About

Sabine Botha is a Research Assistant Professor in the Department of Physics at Arizona State University. She is affiliated with the Biodesign Beus CXFEL Lab, the CXFEL Faculty, and the Biodesign Center for Applied Structural Discovery. Her research focuses on applied structural discovery, leveraging advanced facilities and interdisciplinary collaboration to explore structural phenomena. Her work contributes to the understanding and development of structural analysis techniques within the context of biodesign and applied physics.

Research topics

• Computer Science
• Chemistry
• Optics
• Biology
• Biochemistry
• Crystallography
• Physics
• Medicine
• Computational biology
• Nanotechnology
• Chromatography
• Materials science
• Bioinformatics
• Biophysics
• Virology
• Mechanics
• Pharmacology

Selected publications

• Minimized sample consumption for time-resolved serial crystallography applied to the redox cycle of human NQO1

  Communications Chemistry · 2026-01-29

  articleOpen access

  Sample consumption for serial femtosecond crystallography with X-ray free electron lasers remains a major limitation preventing broader use in macromolecular crystallography. This drawback is exacerbated in time-resolved (TR) experiments, where the amount of sample required per reaction time point is multiplied by the number of time points investigated. To reduce this limitation, we demonstrate a segmented droplet generation strategy coupled to a mix-and-inject approach for TR studies at the European XFEL. The injector produces synchronized droplet trains that enable stable and reproducible injection of protein crystal slurries at significantly reduced flow rates. Using the human flavoenzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a test system, we collected diffraction data after mixing with NADH at 0.3 s and 1.2 s delays. The segmented injection approach achieved up to 97% reduction in sample consumption compared with continuous-flow injection while maintaining data quality suitable for TR crystallography. Reproducible electron density features consistent with low-occupancy NADH binding illustrate both the feasibility and the current limits of studying dynamic redox enzymes using this approach. This work establishes segmented droplet generation as a sample-efficient and XFEL-compatible method for future time-resolved serial crystallography experiments.

  PublisherOA PDFDOI

• PEO-sheathed liquid jets increase sample delivery stability for serial femtosecond X-ray crystallography

  Scientific Reports · 2026-03-25

  articleOpen access

  Viscoelastic jets can be generated by the polyethylene oxide (PEO) sheathing of an aqueous solution using double-flow focusing nozzles (DFFNs) and represent an efficient method to deliver samples that are dispersed in low and medium-viscosity liquids for X-ray diffractive imaging experiments. Due to their micrometre diameter and millimetre length, such jets can be used for pump-probe serial femtosecond crystallography (SFX) in order to access a timescale of a few tens of microseconds. This range is in between the previously achievable ranges accessible at XFELs (picoseconds-to-microsecond time delays) and synchrotrons (a few hundred µs to millisecond delays), respectively. Here, we demonstrate their effectiveness to deliver protein microcrystals (lysozyme and photosystem II) in buffer compositions of various viscosities for SFX and explore capabilities of triple-flow focusing nozzles (TFFNs) that incorporate PEO-sheathing to control challenging-to-jet viscous buffers for time-resolved diffusive mixing experiments.

  PublisherDOI

• Signal extraction in SWAXS data for the compact X-ray light sources: a machine learning approach

  Scientific Reports · 2026-04-07

  articleOpen access

  The development of X-ray free electron lasers has driven significant progress in X-ray science. Given the broad range of their applications, implementing a new generation of this technology at the laboratory scale has been under consideration for several years. This initiative is now under commissioning and construction at Arizona State University, known as the Compact X-ray Light Source (CXLS) and the Compact X-ray Free Electron Laser (CXFEL). Alongside experimental advances in this direction, whether in large or compact X-ray free electron lasers, there is also a growing need for new algorithmic and analytical methods to process the data obtained from such facilities. This work introduces a novel approach for analyzing Small- and Wide-Angle X-ray Scattering (SWAXS) profiles using a data-driven machine learning algorithm. The method is proposed for application to SWAXS datasets collected at both compact and large-scale X-ray facilities. To evaluate the performance of this approach, we analyzed simulated time-resolved SWAXS data from a protein, generated based on the current CXLS experimental parameters, and compared the results with those from the standard singular value decomposition (SVD) technique. Despite the low photon counts in the data, the results demonstrate that our method achieves higher accuracy in extracting structural dynamics information compared to SVD.

  PublisherOA PDFDOI

• Novel polymer fixed-target microfluidic platforms with an ultra-thin moisture barrier for serial macromolecular crystallography

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-07-18 · 2 citations

  preprintOpen access

  Abstract The advent of ultrabright fourth generation X-ray light sources, including X-ray free-electron lasers (XFELs) and diffraction limited synchrotrons, has significantly advanced the field of serial macromolecular protein crystallography (SX). SX experiments demand a continuous supply of fresh microcrystalline sample, ideally while minimizing overall sample consumption. Here, we introduce a novel, robust, and user-friendly polymer film technology that can be assembled in various configurations to encapsulate protein microcrystals and provide sample support for SX. This system provides an efficient hydration barrier over extended durations while maintaining an exceptionally low X-ray background. We have validated this technology by assessing hydration retention under both ambient and ultra-high vacuum conditions, and by evaluating its mechanical stability under XFEL pulses. Furthermore, we have demonstrated the effectiveness of this approach in two room-temperature serial crystallography studies to determine the structure of a 24 kDa Rapid Encystment Phenotype (REP24) protein from Franciscella tularensis .

  PublisherOA PDFDOI

• Data Analysis tools for the Compact X-ray Light Source and Compact X-ray Free Electron Laser facilities at Arizona State University

  Structural Dynamics · 2025-09-01

  articleOpen access1st authorCorresponding

  The Compact X-ray Light Source (Figure 1) and Compact X-ray Free Electron Laser [1], [2] that are currently undergoing commissioning and construction, respectively, at Arizona State University will be the first of their kind with applications in structural biology, medical imaging, atomic, molecular and optical physics as well as studying condensed matter and quantum materials. The unique characteristics of the ultrashort X-ray pulses produced by both sources at kilohertz repetition rates necessitate novel and new data analysis algorithms and tools as well as supportive cyber infrastructure at ASU. Here we will report on the current state of the data analysis support and tools currently under development at CXLS/CXFEL for studying biological macromolecules at these sources for both online (real- time) and offline analysis. These include an AI/ML-based crystal hitfinder, integration of an AI/ML-based image sorter [3], Small Angle X-ray Scattering analysis tools, calibration pipelines etc.

  PublisherOA PDFDOI

• PADT: A novel GUI for serial data classification using Machine Learning approaches

  Structural Dynamics · 2025-03-01

  articleOpen access1st authorCorresponding

  The structure solution of biological structure is being revolutionized by serial X-ray diffraction employing X-ray free-electron laser (XFEL) sources, particularly the advent of serial femtosecond crystallography (SFX). The measurements of micrometer- sized crystals at room temperature allow time-resolved studies to trace the path of biochemical reactions at unprecedented temporal resolution, made possible by X-ray pulses that outrun the effects of radiation damage. These pulses can deliver X-ray doses that are more than a thousand times higher than those that are possible with conventional X-ray sources.1 The large amounts of data produced by these studies (up to multiple TBs for a single experiment) must be quickly processed and analysed. While software for online data monitoring and data reduction have been designed over the past decade (e.g., OM2, Cheetah3, CrystFEL4), these are targeted towards finding crystal hits and not classifying data by spurious, often unquantifiable data artifacts. The state-of-the-art X-ray detectors are undergoing continuous development, and experimental parameters can push them beyond their reliable operating regime for individual frames within a single run of data collection. Including intensities from these frames into the merged structure factors can lead to inaccuracies in the final reported intensities, particularly detrimental for anomalous phasing or time-resolved difference density calculations where highly accurate recordings are required. Here, we report a new data sorting tool that offers a variety of Machine Learning algorithms to sort data trained on either manual sorting by the user, or by profile fitting the expected intensity distribution on the detector based on the experiment. This is integrated into an easy-to-use graphical user interface (GUI), specifically designed to support the detectors, file formats, and software available at the Linac Coherent Light Source (US) and the European XFEL (Germany). The structure solution of biological structure is being revolutionized by serial X-ray diffraction employing X-ray free-electron laser (XFEL) sources, particularly the advent of serial femtosecond crystallography (SFX). The measurements of micrometer- sized crystals at room temperature allow time-resolved studies to trace the path of biochemical reactions at unprecedented temporal resolution, made possible by X-ray pulses that outrun the effects of radiation damage. These pulses can deliver X-ray doses that are more than a thousand times higher than those that are possible with conventional X-ray sources.1 The large amounts of data produced by these studies (up to multiple TBs for a single experiment) must be quickly processed and analysed. While software for online data monitoring and data reduction have been designed over the past decade (e.g., OM2, Cheetah3, CrystFEL4), these are targeted towards finding crystal hits and not classifying data by spurious, often unquantifiable data artifacts. The state-of-the-art X-ray detectors are undergoing continuous development, and experimental parameters can push them beyond their reliable operating regime for individual frames within a single run of data collection. Including intensities from these frames into the merged structure factors can lead to inaccuracies in the final reported intensities, particularly detrimental for anomalous phasing or time-resolved difference density calculations where highly accurate recordings are required. Here, we report a new data sorting tool that offers a variety of Machine Learning algorithms to sort data trained on either manual sorting by the user, or by profile fitting the expected intensity distribution on the detector based on the experiment. This is integrated into an easy-to-use graphical user interface (GUI), specifically designed to support the detectors, file formats, and software available at the Linac Coherent Light Source (US) and the European XFEL (Germany).

  PublisherDOI

• Observation of early events in the photoactivation of Myxobacterial phytochrome using time-resolved serial femtosecond crystallography

  Communications Chemistry · 2025-06-12 · 3 citations

  articleOpen access

  Myxobacteria are non-photosynthetic, soil-dwelling bacteria distinguished by a multicellular stage in their life cycle known as fruiting bodies that are stimulated by light. Myxobacterial phytochromes are candidates for the perception of red-light. The mechanism how light is perceived and converted to a physiological response is unknown. Here, time-resolved serial femtosecond crystallographic (TR-SFX) experiments were conducted on microcrystals of the photosensory core module of the Stigmatella aurantiaca bacteriophytochrome 2 (SaBphP2). Initial events of the Z to E isomerization reaction of the covalently bound, open-chain tetrapyrrole biliverdin (BV) chromophore were determined. At 3 ps after light activation, the BV ring-D assumes a configuration needed for the isomerization. At 100 ps, a mixture of BV in the Z or E configuration is observed in subunit A, while in the other subunit the chromophore remains in the Z configuration. In conjunction with prior results, these structures reveal the molecular mechanism of phytochrome activation in the photomorphogenesis of the myxobacteria and provide the molecular foundation for physiological responses to red light in other bacteria. Although the photocycle of the photosensory core module of the Stigmatella aurantiaca bacteriophytochrome 2 (SaBphP2) has been extensively studied, its early dynamics have not been fully resolved. Here, the authors use time-resolved serial femtosecond crystallography to probe the associated picosecond events and report on the relative population of Z and E isomers after light activation.

  PublisherOA PDFDOI

• Ambient temperature structural studies of Penicillin-binding Protein 2a of <i>Methicillin-Resistant Staphylococcus aureus</i> with XFELs and synchrotrons

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-02-23 · 1 citations

  preprintOpen access

  Abstract Penicillin-binding protein 2a (PBP2a) is a transpeptidase responsible for the β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA), posing significant challenges to antibiotic therapy. PBP2a’s unique structural features, including its highly flexible active site and allosteric regulation, enable it to maintain catalytic activity even in the presence of β-lactam antibiotics. Despite extensive characterization using cryogenic crystallography, key questions remain about its dynamic properties and conformational changes under near-physiological conditions. Room-temperature crystallography methods, particularly serial femtosecond X-ray crystallography (SFX) at XFELs, provide a powerful approach to capture these dynamics. Unlike cryogenic conditions that can constrain protein flexibility, SFX enables the study of conformational variability and interaction networks closer to physiological states. Here, we present the first room-temperature structures of PBP2a obtained using SFX to uncover insights into the enzyme’s flexibility, allosteric communication, and catalytic mechanisms. These findings are built upon optimized large-scale production and crystallization protocols for PBP2a, ensuring high-quality microcrystals suitable for SFX data collection. The room-temperature structures reveal novel interaction patterns, including unique salt bridge networks and dynamic structural elements absent in cryogenic studies. Furthermore, comparative analyses highlight how environmental conditions influence the conformational states of PBP2a, providing new perspectives on its resistance mechanisms. By integrating structural data from EuXFEL and LCLS, this study not only enhances our understanding of PBP2a’s functional dynamics but also underscores the value of room-temperature crystallography in studying antibiotic resistance. These insights could guide the design of next-generation β-lactam antibiotics capable of overcoming PBP2a-mediated resistance.

  PublisherOA PDFDOI

• Functional implications of hexameric dynamics in <scp>SARS</scp>‐<scp>CoV</scp>‐2 Nsp15

  Protein Science · 2025-05-24 · 1 citations

  articleOpen access

  Abstract SARS‐CoV‐2, the virus responsible for the COVID‐19 pandemic, has undergone continuous evolution, leading to the emergence of variants with altered transmissibility and immune evasion. For the non‐structural proteins (Nsps) of SARS‐CoV‐2, there are limited structural analyses of their naturally occurring mutations. Here, we identified four non‐synonymous single‐nucleotide polymorphisms (nsSNPs) in the Epsilon lineage of SARS‐CoV‐2 within Nsp15, an endoribonuclease critical for immune evasion. Of these Epsilon nsSNPs, E266Q is in the catalytic domain. This study investigates the effects of this on enzymatic activity, structural stability, and oligomeric assembly by serial crystallography. By solving the structure of the Nsp15 hexamer at room temperature of both Nsp15‐E266Q and WT in the P2 1 space group to 3 Å, we observed asymmetric motions within its trimer subunits, a feature not visible in previously reported higher‐symmetry space groups. These asymmetric motions resemble substrate‐induced conformational changes reported in RNA‐bound Nsp15 structures, suggesting functional relevance. Biochemical analyses further reveal that Nsp15‐E266Q exhibited significantly higher enzymatic activity and thermal stability compared to the wild‐type protein. These findings highlight how mutations in Nsp15 contribute to viral replication and immune evasion, offering insights into the molecular mechanisms underlying SARS‐CoV‐2 variant evolution and potential therapeutic strategies.

  PublisherOA PDFDOI

• Polarization Tunability of X-ray Radiation and Commissioning of the ASU Compact X-ray Light Source

  2025-01-01

  article

  The Compact X-ray Light Source (CXLS) is an Inverse Compton Scattering source that can produce synchrotron-like x-ray radiation with sub-picosecond pulses and arbitrary polarization states by varying the polarization of its optical undulator.

  PublisherDOI

Frequent coauthors

• Henry N. Chapman

  Max Planck Institute for the Structure and Dynamics of Matter

  43 shared

• Sébastien Boutet

  38 shared

• M. Messerschmidt

  Arizona State University

  33 shared

• Raymond G. Sierra

  SLAC National Accelerator Laboratory

  31 shared

• Anton Barty

  31 shared

• Christopher Kupitz

  Linac Coherent Light Source

  30 shared

• Valerio Mariani

  Linac Coherent Light Source

  30 shared

• Max O. Wiedorn

  Deutsches Elektronen-Synchrotron DESY

  29 shared

Education

• Dr. rer. nat, Chemistry

  Universität Hamburg

  2018

• Meng, Engineering

  University of Oxford

  2012