Title: Solving Challenging Structures using Single-Particle Cryogenic Electron Microscopy
Description: 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.