Machine learning approaches to cryoEM density modification differentially affect biomacromolecule and ligand density quality.

The application of machine learning to cryogenic electron microscopy (cryoEM) data analysis has added a valuable set of tools to the cryoEM data processing pipeline. As these tools become more accessible and widely available, the implications of their use should be assessed. We noticed that machine learning map modification tools can have differential effects on cryoEM densities. In this perspective, we evaluate these effects to show that machine learning tools generally improve densities for biomacromolecules while generating unpredictable results for ligands. This unpredictable behavior manifests both in quantitative metrics of map quality and in qualitative investigations of modified maps. The results presented here highlight the power and potential of machine learning tools in cryoEM, while also illustrating some of the risks of their unexamined use.

Tuning ice thickness using the chameleon for high-quality cryoEM data collection.

Advances in single-particle cryogenic electron microscopy (cryoEM) now allow for routine structure determination of well-behaved biological specimens to high-resolution. Despite advances in the electron microscope, direct electron detectors, and data processing software, the preparation of high-quality grids with thin layers of vitreous ice containing the specimen of interest in random orientations remains a critical bottleneck for many projects. Although numerous efforts have been dedicated to overcoming hurdles frequently encountered during specimen vitrification using traditional blot-and-plunge specimen preparation techniques, the development of blot-free grid preparation devices provide a unique opportunity to carefully tune ice thickness, particle density, and specimen behavior during the vitrification process for improvements in image quality. Here, we describe critical steps of high-quality grid preparation using a SPT Labtech chameleon, evaluation of grid quality/ice thickness using the chameleon software, high-throughput imaging in the electron microscope, and recommend steps for troubleshooting grid preparation when standard parameters fail to yield suitable specimen.

Structural insights into GrpEL1-mediated nucleotide and substrate release of human mitochondrial Hsp70.

Maintenance of protein homeostasis is necessary for cell viability and depends on a complex network of chaperones and co-chaperones, including the heat-shock protein 70 (Hsp70) system. In human mitochondria, mitochondrial Hsp70 (mortalin) and the nucleotide exchange factor (GrpEL1) work synergistically to stabilize proteins, assemble protein complexes, and facilitate protein import. However, our understanding of the molecular mechanisms guiding these processes is hampered by limited structural information. To elucidate these mechanistic details, we used cryoEM to determine the first structures of full-length human mortalin-GrpEL1 complexes in previously unobserved states. Our structures and molecular dynamics simulations allow us to delineate specific roles for mortalin-GrpEL1 interfaces and to identify steps in GrpEL1-mediated nucleotide and substrate release by mortalin. Subsequent analyses reveal conserved mechanisms across bacteria and mammals and facilitate a complete understanding of sequential nucleotide and substrate release for the Hsp70 chaperone system.

The SMC-family Wadjet complex protects bacteria from plasmid transformation by recognition and cleavage of closed-circular DNA.

Self versus non-self discrimination is a key element of innate and adaptive immunity across life. In bacteria, CRISPR-Cas and restriction-modification systems recognize non-self nucleic acids through their sequence and their methylation state, respectively. Here, we show that the Wadjet defense system recognizes DNA topology to protect its host against plasmid transformation. By combining cryoelectron microscopy with cross-linking mass spectrometry, we show that Wadjet forms a complex similar to the bacterial condensin complex MukBEF, with a novel nuclease subunit similar to a type II DNA topoisomerase. Wadjet specifically cleaves closed-circular DNA in a reaction requiring ATP hydrolysis by the structural maintenance of chromosome (SMC) ATPase subunit JetC, suggesting that the complex could use DNA loop extrusion to sense its substrate's topology, then specifically activate the nuclease subunit JetD to cleave plasmid DNA. Overall, our data reveal how bacteria have co-opted a DNA maintenance machine to specifically recognize and destroy foreign DNAs through topology sensing.

Structures of the nitrogenase complex prepared under catalytic turnover conditions.

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multielectron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here, we report cryo-electron microscopy structures of the nitrogenase complex prepared under enzymatic turnover conditions. We observe that asymmetry governs all aspects of the nitrogenase mechanism, including ATP hydrolysis, protein-protein interactions, and catalysis. Conformational changes near the catalytic iron-molybdenum cofactor are correlated with the nucleotide-hydrolysis state of the enzyme.

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy.

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.

Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge.

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

Setting Up Parallel Illumination on the Talos Arctica for High-Resolution Data Collection.

Illuminating a specimen with a parallel electron beam is critical for many experiments in transmission electron microscopy as deviations from this condition cause considerable deterioration of image quality. Carefully establishing parallel illumination is particularly important on two-condenser lens transmission electron microscopes (TEMs) as the parallel illumination condition is limited to a single beam intensity value on these instruments. It was recently shown that a Thermo Fisher Scientific Talos Arctica, a two-condenser lens TEM operating at 200 kV, equipped with a Gatan K2 Summit direct electron detector is capable of resolving frozen-hydrated macromolecules of various sizes and internal symmetries to better than 3 Å resolution using single particle methodologies. A critical aspect of the success of these findings was the careful alignment of the electron microscope to ensure the specimen was illuminated with a parallel electron beam. Here, this chapter describes how to establish parallel illumination conditions in a Talos Arctica TEM for high-resolution cryogenic data collection for structure determination.

Sub-2 Angstrom resolution structure determination using single-particle cryo-EM at 200 keV.

Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination of well-behaved specimens to high-resolution, there nonetheless remains a discrepancy between the resolutions attained for biological specimens and the information limits of modern transmission electron microscopes (TEMs). Instruments operating at 300 kV equipped with DEDs are the current paradigm for high-resolution single-particle cryo-EM, while 200 kV TEMs remain comparatively underutilized for purposes beyond sample screening. Here, we expand upon our prior work and demonstrate that one such 200 kV microscope, the Talos Arctica, equipped with a K2 DED is capable of determining structures of macromolecules to as high as ∼1.7 Šresolution. At this resolution, ordered water molecules are readily assigned and holes in aromatic residues can be clearly distinguished in the reconstructions. This work emphasizes the utility of 200 kV electrons for high-resolution single-particle cryo-EM and applications such as structure-based drug design.

High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM.

Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While the Volta phase plate has enabled visualization of specimens in this size range, this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.7 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity to enable investigation of drug-target interactions. Our ~2.8 Å and ~3.2 Å structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the sub-nanometer cryo-EM structure of a sub-50 kDa protein.

A Multi-model Approach to Assessing Local and Global Cryo-EM Map Quality.

There does not currently exist a standardized indicator of how well cryo-EM-derived models represent the density from which they were generated. We present a straightforward methodology that utilizes freely available tools to generate a suite of independent models and to evaluate their convergence in an EM density. These analyses provide both a quantitative and qualitative assessment of the precision of the models and their representation of the density, respectively, while concurrently providing a platform for assessing both global and local EM map quality. We further use standardized datasets to provide an expected deviation within a suite of models refined against EM maps reported to be at 5 Å resolution or better. Associating multiple atomic models with a deposited EM map provides a rapid and accessible reporter of convergence, a strong indicator of highly resolved molecular detail, and is an important step toward an FSC-independent assessment of map and model quality.

Conformational ensemble of the human TRPV3 ion channel.

Transient receptor potential vanilloid channel 3 (TRPV3), a member of the thermosensitive TRP (thermoTRPV) channels, is activated by warm temperatures and serves as a key regulator of normal skin physiology through the release of pro-inflammatory messengers. Mutations in trpv3 have been identified as the cause of the congenital skin disorder, Olmsted syndrome. Unlike other members of the thermoTRPV channel family, TRPV3 sensitizes upon repeated stimulation, yet a lack of structural information about the channel precludes a molecular-level understanding of TRPV3 sensitization and gating. Here, we present the cryo-electron microscopy structures of apo and sensitized human TRPV3, as well as several structures of TRPV3 in the presence of the common thermoTRPV agonist 2-aminoethoxydiphenyl borate (2-APB). Our results show α-to-π-helix transitions in the S6 during sensitization, and suggest a critical role for the S4-S5 linker π-helix during ligand-dependent gating.

Cryo-EM structure of a mitochondrial calcium uniporter.

Calcium transport plays an important role in regulating mitochondrial physiology and pathophysiology. The mitochondrial calcium uniporter (MCU) is a calcium-selective ion channel that is the primary mediator for calcium uptake into the mitochondrial matrix. Here, we present the cryo-electron microscopy structure of the full-length MCU from Neurospora crassa to an overall resolution of ~3.7 angstroms. Our structure reveals a tetrameric architecture, with the soluble and transmembrane domains adopting different symmetric arrangements within the channel. The conserved W-D-Φ-Φ-E-P-V-T-Y sequence motif of MCU pore forms a selectivity filter comprising two acidic rings separated by one helical turn along the central axis of the channel pore. The structure combined with mutagenesis gives insight into the basis of calcium recognition.

Structural and cellular mechanisms of peptidyl-prolyl isomerase Pin1-mediated enhancement of Tissue Factor gene expression, protein half-life, and pro-coagulant activity.

Tissue Factor is a cell-surface glycoprotein expressed in various cells of the vasculature and is the principal regulator of the blood coagulation cascade and hemostasis. Notably, aberrant expression of Tissue Factor is associated with cardiovascular pathologies such as atherosclerosis and thrombosis. Here, we sought to identify factors that regulate Tissue Factor gene expression and activity. Tissue Factor gene expression is regulated by various transcription factors, including activating protein-1 and nuclear factor-κ B. The peptidyl-prolyl isomerase Pin1 is known to modulate the activity of these two transcription factors, and we now show that Pin1 augments Tissue Factor gene expression in both vascular smooth muscle cells and activated endothelial cells via activating protein-1 and nuclear factor-κ B signaling. Furthermore, the cytoplasmic domain of Tissue Factor contains a well-conserved phospho-Ser258-Pro259 amino-acid motif recognized by Pin1. Using co-immunoprecipitation and solution nuclear magnetic resonance spectroscopy, we show that the WW-domain of Pin1 directly binds the cytoplasmic domain of Tissue Factor. This interaction occurs via the phospho-Ser258-Pro259 sequence in the Tissue Factor cytoplasmic domain and results in increased protein half-life and pro-coagulant activity. Taken together, our results establish Pin1 as an upstream regulator of Tissue Factor-mediated coagulation, thereby opening up new avenues for research into the use of specific Pin1 inhibitors for the treatment of diseases characterized by pathological coagulation, such as thrombosis and atherosclerosis.

Achieving better-than-3-Å resolution by single-particle cryo-EM at 200 keV.

Nearly all single-particle cryo-EM structures resolved to better than 4-Å resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain reconstructions of macromolecular complexes of different sizes to better than 3-Å resolution using a 200-keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules.

Cryo-electron microscopy structure of the lysosomal calcium-permeable channel TRPML3.

The modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signalling. The transient receptor potential mucolipin (TRPML) channel family belongs to the TRP superfamily and is composed of three members: TRPML1-TRPML3. TRPMLs are the major Ca2+-permeable channels on late endosomes and lysosomes (LEL). They regulate the release of Ca2+ from organelles, which is important for various physiological processes, including organelle trafficking and fusion. Loss-of-function mutations in the MCOLN1 gene, which encodes TRPML1, cause the neurodegenerative lysosomal storage disorder mucolipidosis type IV, and a gain-of-function mutation (Ala419Pro) in TRPML3 gives rise to the varitint-waddler (Va) mouse phenotype. Notably, TRPML channels are activated by the low-abundance and LEL-enriched signalling lipid phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), whereas other phosphoinositides such as PtdIns(4,5)P2, which is enriched in plasma membranes, inhibit TRPMLs. Conserved basic residues at the N terminus of the channel are important for activation by PtdIns(3,5)P2 and inhibition by PtdIns(4,5)P2. However, owing to a lack of structural information, the mechanism by which TRPML channels recognize PtdIns(3,5)P2 and increase their Ca2+ conductance remains unclear. Here we present the cryo-electron microscopy (cryo-EM) structure of a full-length TRPML3 channel from the common marmoset (Callithrix jacchus) at an overall resolution of 2.9 Å. Our structure reveals not only the molecular basis of ion conduction but also the unique architecture of TRPMLs, wherein the voltage sensor-like domain is linked to the pore via a cytosolic domain that we term the mucolipin domain. Combined with functional studies, these data suggest that the mucolipin domain is responsible for PtdIns(3,5)P2 binding and subsequent channel activation, and that it acts as a 'gating pulley' for lipid-dependent TRPML gating.

Regulation of nitric oxide signaling by formation of a distal receptor-ligand complex.

The binding of nitric oxide (NO) to the heme cofactor of heme-nitric oxide/oxygen binding (H-NOX) proteins can lead to the dissociation of the heme-ligating histidine residue and yield a five-coordinate nitrosyl complex, an important step for NO-dependent signaling. In the five-coordinate nitrosyl complex, NO can reside on either the distal or proximal side of the heme, which could have a profound influence over the lifetime of the in vivo signal. To investigate this central molecular question, we characterized the Shewanella oneidensis H-NOX (So H-NOX)-NO complex biophysically under limiting and excess NO conditions. The results show that So H-NOX preferably forms a distal NO species with both limiting and excess NO. Therefore, signal strength and complex lifetime in vivo will be dictated by the dissociation rate of NO from the distal complex and the rebinding of the histidine ligand to the heme.

Nitric Oxide-Induced Conformational Changes Govern H-NOX and Histidine Kinase Interaction and Regulation in Shewanella oneidensis.

Nitric oxide (NO) is implicated in biofilm regulation in several bacterial families via heme-nitric oxide/oxygen binding (H-NOX) protein signaling. Shewanella oneidensis H-NOX (So H-NOX) is associated with a histidine kinase (So HnoK) encoded on the same operon, and together they form a multicomponent signaling network whereby the NO-bound state of So H-NOX inhibits So HnoK autophosphorylation activity, affecting the phosphorylation state of three response regulators. Although the conformational changes of So H-NOX upon NO binding have been structurally characterized, the mechanism of HnoK inhibition by NO-bound So H-NOX remains unclear. In the present study, the molecular details of So H-NOX and So HnoK interaction and regulation are characterized. The N-terminal domain in So HnoK was determined to be the site of H-NOX interaction, and the binding interface on So H-NOX was identified using a combination of hydrogen-deuterium exchange mass spectrometry and surface-scanning mutagenesis. Binding kinetics measurements and analytical gel filtration revealed that NO-bound So H-NOX has a tighter affinity for So HnoK compared that of H-NOX in the unliganded state, correlating binding affinity with kinase inhibition. Kinase activity assays with binding-deficient H-NOX mutants further indicate that while formation of the H-NOX-HnoK complex is required for HnoK to be catalytically active, H-NOX conformational changes upon NO-binding are necessary for HnoK inhibition.