Biology subjects

Zee, C.-T.

Publications and source records attributed to Zee, C.-T..

5 recordsLinked to original sources

bioRxiv2022

Structure of a reversible amyloid fibril formed by the CPEB3 prion-like domain reveals a core sequence involved in translational regulation

The cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is a prion-like RNA-binding polypeptide. As a functional prion, CPEB3 is thought to modulate protein synthesis at synapses and enable consolidation of long-term memory in neurons. Here, we report that the prion-like domain 1 of CPEB3 self-assembles into labile amyloid fibrils in vitro. A cryoEM structure of these fibrils reveals an ordered 48-residue core, spanning L103 to F151. CPEB3 constructs lacking this amyloidogenic segment form abnormal puncta in cells when compared to wild type CPEB3, with reduced localization in dormant p-bodies and increased localization in stress granules. Removal of the amyloid core segment in CPEB3 also abolishes its ability to regulate protein synthesis in neurons. Collectively, this evidence suggests that the newly identified amyloidogenic segment within the CPEB3 prion domain is important for its regulated aggregation in cells and suggest its involvement in regulating translational activity and potentially long-term memory formation.

biochemistry↗

bioRxiv2021

Fragment-based ab initio phasing of peptidic nanocrystals by MicroED

Microcrystal electron diffraction (MicroED) is transforming the visualization of molecules from nanocrystals, rendering their three-dimensional atomic structures from previously unamenable samples. Peptidic structures determined by MicroED include naturally occurring peptides, synthetic protein fragments and peptide-based natural products. However, as a diffraction method, MicroED is beholden to the phase problem, and its de novo determination of structures remains a challenge. ARCIMBOLDO, an automated, fragment-based approach to structure determination. It eliminates the need for atomic resolution, instead enforcing stereochemical constraints through libraries of small model fragments, and discerning congruent motifs in solution space to ensure validation. This approach expands the reach of MicroED to presently inaccessible peptidic structures including segments of human amyloids, and yeast and mammalian prions, and portends a more general phasing solution while limiting model bias for a wider set of chemical structures.

biochemistry↗

bioRxiv2021

Inhibition of amyloid formation of the Nucleoprotein of SARS-CoV-2

The SARS-CoV-2 Nucleoprotein (NCAP) functions in RNA packaging during viral replication and assembly. Computational analysis of its amino acid sequence reveals a central low-complexity domain (LCD) having sequence features akin to LCDs in other proteins known to function in liquid-liquid phase separation. Here we show that in the presence of viral RNA, NCAP, and also its LCD segment alone, form amyloid-like fibrils when undergoing liquid-liquid phase separation. Within the LCD we identified three 6-residue segments that drive amyloid fibril formation. We determined atomic structures for fibrils formed by each of the three identified segments. These structures informed our design of peptide inhibitors of NCAP fibril formation and liquid-liquid phase separation, suggesting a therapeutic route for Covid-19. One Sentence SummaryAtomic structures of amyloid-driving peptide segments from SARS-CoV-2 Nucleoprotein inform the development of Covid-19 therapeutics.

biochemistry↗

bioRxiv2018

Homochiral and racemic MicroED structures of a peptide from the ice nucleation protein InaZ

The ice nucleation protein InaZ of Pseudomonas syringae contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. We determine ab initio structures of this repeat segment, resolved to 1.1[A] by microfocus x-ray crystallography and 0.9[A] by the cryoEM method MicroED, from both racemic and homochiral crystals. We evaluate the benefits of racemic protein crystals for structure determination by MicroED and confirm that phase restriction introduced by crystal centrosymmetry increases the number of successful trials during ab initio phasing of electron diffraction data. Both homochiral and racemic GSTSTA form amyloid-like protofibrils with labile, corrugated antiparallel beta sheets that mate face to back. The racemic GSTSTA protofibril represents a new class of amyloid assembly in which all left-handed sheets mate with their all right-handed counterparts. Our determination of racemic amyloid assemblies by MicroED reveals complex amyloid architectures and illustrates the racemic advantage in macromolecular crystallography, now with sub-micron sized crystals.\n\nSynopsisThe atomic asymmetry, left or right handedness, present in macromolecules and first described by Pasteur in his experiments with tartaric acid, is evident even in complex molecular assemblies like amyloid fibrils. Here, using the cryoEM method MicroED, we show that a segment from the ice nucleation protein InaZ assembles into homochiral and racemic water-binding amyloid protofibrils.

biochemistry↗

bioRxiv2018

Analysis of global and site-specific radiation damage in cryo-EM

Micro-crystal electron diffraction (MicroED) is an emerging method in cryo-EM for structure determination using nanocrystals. It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.

biochemistry↗