A5086360909- Advanced Electron Microscopy Techniques and Applications
- Electron and X-Ray Spectroscopy Techniques
- RNA Research and Splicing
- RNA Interference and Gene Delivery
- Advanced X-ray Imaging Techniques
- MicroRNA in disease regulation
- RNA and protein synthesis mechanisms
- Mitochondrial Function and Pathology
- Ubiquitin and proteasome pathways
- Parkinson's Disease Mechanisms and Treatments
- Semiconductor materials and interfaces
- Alzheimer's disease research and treatments
- Neurobiology and Insect Physiology Research
- Surface and Thin Film Phenomena
- Endoplasmic Reticulum Stress and Disease
- Crystallography and Radiation Phenomena
- Ion Channels and Receptors
- Photosynthetic Processes and Mechanisms
- RNA modifications and cancer
- Autophagy in Disease and Therapy
- Integrated Circuits and Semiconductor Failure Analysis
- Advanced Neuroimaging Techniques and Applications
- DNA Repair Mechanisms
- Ion-surface interactions and analysis
- Advanced biosensing and bioanalysis techniques
National Institute of Environmental Health Sciences
2020-2024
National Institutes of Health
2020-2024
Université de Montréal
2011-2024
Research Triangle Park Foundation
2022-2023
Abstract Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging sub-volumes has been successfully used to derive atomic models macromolecules their biological environment. Eliminating biochemical isolation steps required other techniques, this method opens up the cell in-situ structural studies. However, need compensate for errors targeting introduced during mechanical navigation specimen significantly slows down...
Finding the conditions to stabilize a macromolecular target for imaging remains most critical barrier determining its structure by cryo-electron microscopy (cryo-EM). While automation has significantly increased speed of data collection, specimens are still screened manually, laborious and subjective task that often determines success project. Here, we present SmartScope, first framework streamline, standardize, automate specimen evaluation in cryo-EM. SmartScope employs deep-learning-based...
Protein ubiquitination is an essential process that rapidly regulates protein synthesis, function, and fate in dynamic environments. Within its non-proteolytic functions, we showed K63-linked polyubiquitinated conjugates heavily accumulate yeast cells exposed to oxidative stress, stalling ribosomes at elongation. K63-ubiquitinated mostly because of redox inhibition the deubiquitinating enzyme Ubp2; however, role regulation ubiquitin-conjugating enzymes (E2) this pathway remained unclear....
Significance K63 ubiquitination of ribosomes serves as a key regulator protein production during cellular exposure to oxidative stress. Defining the structural and functional mechanisms translation regulation would support current understanding critical reprogramming eukaryotic gene expression. Our paper presents an examination structure ubiquitinated ribosomes, revealing that this modification structurally destabilizes proteins involved in binding factors is required trap at...
The pluripotency factor Lin28 is a highly conserved protein comprising unique combination of RNA-binding motifs, an N-terminal cold-shock domain and C-terminal region containing two retroviral-type CCHC zinc-binding domains. An important function to inhibit the biogenesis let-7 family microRNAs through direct interaction with precursors. Here, we systematically characterize determinants between pre-let-7 g by investigating effect RNA mutations on in vitro binding. We determine that binds...
Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 disease variants have been linked to progressive external ophthalmoplegia ataxia neuropathies among other diseases. Despite biological clinical importance, represents only remaining component human minimal replisome that has yet be structurally characterized. Here, we present 3-dimensional structures W315L. Employing cryo-electron microscopy (cryo-EM), characterize oligomeric assemblies...
Abstract The mitochondrial single-stranded DNA (ssDNA) binding protein, mtSSB or SSBP1, binds to ssDNA prevent secondary structures of that could impede downstream replication repair processes. Clinical mutations in the SSBP1 gene have been linked a range disorders affecting nearly all organs and systems. Yet, molecular determinants governing interaction between remained elusive. Similarly, structural other replisome components, such as polymerase, Polγ, has minimally explored. Here, we...
Abstract Lin28 inhibits the biogenesis of let-7 miRNAs through direct interactions with precursors. Previous studies have described seemingly inconsistent binding sites on pre-let-7 RNAs. Here, we reconcile these data by examining mechanism to terminal loop pre-let-7g (TL-let-7g) using biochemical and biophysical methods. First, investigate TL-let-7g variants short RNA fragments identify three independent for TL-let-7g. We then determine that assembles in a stepwise manner form stable 1:3...
Abstract The human let-7 miRNA family consists of thirteen members that play critical roles in many biological processes, including development timing and tumor suppression, their levels are disrupted several diseases. Dicer is the endoribonuclease responsible for processing precursor (pre-miRNA) to yield mature miRNA, thereby plays a crucial role controlling cellular miRNAs. It well established sequence structural features pre-miRNA hairpins such as 5′-phosphate, apical loop, 2-nt...
Abstract Lewy body dementias are pathologically defined by the deposition of α-synuclein fibrils into inclusions throughout brain. Cerebrospinal fluid(CSF) in disease harbors circulating α-synuclein-fibril seeds, and parental can template core structure amplified fibrils. Using cryo-electron microscopy, we identify six novel fibril assemblies from ten CSF samples (3.8Å to 2.9Å nominal resolutions). Fibrils classified based on two types filament interaction, β-sheet stacking, hydrophobic...
Abstract Propelled by improvements in hardware for data collection and processing, single particle cryo-electron microscopy has rapidly gained relevance structural biology. Yet, finding the conditions to stabilize a macromolecular target imaging remains most critical barrier determining its structure. Attaining optimal specimen requires evaluation of multiple grids microscope as are varied. While automation significantly increased speed collection, optimization is still carried out manually....
Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity highly regulated by factors, and this regulation can impact on levels of miRNAs modulate behavior cell. To better understand underlying mechanisms detailed structural characterization are needed. However, these types studies generally require several milligrams recombinant protein, efficient preparation...
ABSTRACT Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging sub-volumes has been successfully used to derive atomic models macromolecules their biological environment. Eliminating biochemical isolation steps required other techniques, this method opens up the cell in-situ structural studies. However, need compensate for errors targeting introduced during mechanical navigation specimen significantly slows down...
Abstract The replicative mitochondrial DNA polymerase, Polγ, and its protein regulation are essential for the integrity of genome. intricacies Polγ interactions with regulatory proteins, which fine-tuning polymerase function, remain poorly understood. Misregulation heterotrimer, consisting (i) PolG, catalytic subunit (ii) PolG2, accessory subunit, ultimately results in diseases. Here, we used single particle cryo-electron microscopy to resolve structure PolG apoprotein state captured at...
Abstract Protein ubiquitination is an essential process that rapidly regulates protein synthesis, function, and fate in dynamic environments. Among its non-proteolytic functions, K63 ubiquitin accumulates yeast cells exposed to oxidative stress, stalling ribosomes at elongation. conjugates accumulate because of redox inhibition the deubiquitinating enzyme Ubp2, however, role regulation conjugating enzymes this pathway remained unclear. Here we found E2 Rad6 binds modifies elongating during...
Abstract Lewy body dementias are pathologically defined by the deposition of α-synuclein fibrils into inclusions throughout brain. Cerebrospinal fluid(CSF) in disease harbors circulating α-synuclein-fibril seeds, and parental can template core structure amplified fibrils. Using cryo-electron microscopy, we identify six novel fibril assemblies from ten CSF samples (3.8Å to 2.9Å nominal resolutions). Fibrils classified based on two types filament interaction, β-sheet stacking, hydrophobic...
Specimen optimization is currently one of the main limiting steps in cryo-electron microscopy (EM) structure determination pipeline. The ideal specimen a molecule-thin layer macromolecules solution frozen on top holey membrane stabilized by metal support grid. During screening, experienced microscopists visualize at increasing magnifications navigating to areas that are most likely provide information useful guide optimization. Iterating this procedure over different experimental conditions,...
Abstract The human let-7 miRNA family consists of thirteen members that play critical roles in many biological processes, including development timing and tumor suppression, their levels are disrupted several diseases. Dicer is the endoribonuclease responsible for processing precursor (pre-miRNA) to yield mature miRNA, thereby plays a crucial role controlling cellular miRNAs. It well established sequence structural features pre-miRNA hairpins such as 5'-phosphate, apical loop, 2-nt...