Sabine Botha
A5012762561- Enzyme Structure and Function
- Advanced Electron Microscopy Techniques and Applications
- Advanced X-ray Imaging Techniques
- Protein Structure and Dynamics
- Biochemical and Molecular Research
- Photosynthetic Processes and Mechanisms
- Hemoglobin structure and function
- Atomic and Subatomic Physics Research
- Electron Spin Resonance Studies
- bioluminescence and chemiluminescence research
- Computational Drug Discovery Methods
- Time Series Analysis and Forecasting
- X-ray Spectroscopy and Fluorescence Analysis
- Light effects on plants
- Laser-Plasma Interactions and Diagnostics
- Anomaly Detection Techniques and Applications
- Cancer Research and Treatments
- Network Security and Intrusion Detection
- Crystallization and Solubility Studies
- Photoreceptor and optogenetics research
- Mass Spectrometry Techniques and Applications
- Bacteriophages and microbial interactions
- Healthcare and Venom Research
- Bacterial Identification and Susceptibility Testing
- Laser-induced spectroscopy and plasma
Arizona State University
2019-2025
Max Planck Institute for Medical Research
2013-2018
Universität Hamburg
2018
Max Planck Institute for the Structure and Dynamics of Matter
2018
Hamburg Centre for Ultrafast Imaging
2018
Max Planck Society
2014-2017
University of Pretoria
1998
The hemoprotein myoglobin is a model system for the study of protein dynamics. We used time-resolved serial femtosecond crystallography at an x-ray free-electron laser to resolve ultrafast structural changes in carbonmonoxy complex upon photolysis Fe-CO bond. Structural appear throughout within 500 femtoseconds, with C, F, and H helices moving away from heme cofactor E A toward it. These collective movements are predicted by hybrid quantum mechanics/molecular mechanics simulations. Together...
Recent advances in synchrotron sources, beamline optics and detectors are driving a renaissance room-temperature data collection. The underlying impetus is the recognition that conformational differences observed functionally important regions of structures determined using crystals kept at ambient as opposed to cryogenic temperature during In addition, measurements enable time-resolved studies eliminate need find suitable cryoprotectants. Since radiation damage limits high-resolution can be...
Abstract The new European X-ray Free-Electron Laser is the first free-electron laser capable of delivering pulses with a megahertz inter-pulse spacing, more than four orders magnitude higher previously possible. However, to date, it has been unclear whether would indeed be possible measure high-quality diffraction data at pulse repetition rates. Here, we show that structures can obtained using currently available operating conditions XFEL. We present two complete sets, one from well-known...
It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells crystalline form. Here we describe structure determination of Cry3A toxin found naturally crystallized within Bt cells. When whole were streamed into an X-ray free-electron laser beam scattering from other cell components did not obscure diffraction crystals. The resolution limits best images collected same as isolated integrity at moment is unclear; however, given short time (∼ 5 µs)...
Proteins that contain metal cofactors are expected to be highly radiation sensitive since the degree of X-ray absorption correlates with presence high-atomic-number elements and energy. To explore effects local damage in serial femtosecond crystallography (SFX), Clostridium ferredoxin was used as a model system. The protein contains two [4Fe-4S] clusters serve probes for radiation-induced electronic structural changes. High-dose room-temperature SFX datasets were collected at Linac Coherent...
Significance The room temperature structure of natively formed protein nanocrystals consisting 9,000 unit cells has been solved to 2 Å resolution using an unattenuated X-ray free-electron laser (XFEL) beam, representing, by far, the smallest crystals used for determination crystallography date. Radiation damage limits from synchrotron techniques, whereas femtosecond pulses lasers enable much higher tolerable doses, extracting more signal per molecule, allowing study submicrometer crystals....
Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems that are prone to radiation damage. However, phasing XFEL data de novo is complicated by the inherent inaccuracy SFX data, and only a few successful examples, mostly based on exceedingly strong anomalous or isomorphous difference signals, have been reported. Here, it shown from thaumatin microcrystals can be successfully...
Abstract The world’s first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid rate, XFEL may alleviate some of increasing demand for beamtime, particularly membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report SFX experiment, where determined 2.9 Å-resolution...
The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and process photoactivatable adenylate cyclase from Oscillatoria acuminata (OaPAC) upon blue excitation. Structural models with bound active site native OaPAC at cryogenic well room temperature are presented. found one conformation cryogenic- two conformations...
The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for includes: (1) designing a vaccine future protection; (2) de novo drug discovery; (3) identifying existing drugs repurpose them immediate treatments. To assist repurposing design, we determine two apo structures severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial...
Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into path XFEL, wasting a vast amount due to pulsed nature all current XFEL sources. The European (EuXFEL) delivers (fs) pulses in trains spaced 100 ms apart whereas within are currently separated by 889 ns. Therefore, continuous via fast wastes...
The structure solution of biological is being revolutionized by serial X-ray diffraction employing free-electron laser (XFEL) sources, particularly the advent femtosecond crystallography (SFX). measurements micrometer- sized crystals at room temperature allow time-resolved studies to trace path biochemical reactions unprecedented temporal resolution, made possible pulses that outrun effects radiation damage. These can deliver doses are more than a thousand times higher those with...
Over the past two decades, serial X-ray crystallography has enabled structure determination of a wide range proteins. With advent free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and determination. A crucial need to continue advancement is efficient delivery fragile micrometre-sized beam intersection. This paper presents an improved design all-polymer microfluidic `chip' for room-temperature fixed-target that can be tailored broadly meet needs...
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 maintain catalytic activity even presence of antibiotics. Despite extensive characterization using cryogenic crystallography, key questions remain about dynamic properties...
High-resolution ribosome structures determined by X-ray crystallography have provided important insights into the mechanism of translation. Such studies thus far relied on large crystals kept at cryogenic temperatures to reduce radiation damage. Here, application serial femtosecond (SFX) using an free-electron laser (XFEL) obtain diffraction data from microcrystals in liquid suspension ambient temperature is described. 30S ribosomal subunit diffracted beyond 6 Å resolution, demonstrating...
The SARS-CoV-2 main protease of (Mpro) is an important target for related drug repurposing and development studies. Here, we describe the steps structural characterization Mpro, starting from plasmid preparation protein purification. We detail crystallization using sitting drop, microbatch (under oil) approach. Finally, cover data collection structure determination serial femtosecond crystallography. For complete details on use execution this protocol, please refer to Durdagi et al. (2021).
A 3D-printed modular droplet injector successfully delivered microcrystals of human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin with electrical stimulation in a serial crystallography experiment at 120 Hz repetition rate.
X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during pulse, with preferential 'bleaching' heavy atoms. This paper investigates effects electronic damage on experimental data collected Gd derivative...
Abstract Multimeric protein assemblies are abundant in nature. Streptavidin is an attractive that provides a paradigm system to investigate the intra- and intermolecular interactions of multimeric complexes. Also, it offers versatile tool for biotechnological applications. Here, we present two apo-streptavidin structures, first one ambient temperature Serial Femtosecond X-ray crystal (Apo-SFX) structure at 1.7 Å resolution second cryogenic (Apo-Cryo) 1.1 resolution. These structures mostly...
Serial femtosecond crystallography (SFX) is an emerging method for data collection at free-electron lasers (FELs) in which single diffraction snapshots are taken from a large number of crystals. The partial intensities collected this way then combined scheme called Monte Carlo integration, provides the full intensities. However, apart having to perform merging, integration must also average out all variations crystal quality, size, X-ray beam properties and other factors, necessitating...
Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macromolecules at room temperature. Despite impressive exposition structural details with this novel crystallographic approach, methods currently available introduce crystals into path beam sometimes exhibit serious drawbacks. Samples requiring liquid injection crystal slurries consume large quantities (at times up gram protein per data set), may not be...