Applying 3D ED/MicroED workflows toward the next frontiers.

We report on the latest advancements in Microcrystal Electron Diffraction (3D ED/MicroED), as discussed during a symposium at the National Center for CryoEM Access and Training housed at the New York Structural Biology Center. This snapshot describes cutting-edge developments in various facets of the field and identifies potential avenues for continued progress. Key sections discuss instrumentation access, research applications for small molecules and biomacromolecules, data collection hardware and software, data reduction software, and finally reporting and validation. 3D ED/MicroED is still early in its wide adoption by the structural science community with ample opportunities for expansion, growth, and innovation.

Scaling up cryo-EM for biology and chemistry: The journey from niche technology to mainstream method.

Cryoelectron microscopy (cryo-EM) methods have made meaningful contributions in a wide variety of scientific research fields. In structural biology, cryo-EM routinely elucidates molecular structure from isolated biological macromolecular complexes or in a cellular context by harnessing the high-resolution power of the electron in order to image samples in a frozen, hydrated environment. For structural chemistry, the cryo-EM method popularly known as microcrystal electron diffraction (MicroED) has facilitated atomic structure generation of peptides and small molecules from their three-dimensional crystal forms. As cryo-EM has grown from an emerging technology, it has undergone modernization to enable multimodal transmission electron microscopy (TEM) techniques becoming more routine, reproducible, and accessible to accelerate research across multiple disciplines. We review recent advances in modern cryo-EM and assess how they are contributing to the future of the field with an eye to the past.

Fully human monoclonal antibody targeting activated ADAM10 on colorectal cancer cells.

Metastasis and chemoresistance in colorectal cancer are mediated by certain poorly differentiated cancer cells, known as cancer stem cells, that are maintained by Notch downstream signaling initiated upon Notch cleavage by the metalloprotease ADAM10. It has been shown that ADAM10 overexpression correlates with aberrant signaling from Notch, erbBs, and other receptors, as well as a more aggressive metastatic phenotype, in a range of cancers including colon, gastric, prostate, breast, ovarian, uterine, and leukemia. ADAM10 inhibition, therefore, stands out as an important and new approach to deter the progression of advanced CRC. For targeting the ADAM10 substrate-binding region, which is located outside of the catalytic domain of the protease, we generated a human anti-ADAM10 monoclonal antibody named 1H5. Structural and functional characterization of 1H5 reveals that it binds to the substrate-binding cysteine-rich domain and recognizes an activated ADAM10 conformation present on tumor cells. The mAb inhibits Notch cleavage and proliferation of colon cancer cell lines in vitro and in mouse models. Consistent with its binding to activated ADAM10, the mAb augments the catalytic activity of ADAM10 towards small peptide substrates in vitro. Most importantly, in a mouse model of colon cancer, when administered in combination with the therapeutic agent Irinotecan, 1H5 causes highly effective tumor growth inhibition without any discernible toxicity effects. Our singular approach to target the ADAM10 substrate-binding region with therapeutic antibodies could overcome the shortcomings of previous intervention strategies of targeting the protease active site with small molecule inhibitors that exhibit musculoskeletal toxicity.

Allosteric interactions prime androgen receptor dimerization and activation.

The androgen receptor (AR) is a nuclear receptor that governs gene expression programs required for prostate development and male phenotype maintenance. Advanced prostate cancers display AR hyperactivation and transcriptome expansion, in part, through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR domains and cofactors cooperate to bind DNA has remained elusive. Using single-particle cryo-electron microscopy, we isolated three conformations of AR bound to DNA, showing that AR forms a non-obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome and reinforced by AR's oncoprotein cofactor, ERG, and by DNA-binding motifs. Finally, we present evidence that this plastic dimer interface may have been adopted for transactivation at the expense of DNA binding. Our work highlights how fine-tuning AR's cooperative interactions translate to consequences in development and disease.

Structural insights into DNMT5-mediated ATP-dependent high-fidelity epigenome maintenance.

Epigenetic evolution occurs over million-year timescales in Cryptococcus neoformans and is mediated by DNMT5, the first maintenance type cytosine methyltransferase identified in the fungal or protist kingdoms, the first dependent on adenosine triphosphate (ATP), and the most hemimethyl-DNA-specific enzyme known. To understand these novel properties, we solved cryo-EM structures of CnDNMT5 in three states. These studies reveal an elaborate allosteric cascade in which hemimethylated DNA binding first activates the SNF2 ATPase domain by a large rigid body rotation while the target cytosine partially flips out of the DNA duplex. ATP binding then triggers striking structural reconfigurations of the methyltransferase catalytic pocket to enable cofactor binding, completion of base flipping, and catalysis. Bound unmethylated DNA does not open the catalytic pocket and is instead ejected upon ATP binding, driving high fidelity. This unprecedented chaperone-like, enzyme-remodeling role of the SNF2 ATPase domain illuminates how energy is used to enable faithful epigenetic memory.

Structure and mechanism of Mycobacterium smegmatis polynucleotide phosphorylase.

Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3'-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction PNPase acts as a polymerase, using NDPs as substrates to add NMPs to the 3'-OH terminus of RNA chains while expelling inorganic phosphate. The apparent essentiality of PNPase for growth of M. tuberculosis militates for mycobacterial PNPase as a potential drug target. A cryo-EM structure of Mycobacterium smegmatis PNPase (MsmPNPase) reveals a characteristic ring-shaped homotrimer in which each protomer consists of two RNase PH-like domains and an intervening α-helical module on the inferior surface of the ring. The C-terminal KH and S1 domains, which impart RNA specificity to MsmPNPase, are on the opposite face of the core ring and are conformationally mobile. Single particle reconstructions of MsmPNPase in the act of poly(A) synthesis highlight a 3'-terminal (rA)4 oligonucleotide and two magnesium ions in the active site and an adenine nucleobase in the central tunnel. We identify amino acids that engage the 3' segment of the RNA chain (Phe68, Arg105, Arg112, Arg430, Arg431) and the two metal ions (Asp526, Asp532, Gln546, Asp548) and we infer those that bind inorganic phosphate (Thr470, Ser471, His435, Lys534). Alanine mutagenesis pinpointed RNA and phosphate contacts as essential (Arg105, Arg431, Lys534, Thr470+Ser471), important (Arg112, Arg430), or unimportant (Phe68) for PNPase activity. Severe phosphorolysis and polymerase defects accompanying alanine mutations of the enzymic metal ligands suggest a two-metal mechanism of catalysis by MsmPNPase.

Molecular mechanisms of assembly and TRIP13-mediated remodeling of the human Shieldin complex.

The Shieldin complex, composed of REV7, SHLD1, SHLD2, and SHLD3, protects DNA double-strand breaks (DSBs) to promote nonhomologous end joining. The AAA+ ATPase TRIP13 remodels Shieldin to regulate DNA repair pathway choice. Here we report crystal structures of human SHLD3-REV7 binary and fused SHLD2-SHLD3-REV7 ternary complexes, revealing that assembly of Shieldin requires fused SHLD2-SHLD3 induced conformational heterodimerization of open (O-REV7) and closed (C-REV7) forms of REV7. We also report the cryogenic electron microscopy (cryo-EM) structures of the ATPγS-bound fused SHLD2-SHLD3-REV7-TRIP13 complexes, uncovering the principles underlying the TRIP13-mediated disassembly mechanism of the Shieldin complex. We demonstrate that the N terminus of REV7 inserts into the central channel of TRIP13, setting the stage for pulling the unfolded N-terminal peptide of C-REV7 through the central TRIP13 hexameric channel. The primary interface involves contacts between the safety-belt segment of C-REV7 and a conserved and negatively charged loop of TRIP13. This process is mediated by ATP hydrolysis-triggered rotatory motions of the TRIP13 ATPase, thereby resulting in the disassembly of the Shieldin complex.

Automation of Continuous-Rotation Data Collection for MicroED.

Automated coordination of microscope and camera functions for MicroED data collection simplifies the procedure for robust dataset acquisition and enables unattended sequential collection of many crystal targets. This chapter discusses the prerequisites for an algorithm of data collection automation for continuous-rotation MicroED and presents a practical protocol for achieving this goal using the popular TEM control software program SerialEM.

MicroED data collection with SerialEM.

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.

Nanoemulsion-Based Delivery of Fluorescent PARP Inhibitors in Mouse Models of Small Cell Lung Cancer.

The preclinical potential of many diagnostic and therapeutic small molecules is limited by their rapid washout kinetics and consequently modest pharmacological performances. In several cases, these could be improved by loading the small molecules into nanoparticulates, improving blood half-life, in vivo uptake and overall pharmacodynamics. In this study, we report a nanoemulsion (NE) encapsulated form of PARPi-FL. As a proof of concept, we used PARPi-FL, which is a fluorescently labeled sensor for olaparib, a FDA-approved small molecule inhibitor of the nuclear enzyme poly(ADP-ribose)polymerase 1 (PARP1). Encapsulated PARPi-FL showed increased blood half-life, and delineated subcutaneous xenografts of small cell lung cancer (SCLC), a fast-progressing disease where efficient treatment options remain an unmet clinical need. Our study demonstrates an effective method for expanding the circulation time of a fluorescent PARP inhibitor, highlighting the pharmacokinetic benefits of nanoemulsions as nanocarriers and confirming the value of PARPi-FL as an imaging agent targeting PARP1 in small cell lung cancer.

Integrative structure and functional anatomy of a nuclear pore complex.

Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.

MicroED Structure of Au146(p-MBA)57 at Subatomic Resolution Reveals a Twinned FCC Cluster.

Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. Herein we present the structure of the largest aqueous gold cluster, Au146(p-MBA)57 (p-MBA: para-mercaptobenzoic acid), solved by electron micro-diffraction (MicroED) to subatomic resolution (0.85 Å) and by X-ray diffraction at atomic resolution (1.3 Å). 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.

Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.

Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.

Modeling truncated pixel values of faint reflections in MicroED images.

The weak pixel counts surrounding the Bragg spots in a diffraction image are important for establishing a model of the background underneath the peak and estimating the reliability of the integrated intensities. Under certain circumstances, particularly with equipment not optimized for low-intensity measurements, these pixel values may be corrupted by corrections applied to the raw image. This can lead to truncation of low pixel counts, resulting in anomalies in the integrated Bragg intensities, such as systematically higher signal-to-noise ratios. A correction for this effect can be approximated by a three-parameter lognormal distribution fitted to the weakly positive-valued pixels at similar scattering angles. The procedure is validated by the improved refinement of an atomic model against structure factor amplitudes derived from corrected micro-electron diffraction (MicroED) images.

The collection of MicroED data for macromolecular crystallography.

The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.

MicroED data collection and processing.

MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals. A major limiting factor in X-ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges.

Determination of molecular structures of HIV envelope glycoproteins using cryo-electron tomography and automated sub-tomogram averaging.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) (2). Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor (5,9). The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine. The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at ˜20 Šresolution (9,14). This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH (http://biowulf.nih.gov). This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

Three-dimensional structures of soluble CD4-bound states of trimeric simian immunodeficiency virus envelope glycoproteins determined by using cryo-electron tomography.

The trimeric envelope glycoprotein (Env) spikes displayed on the surfaces of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) virions are composed of three heterodimers of the viral glycoproteins gp120 and gp41. Although binding of gp120 to cell surface CD4 and a chemokine receptor is known to elicit conformational changes in gp120 and gp41, changes in quaternary structure of the trimer have only recently been elucidated. For the HIV-1 BaL isolate, CD4 attachment results in a striking rearrangement of the trimer from a "closed" to an "open" conformation. The effect of CD4 on SIV trimers, however, has not been described. Using cryo-electron tomography, we have now determined molecular architectures of the soluble CD4 (sCD4)-bound states of SIV Env trimers for three different strains (SIVmneE11S, SIVmac239, and SIV CP-MAC). In marked contrast to HIV-1 BaL, SIVmneE11S and SIVmac239 Env showed only minor conformational changes following sCD4 binding. In SIV CP-MAC, where trimeric Env displays a constitutively "open" conformation similar to that seen for HIV-1 BaL Env in the sCD4-complexed state, we show that there are no significant further changes in conformation upon the binding of either sCD4 or 7D3 antibody. The density maps also show that 7D3 and 17b antibodies target epitopes on gp120 that are on opposites sides of the coreceptor binding site. These results provide new insights into the structural diversity of SIV Env and show that there are strain-dependent variations in the orientation of sCD4 bound to trimeric SIV Env.

Effects of UVA irradiation, aryl azides, and reactive oxygen species on the orthogonal inactivation of the human immunodeficiency virus (HIV-1).

Previously we reported that hydrophobic aryl azides partition into hydrophobic regions of the viral membrane of enveloped viruses and inactivate the virus upon UVA irradiation for 2 min. Prolonged irradiation (15 min) resulted in viral protein aggregation as visualized via Western blot analysis, due to reactive oxygen species (ROS) formation, with preservation of the surface antigenic epitopes. Herein, we demonstrate that these aggregates show detergent resistance and that this property may be useful towards the creation of a novel orthogonal virus inactivation strategy for use in preparing experimental vaccines. When ROS-modified HIV virus preparations were treated with 1% Triton X-100, there was an increase in the percent of viral proteins (gp41, p24) in the viral pellet after ultracentrifugation through sucrose. Transmission electron microscopy (TEM) of these detergent-resistant pellets shows some recognizable virus fragments, and immunoprecipitation studies of the gp41 aggregates suggest the aggregation is covalent in nature, involving short-range interactions.