The cryoEM method Microcrystal Electron Diffraction (MicroED) involves transmission electron microscope (TEM) and electron detector working in synchrony to collect electron diffraction data by continuous rotation. We previously reported several protein, peptide, and small molecule structures by MicroED using manual control of the microscope and detector to collect data. Here we present a procedure to automate this process using a script developed for the popular open-source software package SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. Depending on crystal and substrate geometry, more than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses.

Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. We report the structure of Au146(p-MBA)57 at subatomic resolution (0.85 {\AA}) using electron diffraction (MicroED) and atomic resolution by X-ray diffraction. The 146 gold atoms may be decomposed into two constituent sets consisting of 119 core and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure whereas the surface gold atoms follow a C2 rotational symmetry about an axis bisecting the twinning plane. The protective layer of 57 p-MBAs fully encloses the cluster and comprises bridging, monomeric, and dimeric staple motifs. Au146(p-MBA)57 is the largest cluster observed exhibiting a bulk-like FCC structure as well as the smallest gold particle exhibiting a stacking fault.

Sodium (Na) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na are proposed to play critical roles in determining the conductance and the selectivity of Na channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na ion at the entrance of the channel pore. A process of dilation coupled with Na movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics.

Theoretical calculations suggest that crystals exceeding 100 nm thickness are excluded by dynamical scattering from successful structure determination using microcrystal electron diffraction (MicroED). These calculations are at odds with experimental results where MicroED structures have been determined from significantly thicker crystals. Here we systematically evaluate the influence of thickness on the accuracy of MicroED intensities and the ability to determine structures from protein crystals one micrometer thick. To do so, we compare ab initio structures of a human prion protein segment determined from thin crystals to those determined from crystals up to one micrometer thick. We also compare molecular replacement solutions from crystals of varying thickness for a larger globular protein, proteinase K. Our results indicate that structures can be reliably determined from crystals at least an order of magnitude thicker than previously suggested by simulation, opening the possibility for an even broader range of MicroED experiments.

HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.

Engineered tissues can be used to understand fundamental features of biology, develop organotypic in vitro model systems, and as engineered tissue constructs for replacing damaged tissue in vivo. However, a key limitation is an inability to test the wide range of parameters that might impact the engineered tissue in a high-throughput manner and in an environment that mimics the three-dimensional (3D) native architecture. We developed a microfabricated platform to generate arrays of microtissues embedded within 3D micropatterned matrices. Microcantilevers simultaneously constrain microtissue formation and report forces generated by the microtissues in real time, opening the possibility to use high-throughput, low-volume screening for studies on engineered tissues. Thanks to the micrometer scale of the microtissues, this platform is also suitable for high-throughput monitoring of drug-induced effect on architecture and contractility in engineered tissues. Moreover, independent variations of the mechanical stiffness of the cantilevers and collagen matrix allow the measurement and manipulation of the mechanics of the microtissues. Thus, our approach will likely provide valuable opportunities to elucidate how biomechanical, electrical, biochemical, and genetic/epigenetic cues modulate the formation and maturation of 3D engineered tissues. In this chapter, we describe the microfabrication, preparation, and experimental use of such microfabricated tissue gauges.

Fluorescence microscopy, a key driver for progress in the life sciences, faces limitations due to the microscope’s optics, fluorophore chemistry, and photon exposure limits, necessitating trade-offs in imaging speed, resolution, and depth. Here, we introduce MicroSplit, a computational multiplexing technique based on deep learning that allows multiple cellular structures to be imaged in a single fluorescent channel and then unmixed computationally, allowing faster imaging and reduced photon exposure. We show that MicroSplit efficiently separates up to four superimposed noisy structures into distinct denoised fluorescent image channels. Furthermore, using Variational Splitting Encoder-Decoder (VSE) networks, our approach can sample diverse predictions from a trained posterior of solutions. The diversity of these samples scales with the uncertainty in a given input, allowing us to estimate the true prediction errors by computing the variability between posterior samples. We demonstrate the robustness of MicroSplit across various datasets and noise levels and show its utility to image more, image faster, and improve downstream analysis. We provide MicroSplit along with all associated training and evaluation datasets as open resources, enabling life scientists to benefit from the potential of computational multiplexing and accelerate the pace of scientific discovery.

We present a method for microtubule tracking in electron microscopy volumes. Our method first identifies a sparse set of voxels that likely belong to microtubules. Similar to prior work, we then enumerate potential edges between these voxels, which we represent in a candidate graph. Tracks of microtubules are found by selecting nodes and edges in the candidate graph by solving a constrained optimization problem incorporating biological priors on microtubule structure. For this, we present a novel integer linear programming formulation, which results in speed-ups of three orders of magnitude and an increase of 53% in accuracy compared to prior art (evaluated on three 1 . 2 × 4 × 4µm volumes of Drosophila neural tissue). We also propose a scheme to solve the optimization problem in a block-wise fashion, which allows distributed tracking and is necessary to process very large electron microscopy volumes. Finally, we release a benchmark dataset for microtubule tracking, here used for training, testing and validation, consisting of eight 30 x 1000 x 1000 voxel blocks (1 . 2 × 4 × 4µm) of densely annotated microtubules in the CREMI data set (https://github.com/nilsec/micron).

Tethered midbody remnants dancing across apical microvilli, encountering the centrosome, and beckoning forth a cilium-who would have guessed this is how polarized epithelial cells coordinate the end of mitosis and the beginning of ciliogenesis? New evidence from Bernabé-Rubio et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201601020) supports this emerging model.

Purine nucleotides are essential for mammalian development1,2. Purine monophosphates support cell signaling and proliferation and are synthesized by cells through either de novo synthesis or a salvage pathway3. We previously identified a midgestational metabolic transition in mice (gestational days gd10.5–11.5) characterized by changes in purine metabolism4. Midgestation is a period of rapid growth for placenta and embryo, yet it remains unclear how the placental tissues expand without directly competing with the embryo for biosynthetic resources. Here, we show that this midgestational metabolic transition is associated with a marked reduction in embryonic expression of purine salvage enzymes, which constrains embryonic metabolism and leads to different strategies for purine synthesis between the placenta and embryo. Midgestation embryos are unable to engage the purine salvage pathway even when de novo purine synthesis is blocked either in vivo or in ex utero embryo culture, whereas placental tissue and trophoblasts retain the capacity to use either pathway. Disruption of de novo purine synthesis in mice causes reduced embryonic growth, impaired axial elongation, and abnormal brain and placental development, which are only partially rescued by supplementation with purine salvage precursors. In human placenta, trophoblast stem cells readily switch between the de novo and salvage pathways based on nutrient availability, and syncytiotrophoblasts (STB) preferentially rely on the salvage pathway. We identified guanosine monophosphate (GMP) as a metabolic checkpoint regulating STB differentiation, with insufficient GMP levels causing degradation of the small GTPase Rheb and failure of mTOR activation. Supplementation of purine salvage substrates restored GMP synthesis and STB differentiation in humans, but not mice. Further, in vivo measurements in humans revealed that maternal circulating hypoxanthine decreases during pregnancy and is further reduced in women with clinically small placentas, highlighting the role of hypoxanthine in supporting placental growth. These results uncover compartmentalized purine salvage between the embryo and placenta as a mechanism that limits competition for biosynthetic resources and enables coordinated growth during mammalian development.