Solving Challenging Structures using Single-Particle Cryogenic Electron Microscopy

Single-particle cryogenic electron microscopy (cryo-EM) has become a powerful mainstay tool in high resolution structural biology thanks to advances in hardware, software and sample preparation technology. In my thesis, I utilized this technique to unravel the function of various challenging biological macromolecules. My first focus was bacterial ribosomal biogenesis: understanding how bacteria assemble their ribosomes. Ribosomes are the factories of the cell, responsible for manufacturing all proteins. Ribosomes themselves are huge, with the bacterial version made of 52 proteins and 4566 RNA nucleotides. How these components assemble has long been a mystery. Early groundbreaking work sketched out a biogenesis pathway using purified components in vitro – but under non-physiological conditions. We sought to understand how the bacterial ribosome – specifically the large subunit 50S – is built inside the cell. To achieve this, we engineered a conditional knock-out bacterial strain that lacked one specific ribosomal protein (L17). This caused the cells to accumulate incomplete intermediates along the 50S biogenesis pathway. These intermediates were purified and examined with mass spectrometry and single-particle cryo-EM. Two major hurdles arose in this project: firstly, the biogenesis intermediates exhibited a preferred orientation when vitrified for cryo-EM analysis. This means that instead of showing many different views required for reconstruction of the 3D structure, the intermediates only adopted one view on the cryo-EM grid. To overcome this problem, we engineered a method to induce additional views on the microscope by tilting the stage. Using another test protein that also exhibited preferred orientation (hemagglutinin), we optimized and characterized this new tilt methodology and showed it was generally applicable to overcoming preferred orientation, regardless of type of specimen. We also created a software tool, called 3DFSC (3dfsc.salk.edu), for other microscopists to calculate the degree of directional anisotropy in their structures due to preferred orientation. Using this tilt strategy finally enabled the structural elucidation of our 50S intermediates. The second challenge in the project was the large amount of heterogeneity present in the sample. Through hierarchical 3D classification schemes using the latest software tools, we obtained 14 different 50S intermediate structures, all from imaging a single cryo-EM grid. By analyzing the missing components of each intermediate, and corroborating these observations with mass spectrometry data, we outlined the first in vivo 50S assembly pathway, and showed that ribosome assembly occurs step-wise and in parallel pathways. My second focus was on pushing the resolution limits of single-particle cryo-EM using adeno-associated virus (AAV) serotype 2 homogeneous virus-like particles (VLPs) that lack DNA. Exploiting several technical advances to improve resolution, including use of gold grids, per-particle CTF refinement, and correction for Ewald sphere curvature, we managed to obtain a 1.86 Å resolution reconstruction of the AAV2L336C variant VLP, the highest resolution icosahedral virus reconstruction solved by single-particle cryo-EM to date. Using our structure, we were able to show improvements using Ewald sphere curvature correction and shed light on the mechanistic basis as to why the L336C mutation resulted in defects in genome packaging and infectivity compared to the WT viral particles. My third focus was the understanding of small membrane proteins involved in infectious diseases. Membrane proteins are a challenge to work with due to the need for them to be extracted from the lipid bilayer for studies as compared to soluble proteins. Infectious diseases have a huge burden on society, with the top three infectious agents accounting for 2.7 million deaths in 2016. The third most deadly infectious disease is malaria, a mosquito-borne parasite which kills 450,000 people annually. One drug used early on for treating malaria was chloroquine but its usefulness waned due to development of resistance. Chloroquine resistance is mediated by the chloroquine resistance transporter (PfCRT). Although small (49 kDa) for single-particle cryo-EM, we solved its structure by using fragment antibody technology to add mass and help with image alignment and 3D reconstruction. The 3.2 Å structure resembles other drug metabolite transporters, and the chloroquine resistance mutations map to a ring around the central cavity, suggesting this central pore as the drug binding site. Tuberculosis (TB) is the top killer, above malaria and HIV/AIDS, being responsible for 1.3 million deaths. In TB, a common antibiotic target is the bacterium’s cell wall synthesis machinery. One family of such enzymes is the arabinosyltransferases, which synthesize the critical arabinose sugars. Using single-particle cryo-EM, we solved two high resolution structures of one such essential enzyme, AftD. Due to the low yield of the protein, a picoliter automated sample dispensing robot was crucial to allow for initial cryo-EM analysis. We then performed mutagenesis studies in M. smegmatis, a TB model organism, which uncovered the critical amino acid residues in the active site and determined that a bound acyl-carrier-protein was likely involved in allosteric inhibition of AftD’s active site. Another member of the family, EmbB, is the target of a widely used frontline TB drug called ethambutol. We have solved the high resolution structures of the apo and putative drug-bound states of EmbB, allowing us to map out, for the first time, both the active site and drug-resistance mutations of this crucial enzyme. The atomic structures of the functional pockets of Mycobacterial AftD and malarial PfCRT will hopefully enable structure-based drug design to improve existing drugs or potentially even develop new treatments against these infectious maladies. In conclusion, the continual and breathtaking improvements in single-particle cryo-EM methodology has been instrumental in allowing the elucidation of the aforementioned biological macromolecules from ribosome biogenesis intermediates, to AAV2 vehicle, Plasmodium drug resistance transporter to mycobacterial glycosyltransferases – structures of which help explain biological function

Fireball/Blastwave Model and Soft Gamma-ray Repeaters

Soft gamma-ray repeaters are at determined distances and their positions are known accurately. If observed, afterglows from their soft gamma-ray bursts will provide important clues to the study of the so called "classical gamma-ray bursts". On applying the popular fireball/blastwave model of classical gamma-ray bursts to soft gamma-ray repeaters, it is found that their X-ray and optical afterglows are detectable. Monitoring of the three repeaters is solicited.Comment: Already published in 1998 in "Chinese Physics Letters", replaced with the published version. See astro-ph/0502452 for a more detailed versio

Geographical Origin Traceability of Red Wines Based on Chemometric Classification via Organic Acid Profiles

A preliminary study on the chemometric classification of red wines produced from different grape varieties and geographical origins was performed based on their chromatographic profiles of organic acids. Tartaric, malic, citric, lactic, acetic, and succinic acids in wines were detected via high performance liquid chromatography (HPLC). Employing multivariate statistical methods including principal component analysis (PCA) and linear discriminant analysis (LDA), pattern recognition models were built for the classification of the investigated wines regarding the grape varieties and geographical origins. The PCA clearly grouped the wines according to variety, and the LDA further offered 100% classification ability toward geographical identification of the wines and the leave-one-out cross-validated assignments were 100%, 86.7%, and 100% correct for Cabernet Sauvignon, Merlot, and Pinot Noir wines, respectively. The results reveal the potential of using chromatographic profiles of organic acid as the characteristic indices for chemometric classification of red wines

Full velocities and propagation directions of coronal mass ejections inferred from simultaneous full-disk imaging and Sun-as-a-star spectroscopic observations

Coronal mass ejections (CMEs) are violent ejections of magnetized plasma from the Sun, which can trigger geomagnetic storms, endanger satellite operations and destroy electrical infrastructures on the Earth. After systematically searching Sun-as-a-star spectra observed by the Extreme-ultraviolet Variability Experiment (EVE) onboard the Solar Dynamics Observatory (SDO) from May 2010 to May 2022, we identified eight CMEs associated with flares and filament eruptions by analyzing the blue-wing asymmetry of the O III 52.58 nm line profiles. Combined with images simultaneously taken by the 30.4 nm channel of the Atmospheric Imaging Assembly onboard SDO, the full velocity and propagation direction for each of the eight CMEs are derived. We find a strong correlation between geomagnetic indices (Kp and Dst) and the angle between the CME propagation direction and the Sun-Earth line, suggesting that Sun-as-a-star spectroscopic observations at EUV wavelengths can potentially help to improve the prediction accuracy of the geoeffectiveness of CMEs. Moreover, an analysis of synthesized long-exposure Sun-as-a-star spectra implies that it is possible to detect CMEs from other stars through blue-wing asymmetries or blueshifts of spectral lines.Comment: Accepted by Ap

Synthesizing Speech Test Cases with Text-to-Speech? An Empirical Study on the False Alarms in Automated Speech Recognition Testing

Recent studies have proposed the use of Text-To-Speech (TTS) systems to automatically synthesise speech test cases on a scale and uncover a large number of failures in ASR systems. However, the failures uncovered by synthetic test cases may not reflect the actual performance of an ASR system when it transcribes human audio, which we refer to as false alarms. Given a failed test case synthesised from TTS systems, which consists of TTS-generated audio and the corresponding ground truth text, we feed the human audio stating the same text to an ASR system. If human audio can be correctly transcribed, an instance of a false alarm is detected. In this study, we investigate false alarm occurrences in five popular ASR systems using synthetic audio generated from four TTS systems and human audio obtained from two commonly used datasets. Our results show that the least number of false alarms is identified when testing Deepspeech, and the number of false alarms is the highest when testing Wav2vec2. On average, false alarm rates range from 21% to 34% in all five ASR systems. Among the TTS systems used, Google TTS produces the least number of false alarms (17%), and Espeak TTS produces the highest number of false alarms (32%) among the four TTS systems. Additionally, we build a false alarm estimator that flags potential false alarms, which achieves promising results: a precision of 98.3%, a recall of 96.4%, an accuracy of 98.5%, and an F1 score of 97.3%. Our study provides insight into the appropriate selection of TTS systems to generate high-quality speech to test ASR systems. Additionally, a false alarm estimator can be a way to minimise the impact of false alarms and help developers choose suitable test inputs when evaluating ASR systems. The source code used in this paper is publicly available on GitHub at https://github.com/julianyonghao/FAinASRtest.Comment: 12 pages, Accepted at ISSTA202

Overall Evolution of Realistic Gamma-ray Burst Remnant and Its Afterglow

Conventional dynamic model of gamma-ray burst remnants is found to be incorrect for adiabatic blastwaves during the non-relativistic phase. A new model is derived, which is shown to be correct for both radiative and adiabatic blastwaves during both ultra-relativistic and non-relativistic phase. Our model also takes the evolution of the radiative efficiency into account. The importance of the transition from the ultra-relativistic phase to the non-relativistic phase is stressed.Comment: 9 pages, aasms4 style, 3 ps figures, minor changes, will be published in Chin. Phys. Let

Serological survey of Aujeszkys disease in Peninsular Malaysia in 2016

Aujeszky’s disease (AD) is a common swine disease that widespread throughout the world. The symptoms include nervous signs, respiration and reproduction problems that lead to great economic losses to the industry. AD is endemic in Malaysia, where outbreaks had been reported in previous years. In Malaysia, approximately 95% of the pig farms are vaccinated for AD. Despite the regular vaccination, AD serological status remains unknown in this country. This study provides AD serological status in Peninsular Malaysia in 2016 based on the samples submitted to Faculty of Veterinary Medicine, University Putra Malaysia (UPM). A total of 1154 serum samples from 36 farms were submitted for AD ELISA diagnostic test; grouped as 8 weeks, 12 weeks, 16 weeks, 20 weeks, gilts and sows with different parity. The samples were subjected to AD antibody detection with IDEXX Pseudorabies Virus gpI Antibody Test Kit. Among the 36 farms submitted to UPM, 8 farms were detected with positive gI antibody indicated that these farms were still facing challenges from AD field virus. Among these eight seropositive farms, three farms were having seroprevalence in the range of 33.33% to 37.14%. In general, vaccination of AD is ideal and stable in Malaysia but we still need to be alert with the field challenge as it will be a threat to the industry

DESIGN, ENGINEERING, AND ASSESSMENT OF MOBILE MINEFIELDS

Naval mine warfare typically supports a sea denial strategy through the denial and/or delay of the enemy’s use of the water space or by controlling sea traffic in a designated area. Sea mines have been effective for decades. However, with technological progress, mine countermeasure (MCM) efforts have reduced the risks of a minefield by detecting and/or neutralizing mines to establish and maintain a Q-route for safe passage. The concept of a mobile minefield is proposed to increase the difficulty of the enemy’s MCM and improve the survivability of the minefield by adding mobility. This research explores both the physical design concepts and the operational effectiveness of mobile mines based on simulations and models. The simulation results show that, compared to static mines, mobile mines improved the number of enemy ships destroyed by at least 200% and increased the time it took the enemy to transition through the minefield by 50%. The results suggest that the mobile minefield would be operationally useful for the Department of the Navy and this technology is worth pursing and exploring.Distribution Statement A. Approved for public release: Distribution is unlimited.Captain, Singapore ArmyCaptain, Singapore ArmyMajor, Singapore ArmyLieutenant, Taiwan NavyMajor, United States ArmyCivilian, Department of the NavyLieutenant, United States NavyCivilian, Singapore Technologies Engineering, SingaporeMajor, Singapore ArmyMajor, Singapore ArmyMajor, Singapore ArmyCommander, United States NavyCivilian, Defense Science and Technology Agency (DSTA), SingaporeMajor, Singapore ArmyMajor, Republic of Singapore Air ForceTenente-Coronel, Brazilian Air ForceLieutenant, United States NavyCivilian, Department of the ArmyMajor, Singapore ArmyMajor, Israel Defense ForcesCivilian, Defense Science Organisation, SingaporeCaptain, Singapore Arm

Dynamical Evolution of Gamma-Ray Burst Remnants with Evolving Radiative Efficiency

In previous works, a generic dynamical model has been suggested by Huang et al., which is shown to be correct for both adiabatic and radiative blastwaves, and in both ultra-relativistic and non-relativistic phases. In deriving their equations, Huang et al. have assumed that the radiative efficiency of the fireball is constant. They then applied their model directly to realistic cases where the radiative efficiency evolves with time. In this paper, we abandon the above assumption and re-derive a more accurate dynamical equation for gamma-ray burst remnants. Numerical results show that Huang et al.'s model is accurate enough in general cases.Comment: 10 pages, 5 embedded eps figures, to appear in: Chin. J. Astron. Astrophys. (2002), Vol