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Over the past 10 years, the bioenergy field has realized significant achievements that have encouraged many follow on efforts centered on biosynthetic production of fuel-like compounds. Key to the success of these efforts has been transformational developments in feedstock characterization and metabolic engineering of biofuel-producing microbes. Lagging far behind these advancements are analytical methods to characterize and quantify systems of interest to the bioenergy field. In particular, the utilization of proteomics, while valuable for identifying novel enzymes and diagnosing problems associated with biofuel-producing microbes, is limited by a lack of robustness and limited throughput. Nano-flow liquid chromatography coupled to high-mass accuracy, high-resolution mass spectrometers has become the dominant approach for the analysis of complex proteomic samples, yet such assays still require dedicated experts for data acquisition, analysis, and instrument upkeep. The recent adoption of standard flow chromatography (ca. 0.5 mL/min) for targeted proteomics has highlighted the robust nature and increased throughput of this approach for sample analysis. Consequently, we assessed the applicability of standard flow liquid chromatography for shotgun proteomics using samples from Escherichia coli and Arabidopsis thaliana, organisms commonly used as model systems for lignocellulosic biofuels research. Employing 120 min gradients with standard flow chromatography, we were able to routinely identify nearly 800 proteins from E. coli samples; while for samples from Arabidopsis, over 1,000 proteins could be reliably identified. An examination of identified peptides indicated that the method was suitable for reproducible applications in shotgun proteomics. Standard flow liquid chromatography for shotgun proteomics provides a robust approach for the analysis of complex samples. To the best of our knowledge, this study represents the first attempt to validate the standard flow approach for shotgun proteomics.

The γ-tubulin ring complex (γTuRC) nucleates microtubules in the cell. The functional, closed state of yeast γTuRC is now visualized, and its microtubule-nucleating activity is found to be species specific. The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in cells. γγTuRC is assembled from repeating γγ-tubulin small complex (γTuSC) subunits and is thought to function as a template by presenting a γ-tubulin ring that mimics microtubule geometry. However, a previous yeast γTuRC structure showed γTuSC in an open conformation that prevents matching to microtubule symmetry. By contrast, we show here that γ-tubulin complexes are in a closed conformation when attached to microtubules. To confirm the functional importance of the closed γTuSC ring, we trapped the closed state and determined its structure, showing that the γ-tubulin ring precisely matches microtubule symmetry and providing detailed insight into γTuRC architecture. Importantly, the closed state is a stronger nucleator, thus suggesting that this conformational switch may allosterically control γTuRC activity. Finally, we demonstrate that γTuRCs have a strong preference for tubulin from the same species.

The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in cells. γγTuRC is assembled from repeating γγ-tubulin small complex (γTuSC) subunits and is thought to function as a template by presenting a γ-tubulin ring that mimics microtubule geometry. However, a previous yeast γTuRC structure showed γTuSC in an open conformation that prevents matching to microtubule symmetry. By contrast, we show here that γ-tubulin complexes are in a closed conformation when attached to microtubules. To confirm the functional importance of the closed γTuSC ring, we trapped the closed state and determined its structure, showing that the γ-tubulin ring precisely matches microtubule symmetry and providing detailed insight into γTuRC architecture. Importantly, the closed state is a stronger nucleator, thus suggesting that this conformational switch may allosterically control γTuRC activity. Finally, we demonstrate that γTuRCs have a strong preference for tubulin from the same species.

The γ-tubulin ring complex (γTuRC) is a protein complex of relative molecular mass ~2.2 × 10 6 that nucleates microtubules at the centrosome. Here we use electron-microscopic tomography and metal shadowing to examine the structure of isolated Drosophila γTuRCs and the ends of microtubules nucleated by γTuRCs and by centrosomes. We show that the γTuRC is a lockwasher-like structure made up of repeating subunits, topped asymmetrically with a cap. A similar capped ring is also visible at one end of microtubules grown from isolated γTuRCs and from centrosomes. Antibodies against γ-tubulin label microtubule ends, but not walls, in centrosomes. These data are consistent with a template-mediated mechanism for microtubule nucleation by the γTuRC.

THE microtubule cytoskeleton of animal cells does not assemble spontaneously, but instead requires the centrosome. This organelle consists of a pair of centrioles surrounded by a complex collection of proteins known as the pericentriolar material (PCM) 1 . The PCM is required for microtubule nucleation 2 . The minus, or slow-growing, ends of microtubules are embedded in the PCM and the plus, or fast-growing, ends project outwards into the cytoplasm during interphase, or into the spindle apparatus during mitosis, γ-Tubulin is the only component of the PCM that is so far implicated in microtubule nucleation 3–6 . Here we use immuno-electron microscopic tomography to show that γ-tubulin is localized in ring structures in the PCM of purified centrosomes without microtubules. When these centrosomes are used to nucleate microtubule growth, γ-tubulin is localized at the minus ends of the microtubules. We conclude that microtubule-nucleating sites within the PCM are ring-shaped templates that contain multiple copies of γ-tubulin.

The CELLULOSE SYNTHASE-LIKE F6 (CslF6) gene was previously shown to mediate the biosynthesis of mixed-linkage glucan (MLG), a cell wall polysaccharide that is hypothesized to be tightly associated with cellulose and also have a role in cell expansion in the primary cell wall of young seedlings in grass species. We have recently shown that loss-of-function cslf6 rice mutants do not accumulate MLG in most vegetative tissues. Despite the absence of a structurally important polymer, MLG, these mutants are unexpectedly viable and only show a moderate growth compromise compared to wild type. Therefore these mutants are ideal biological systems to test the current grass cell wall model. In order to gain a better understanding of the role of MLG in the primary wall, we performed in-depth compositional and structural analyses of the cell walls of 3 day-old rice seedlings using various biochemical and novel microspectroscopic approaches. We found that cellulose content as well as matrix polysaccharide composition was not significantly altered in the MLG deficient mutant. However, we observed a significant change in cellulose microfibril bundle organization in mesophyll cell walls of the cslf6 mutant. Using synchrotron source Fourier Transform Mid-Infrared (FTM-IR) Spectromicroscopy for high-resolution imaging, we determined that the bonds associated with cellulose and arabinoxylan, another major component of the primary cell walls of grasses, were in a lower energy configuration compared to wild type, suggesting a slightly weaker primary wall in MLG deficient mesophyll cells. Taken together, these results suggest that MLG may influence cellulose deposition in mesophyll cell walls without significantly affecting anisotropic growth thus challenging MLG importance in cell wall expansion.

Microtubules are dynamic polymers with fundamental roles in eukaryotes. Despite their importance, how new microtubules form is poorly understood. Textbooks focus on a nucleation-elongation mechanism in which monomers rapidly equilibrate with an unstable oligomer (nucleus) that limits the rate of polymer formation; once formed, the polymer elongates efficiently from this nucleus by monomer addition. Such models faithfully describe actin assembly, but fail to account for how more complex polymers like hollow microtubules assemble. Here we articulate a new model for microtubule formation that has three key features: microtubules initiate via rectangular, sheet-like structures which grow faster the larger they become; the dominant pathway proceeds via accretion, stepwise addition of longitudinal or lateral layers; a 'straightening penalty' to account for the energetic cost of Tubulin's curved-to-straight conformational transition. This model can quantitatively fit experimental assembly data, providing new insights into biochemical determinants and assembly pathways for microtubule nucleation. Competing Interest Statement The authors have declared no competing interest. Footnotes * To make the paper easier to follow/undrestand, we eliminated what had been the introductory figure discussing phenomenological scaling, and all panels of subsequent figures that relied on concepts from that introductory figure. To support the robustness of our claims, we analyzed a second, independently collected dataset. We made a number of other changes to text and figures with the goal of streamlining to improve readability, and to better explain some of the logic.

The gamma-tubulin ring complex (gammaTuRC) is a protein complex of relative molecular mass approximately 2.2 x 10(6) that nucleates microtubules at the centrosome. Here we use electron-microscopic tomography and metal shadowing to examine the structure of isolated Drosophila gammaTuRCs and the ends of microtubules nucleated by gammaTuRCs and by centrosomes. We show that the gammaTuRC is a lockwasher-like structure made up of repeating subunits, topped asymmetrically with a cap. A similar capped ring is also visible at one end of microtubules grown from isolated gammaTuRCs and from centrosomes. Antibodies against gamma-tubulin label microtubule ends, but not walls, in centrosomes. These data are consistent with a template-mediated mechanism for microtubule nucleation by the gammaTuRC.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

Microtubule nucleation is spatiotemporally regulated in cells by several molecules, including the template γ-tubulin and the polymerase XMAP215. Recently, XMAP215 and the γ-tubulin ring complex were reported to function synergistically, and this synergy was hypothesized to be due to direct binding between XMAP215 and γ-tubulin. Here, we address this hypothesis by 1) probing domain requirements for XMAP215 to promote microtubule nucleation and 2) testing whether XMAP215 functions synergistically with γ-tubulin in the absence of the other ring complex proteins. We confirm that γ-tubulin and XMAP215 are classically defined nucleators that reduce the nucleation lag seen in bulk tubulin assembly. Then, using deletion constructs, we show that XMAP215's ability to nucleate microtubules in purified solutions correlates with its ability to elongate existing microtubules and does not depend on the number of TOG domains. Finally, we show that XMAP215 and γ-tubulin promote αβ-tubulin assembly in an additive, not synergistic, manner. Thus, their modes of action during microtubule nucleation are distinct, and the synergy reported between XMAP215 and the γ-tubulin ring complex is not due to γ-tubulin alone. Competing Interest Statement The authors have declared no competing interest.