generating segmented aqueous-in-oil droplets was developed. The device surface properties were characterized both with and without a surface coating for improved droplet generation stability. Additionally, the droplet generation frequency was characterized. The 3D printed device interfaced with gas dynamic virtual nozzles (GDVNs) at the Linac Coherent Light Source (LCLS), and a relationship between the aqueous phase volume and the resulting crystal hit rate was developed. Furthermore, at the European XFEL (EuXFEL) a similar quantity and quality of diffraction data was collected for segmented sample delivery using ~60% less sample volume than continuous injection, and a structure of 3-deoxy-D-manno- octulosonate 8-phosphate synthase (KDO8PS) delivered by segmented injection was solved that revealed new structural details to a resolution of 2.8 Å.
For MISC, a 3D printed hydrodynamic focusing mixer for fast mixing by diffusion was developed to automate device fabrication and simplify device assembly. The mixer was characterized with numerical models and fluorescence microscopy. A variety of devices were developed to reach reaction intermediate time points, 𝑡", on the order of 100 – 103 ms. These devices include 3D printed mixers coupled to glass or 3D printed GDVNs and two designs of mixers with GDVNs integrated into the one device. A 3D printed mixer coupled to a glass GDVN was utilized at LCLS to study the oxidation of cytochrome c oxidase (CcO), and a structure of the CcO Pr intermediate was determined at 𝑡" = 8 s.
ContributorsEchelmeier, Austin (Author) / Ros, Alexandra (Thesis advisor) / Levitus, Marcia (Committee member) / Weierstall, Uwe (Committee member) / Arizona State University (Publisher)
Created2019
Description
Serial femtosecond crystallography (SFX) uses diffraction patterns from crystals delivered in a serial fashion to an X-Ray Free Electron Laser (XFEL) for structure determination. Typically, each diffraction pattern is a snapshot from a different crystal. SFX limits the effect of radiation damage and enables the use of nano/micro crystals for structure determination. However, analysis of SFX data is challenging since each snapshot is processed individually.
Many photosystem II (PSII) dataset have been collected at XFELs, several of which are time-resolved (containing both dark and laser illuminated frames). Comparison of light and dark datasets requires understanding systematic errors that can be introduced during data analysis. This dissertation describes data analysis of PSII datasets with a focus on the effect of parameters on later results. The influence of the subset of data used in the analysis is also examined and several criteria are screened for their utility in creating better subsets of data. Subsets are compared with Bragg data analysis and continuous diffuse scattering data analysis.
A new tool, DatView aids in the creation of subsets and visualization of statistics. DatView was developed to improve the loading speed to visualize statistics of large SFX datasets and simplify the creation of subsets based on the statistics. It combines the functionality of several existing visualization tools into a single interface, improving the exploratory power of the tool. In addition, it has comparison features that allow a pattern-by-pattern analysis of the effect of processing parameters. \emph{DatView} improves the efficiency of SFX data analysis by reducing loading time and providing novel visualization tools.
Many photosystem II (PSII) dataset have been collected at XFELs, several of which are time-resolved (containing both dark and laser illuminated frames). Comparison of light and dark datasets requires understanding systematic errors that can be introduced during data analysis. This dissertation describes data analysis of PSII datasets with a focus on the effect of parameters on later results. The influence of the subset of data used in the analysis is also examined and several criteria are screened for their utility in creating better subsets of data. Subsets are compared with Bragg data analysis and continuous diffuse scattering data analysis.
A new tool, DatView aids in the creation of subsets and visualization of statistics. DatView was developed to improve the loading speed to visualize statistics of large SFX datasets and simplify the creation of subsets based on the statistics. It combines the functionality of several existing visualization tools into a single interface, improving the exploratory power of the tool. In addition, it has comparison features that allow a pattern-by-pattern analysis of the effect of processing parameters. \emph{DatView} improves the efficiency of SFX data analysis by reducing loading time and providing novel visualization tools.
ContributorsStander, Natasha (Author) / Fromme, Petra (Thesis advisor) / Zatsepin, Nadia (Thesis advisor) / Kirian, Richard (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2019
Description
Novel electric field-assisted microfluidic platforms were developed to exploit unique migration phenomena, particle manipulation, and enhanced droplet control. The platforms can facilitate various analytical challenges such as size-based separations, and delivery of protein crystals for structural discovery with both high selectivity and sensitivity. The vast complexity of biological analytes requires efficient transport and fractionation approaches to understand variations of biomolecular processes and signatures. Size heterogeneity is one characteristic that is especially important to understand for sub-micron organelles such as mitochondria and lipid droplets. It is crucial to resolve populations of sub-cellular or diagnostically relevant bioparticles when these often cannot be resolved with traditional methods. Herein, novel microfluidic tools were developed for the unique migration mechanism capable of separating sub-micron sized bioparticles by size. This based on a deterministic ratchet effect in a symmetrical post array with dielectrophoresis (DEP) for the fast migration allowing separation of polystyrene beads, mitochondria, and liposomes in tens of seconds. This mechanism was further demonstrated using high throughput DEP-based ratchet devices for versatile, continuous sub-micron size particle separation with large sample volumes. Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) revolutionized protein structure determination. In SFX experiments, a majority of the continuously injected liquid crystal suspension is wasted due to the unique X-ray pulse structure of XFELs, requiring a large amount (up to grams) of crystal sample to determine a protein structure. To reduce the sample consumption in such experiments, 3D printed droplet-based microfluidic platforms were developed for the generation of aqueous droplets in an oil phase. The implemented droplet-based sample delivery method showed 60% less sample volume consumption compared to the continuous injection at the European XFEL. For the enhanced control of aqueous droplet generation, the device allowed dynamic triggering of droplets for further improvement in synchronization between droplets and the X-ray pulses. This innovative technique of triggering droplets can play a crucial role in saving protein crystals in future SFX experiments. The electric field-assisted unique migration and separation phenomena in microfluidic platforms will be the key solution for revolutionizing the field of organelle separation and structural analysis of proteins.
ContributorsKim, Dai Hyun (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2020
Description
This thesis focuses on serial crystallography studies with X-ray free electron lasers
(XFEL) with a special emphasis on data analysis to investigate important processes
in bioenergy conversion and medicinal applications.
First, the work on photosynthesis focuses on time-resolved femtosecond crystallography
studies of Photosystem II (PSII). The structural-dynamic studies of the water
splitting reaction centering on PSII is a current hot topic of interest in the field, the
goal of which is to capture snapshots of the structural changes during the Kok cycle.
This thesis presents results from time-resolved serial femtosecond (fs) crystallography
experiments (TR-SFX) where data sets are collected at room temperature from a
stream of crystals that intersect with the ultrashort femtosecond X-ray pulses at an
XFEL with the goal to obtain structural information from the transient state (S4)
state of the cycle where the O=O bond is formed, and oxygen is released. The most
current techniques available in SFX/TR-SFX to handle hundreds of millions of raw
diffraction patterns are discussed, including selection of the best diffraction patterns,
allowing for their indexing and further data processing. The results include two 4.0 Å
resolution structures of the ground S1 state and triple excited S4 transient state.
Second, this thesis reports on the first international XFEL user experiments in
South Korea at the Pohang Accelerator Laboratory (PAL-XFEL). The usability of this
new XFEL in a proof-of-principle experiment for the study of microcrystals of human
taspase1 (an important cancer target) by SFX has been tested. The descriptions of
experiments and discussions of specific data evaluation challenges of this project in
light of the taspase1 crystals’ high anisotropy, which limited the resolution to 4.5 Å,
are included in this report
In summary, this thesis examines current techniques that are available in the
SFX/TR-SFX domain to study crystal structures from microcrystals damage-free,
with the future potential of making movies of biological processes.
(XFEL) with a special emphasis on data analysis to investigate important processes
in bioenergy conversion and medicinal applications.
First, the work on photosynthesis focuses on time-resolved femtosecond crystallography
studies of Photosystem II (PSII). The structural-dynamic studies of the water
splitting reaction centering on PSII is a current hot topic of interest in the field, the
goal of which is to capture snapshots of the structural changes during the Kok cycle.
This thesis presents results from time-resolved serial femtosecond (fs) crystallography
experiments (TR-SFX) where data sets are collected at room temperature from a
stream of crystals that intersect with the ultrashort femtosecond X-ray pulses at an
XFEL with the goal to obtain structural information from the transient state (S4)
state of the cycle where the O=O bond is formed, and oxygen is released. The most
current techniques available in SFX/TR-SFX to handle hundreds of millions of raw
diffraction patterns are discussed, including selection of the best diffraction patterns,
allowing for their indexing and further data processing. The results include two 4.0 Å
resolution structures of the ground S1 state and triple excited S4 transient state.
Second, this thesis reports on the first international XFEL user experiments in
South Korea at the Pohang Accelerator Laboratory (PAL-XFEL). The usability of this
new XFEL in a proof-of-principle experiment for the study of microcrystals of human
taspase1 (an important cancer target) by SFX has been tested. The descriptions of
experiments and discussions of specific data evaluation challenges of this project in
light of the taspase1 crystals’ high anisotropy, which limited the resolution to 4.5 Å,
are included in this report
In summary, this thesis examines current techniques that are available in the
SFX/TR-SFX domain to study crystal structures from microcrystals damage-free,
with the future potential of making movies of biological processes.
ContributorsKetawala, Gihan Kaushyal (Author) / Fromme, Petra (Thesis advisor) / Liu, Wei (Committee member) / Kirian, Richard (Committee member) / Arizona State University (Publisher)
Created2020