Faculty Bibliography

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The human GABA_B receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction¹. A unique GPCR that is known to require heterodimerization for function^2-6, the GABA_B receptor has two subunits, GABA_B1 and GABA_B2, that are structurally homologous but perform distinct and complementary functions. GABA_B1 recognizes orthosteric ligands^7,8, while GABA_B2 couples with G proteins^9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail¹⁵. Although the VFT heterodimer structure has been resolved¹⁶, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABA_B receptor at atomic resolution, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction¹. A unique GPCR that is known to require heterodimerization for function^2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands^7,8, while GABAB2 couples with G proteins^9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail¹⁵. Although the VFT heterodimer structure has been resolved¹⁶, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor at atomic resolution, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.
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The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic β-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells.

BACKGROUND INFORMATION/BACKGROUND:The current consensus on cilia development posits that the ciliary transition zone (TZ) is formed via extension of nine centrosomal microtubules. In this model, TZ structure remains unchanged in microtubule number throughout the cilium life cycle. This model does not however explain structural variations of TZ structure seen in nature and could also lend itself to the misinterpretation that deviations from nine-doublet microtubule ultrastructure represent an abnormal phenotype. Thus, a better understanding of events that occur at the TZ in vivo during metazoan development is required. RESULTS:To address this issue, we characterized ultrastructure of two types of sensory cilia in developing Caenorhabditis elegans. We discovered that, in cephalic male (CEM) and inner labial quadrant (IL2Q) sensory neurons, ciliary TZs are structurally plastic and remodel from one structure to another during animal development. The number of microtubule doublets forming the TZ can be increased or decreased over time, depending on cilia type. Both cases result in structural TZ intermediates different from TZ in cilia of adult animals. In CEM cilia, axonemal extension and maturation occurs concurrently with TZ structural maturation. CONCLUSIONS AND SIGNIFICANCE/CONCLUSIONS:Our work extends the current model to include the structural plasticity of metazoan transition zone, which can be structurally delayed, maintained or remodelled in cell type-specific manner.

Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

The scope and complexity of cryogenic electron microscopy (cryoEM) data has greatly increased, and will continue to do so, due to recent and ongoing technical breakthroughs that have led to much improved resolutions for macromolecular structures solved using this method. This big data explosion includes single particle data as well as tomographic tilt series, both generally acquired as direct detector movies of ∼10-100 frames per image or per tilt-series. We provide a brief survey of the developments leading to the current status, and describe existing cryoEM pipelines, with an emphasis on the scope of data acquisition, methods for automation, and use of cloud storage and computing.

Cryo electron microscopy facilities running multiple instruments and serving users with varying skill levels need a robust and reliable method for benchmarking both the hardware and software components of their single particle analysis workflow. The workflow is complex, with many bottlenecks existing at the specimen preparation, data collection and image analysis steps; the samples and grid preparation can be of unpredictable quality, there are many different protocols for microscope and camera settings, and there is a myriad of software programs for analysis that can depend on dozens of settings chosen by the user. For this reason, we believe it is important to benchmark the entire workflow, using a standard sample and standard operating procedures, on a regular basis. This provides confidence that all aspects of the pipeline are capable of producing maps to high resolution. Here we describe benchmarking procedures using a test sample, rabbit muscle aldolase.

Automated data acquisition is used widely for single-particle reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope. Automation has become integral to this method because of the very large number of particle images required in order to overcome the typically low signal-to-noise ratio of these images. For optimal efficiency, automated data acquisition software packages typically employ some beam-image shift targeting as this method is both fast and accurate (±0.1 µm). In contrast, using only stage movement, relocation to a targeted area under low-dose conditions can only be achieved in combination with multiple iterations or long relaxation times, both reducing efficiency. Nevertheless it is well known that applying beam-image shift induces beam-tilt and with it a potential structure phase error with a phase error π/4 the highest acceptable value. This theory has been used as an argument against beam-image shift for high resolution data collection. Nevertheless, in practice many small beam-image shift datasets have resulted in 3D reconstructions beyond the π/4 phase error limit. To address this apparent contradiction, we performed cryo-EM single-particle reconstructions on a T20S proteasome sample using applied beam-image shifts corresponding to beam tilts from 0 to 10 mrad. To evaluate the results we compared the FSC values, and examined the water density peaks in the 3D map. We conclude that the phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the π/4 resolution limit.

Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing.