Angstrom Resolution Research Articles

How to Access the Cryo-EM Facility in KEK

In March 2018, KEK obtained a 200 kV cryogenic electron microscopy（cryo-EM; a Talos Arctica G2 with a Falcon 3EC direct electron detector）and prepared a pipeline for solving high-resolution structures of macromolecular biomolecules, such as proteins, DNA, and RNA. In October 2018, the cryo-EM facility in KEK opened to academic and industrial users. Our mission is three-fold：to provide cryo-EM machine time for external users, to support grid-prep and data collection（and single particle analysis, if needed）, and to help users acquire cryo-EM skills. One remarkable result is the single-particle analysis of an 860-kDa enzyme at 2.05 angstrom resolution, which suggested that we had established a proper protocol for cryo-EM operation and single-particle analysis. We will continue to support external users and carry out cryo-EM single particle analysis of macromolecular biomolecules.

Ultrafast Photon-Induced Tunneling Microscopy.

Unification of the techniques of ultrafast science and scanning tunneling microscopy (STM) has the potential of tracking electronic motion in molecules simultaneously in real space and real time. Laser pulses can couple to an STM junction either in the weak-field or in the strong-field interaction regime. The strong-field regime entails significant modification (dressing) of the tunneling barrier of the STM junction, whereas the weak-field or the photon-driven regime entails perturbative interaction. Here, we describe how photons carried in an ultrashort pulse interact with an STM junction, defining the basic fundamental framework of ultrafast photon-induced tunneling microscopy. Selective dipole coupling of electronic states by photons is shown to be controllable by adjusting the DC bias at the STM junction. An ultrafast tunneling microscopy involving photons is established. Consolidation of the technique calls for innovative approaches to detect photon-induced tunneling currents at the STM junction. We introduce and characterize here three techniques involving dispersion, polarization, and frequency modulation of the laser pulses to lock-in detect the laser-induced tunneling current. We show that photon-induced tunneling currents can simultaneously achieve angstrom scale spatial resolution and sub-femtosecond temporal resolution. Ultrafast photon-induced tunneling microscopy will be able to directly probe electron dynamics in complex molecular systems, without the need of reconstruction techniques.

Structural Basis of VSIG3: The Ligand for VISTA.

B7 family members and their receptors play key roles in regulating T cell responses, and constitute very attractive targets for developing immunotherapeutic drugs. V-Set and Immunoglobulin domain containing 3 (VSIG3), a ligand for the novel B7 family immune checkpoint V-domain immunoglobulin suppressor of T cell activation (VISTA), can significantly inhibit T cell functions. Inhibitors targeting the VISTA/VSIG3 pathway are of great significance in tumor immunology. Here, we show the crystal structure of the extracellular domain (ECD) of the human VSIG3 protein at 2.64 angstrom resolution, and we produce recombinant human VSIG-3 ECD in both CHO cells and E. coli. Furthermore, we demonstrated the interaction of VISTA and VSIG3 by coimmunoprecipitation (Co-IP). Based on protein-protein docking for VISTA and VSIG3, we report a small molecule inhibitor of VSIG3 K284-3046 and evaluate its biological activities in vitro. This study was the first to reveal the crystal structure of VSIG3, and provides the structural basis for designing antibodies or compounds for the unique VSIG3/VISTA coinhibitory pathway in the treatment of cancers, autoimmune diseases and may be beneficial of designing vaccines.

Mycobacterium tuberculosis puromycin hydrolase displays a prolyl oligopeptidase fold and an acyl aminopeptidase activity.

Puromycin-hydrolizing peptidases have been described as members of the prolyl oligopeptidase peptidase family. These enzymes are present across all domains of life but still little is known of the homologs found in the pathogenic bacterium Mycobacterium tuberculosis. The crystal structure of a M. tuberculosis puromycin hydrolase peptidase has been determined at 3 Angstrom resolution, revealing a conserved prolyl oligopeptidase fold, defined by α/β-hydrolase and β-propeller domains with two distinctive loops that occlude access of large substrates to the active site. The enzyme displayed amino peptidase activity with a substrate specificity preference for hydrophobic residues in the decreasing order of phenylalanine, leucine, alanine and proline. The enzyme's active site is lined by residues Glu564 for the coordination of the substrates amino terminal moiety and His561, Val608, Tyr78, Trp306, Phe563 and Ty567 for the accommodation of hydrophobic substrates. The availability of a crystal structure for puromycin hydrolase of M. tuberculosis shall facilitate the development of inhibitors with therapeutic applications.

Elongational stalling activates mitoribosome-associated quality control.

The human mitochondrial ribosome (mitoribosome) and associated proteins regulate the synthesis of 13 essential subunits of the oxidative phosphorylation complexes. We report the discovery of a mitoribosome-associated quality control pathway that responds to interruptions during elongation, and we present structures at 3.1- to 3.3-angstrom resolution of mitoribosomal large subunits trapped during ribosome rescue. Release factor homolog C12orf65 (mtRF-R) and RNA binding protein C6orf203 (MTRES1) eject the nascent chain and peptidyl transfer RNA (tRNA), respectively, from stalled ribosomes. Recruitment of mitoribosome biogenesis factors to these quality control intermediates suggests additional roles for these factors during mitoribosome rescue. We also report related cryo-electron microscopy structures (3.7 to 4.4 angstrom resolution) of elongating mitoribosomes bound to tRNAs, nascent polypeptides, the guanosine triphosphatase elongation factors mtEF-Tu and mtEF-G1, and the Oxa1L translocase.

Crystalizing Structured RNAs Using a Surface‐Entropy‐Reduced Fab as a Crystallization Chaperone

Our lab studies the molecular mechanisms of regulatory RNAs elements by determining their atomic resolution structure using x‐ray crystallography. However, RNA crystallization presents many challenges. One strategy to meet these challenges is the use of a protein chaperone to assist RNA crystallization. Antibody fragments (Fabs) provide one type of crystallization chaperone, and we have used these successfully for RNA crystallization in two ways. In one approach, a Fab‐binding‐RNA epitope is grafted into the RNA of interest to create a binding site for the cognate Fab crystallization chaperone. To improve this method, I performed surface entropy reduction on the Fab framework to make a version of our crystallization chaperone that is more suited to well‐ordered crystal packing. Using structural analysis software as a guide, I made twelve surface‐entropy‐reduced (SER) mutant versions of the Fab framework, in which patches of bulky, flexible residues are mutated to alanine. To test the performance of these mutants, I carried out crystallization trials of their complexes with the yjdF RNA, a novel bacterial riboswitch that binds to aza‐aromatic compounds. The wild type Fab (wt BL3‐6) and many of the mutants were able to crystalize with the riboswitch and gave diffraction datasets with 3.5 – 3 angstrom resolution range, and the structure is currently being solved. Using the mutants, I have also crystalized a second RNA target, PEMV2, which is a viral cap‐independent‐translation‐element. This RNA crystalized with wild type but could only diffract to 2.75 angstrom resolution with the aid of a mutant. I am in the process of solving this structure which will give valuable hints to the mechanism of action of this class of viral cap‐independent‐translation‐elements. As I refine this crystallization chaperone technology, I will also apply it to a notoriously difficult crystallization target, domain V of the human polio virus IRES.Support or Funding InformationNIH, HHMI, The University of Chicago

Structural Determination of the CCT:PhLP1:Gβ5 Folding Complex

Before the Gβ subunit of the G‐protein heterotrimer can be assembled into signaling complexes, it must first be folded by the cytosolic chaperonin CCT and by the co‐chaperone Phosducin‐like Protein 1 (PhLP1). PhLP1 has been observed to release the Gβ1 isoform from CCT after folding. In contrast, PhLP1 increases the affinity of the Gβ5 isoform for CCT. Here we present a structure of CCT in complex with PhLP1 and Gβ5 at less than 4 angstrom resolution. Using a combination of cryo‐electron microscopy and crosslinking coupled with mass spectrometry, we found this structure differs markedly from the previously published complex of CCT with PhLP1 and Gβ1. In the case of Gβ5, the substrate sits deep in the CCT folding chamber between the CCT rings, whereas Gβ1 sits high in the folding chamber associated with the apical domain of the CCT3 subunit. The position of PhLP1 is also distinct, presumably to accommodate the differences in substrate position. These structural observations provide insight into the different roles PhLP1 plays in folding Gβ1 and Gβ5 and their subsequent dimerization with Gγ subunits or RGS proteins, respectively.Support or Funding InformationR01‐EY012287

Crystallographic Structural Elucidation of E. coli HU and a Four‐Way Junction

Histone‐like protein HU binds with high affinity (Kd = 30 nM in gel and Kd = 10 nM in solution) to a Holliday or Four‐Way junction (HJ), a DNA recombination intermediate. While previous Förster resonance energy transfer (FRET) studies have determined possible structural models for the HU‐junction interaction, X‐ray crystallographic data is needed to confirm these models. In our studies, we are using J20, a version of the well‐characterized J3 Holliday junction, that has been modified to have only ten base pairs per arm. Based on our anisotropy measurements, the shorter arms reduce the relative flexibility of the junction, potentially making this junction a better substrate for our crystallization studies. Our investigation aims to determine the X‐ray crystal structure of the HU‐HJ interaction by elucidating the ideal conditions for generating HU‐J20 crystals. Initial investigations of crystallization conditions have produced crystals that diffract to 20 Å resolution, therefore, our current efforts are focused on generating crystals that will diffract at higher Angstrom resolution. In order to do so, we will screen around our successful conditions and slightly vary the pH, viscosity, and protein concentrations of our crystallography solutions. Conditions for optimizing crystal generation and diffraction will be discussed.

Insight into the binding affinity of thiourea in the calcium binding pocket of proteinase K, through high resolution X-ray crystallography.

Proteinase K is a stable serine protease, crystallized and extensively used in the study of molecular interactions at the atomic level. During the current study, crystal structure of proteinase K with thiourea (TU) was solved at 1.45Å (angstrom) resolution. Proteinase K showed its binding affinity with thiourea after soaking with 200mM (millimolar) concentration of thiourea solution for 6h. The binding affinity of proteinase K was evaluated with three different molecules i.e., thiourea, acetamide, and thiosemicarbazide. Interestingly, only the thiourea went into the calcium-binding region, and showed interactions with those amino acids which have also displayed interactions with calcium previously. Pro175 (proline 175), Ser197 (Serine 197), Val198 (valine 198), and Asp200 (aspartic acid 200) were the key amino acids involved in the binding of thiourea with proteinase K. Thiourea showed strong hydrogen bondings with Pro175 (2.85Å), Ser197 (2.88Å), and Asp200 (2.90Å, and 3.30Å), as the key interactions involved in the binding of thiourea with proteinase K. This study provides an insight into the binding mechanism of thiourea with calcium-binding pocket of proteinase K, and thus can be extrapolated to other calcium-binding proteins.

Atomic-scale mapping of hydrophobic layers on graphene and few-layer MoS2 and WSe2 in water

The structure and the role of the interfacial water in mediating the interactions of extended hydrophobic surfaces are not well understood. Two-dimensional materials provide a variety of large and atomically flat hydrophobic surfaces to facilitate our understanding of hydrophobic interactions. The angstrom resolution capabilities of three-dimensional AFM are exploited to image the interfacial water organization on graphene, few-layer MoS2 and few-layer WSe2. Those interfaces are characterized by the existence of a 2 nm thick region above the solid surface where the liquid density oscillates. The distances between adjacent layers for graphene, few-layer MoS2 and WSe2 are ~0.50 nm. This value is larger than the one predicted and measured for water density oscillations (~0.30 nm). The experiments indicate that on extended hydrophobic surfaces water molecules are expelled from the vicinity of the surface and replaced by several molecular-size hydrophobic layers.

Iron Superoxide Dismutase is Upregulated in Response to Antibiotic Challenge in Multi‐Drug Resistant Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic gram‐negative human pathogen. Underscoring the threat to global health is the observation of isolates of A. baumannii that are resistant to all known antibiotics. Multidrug resistant A. baumannii is a leading cause of nosocomial infections and can lead to increased mortality and length of stay. To address this public health problem, there is a need to develop new therapeutics. Our approach is to identify potential drug targets by characterizing molecular mechanisms that contribute to the antibiotic resistance of A. baumannii. To achieve this, we leverage a collated bank of 247 isolates (72% are multi‐drug resistant) of A. baumannii from patients at Nashville General Hospital. Isolate MMC4 (Meharry Medical College Isolate 4) is resistant to >25 antibiotics and hence represents an ideal system for dissecting the molecular origins of antibiotic resistance. Here we report the identification of superoxide dismutase B (SodB) as an important antibiotic resistance factor along with its biochemical characterization and high‐resolution crystal structure. Isolate MMC4 was subjected to proteomic analysis in the presence and absence of antibiotic challenge using multidimensional protein identification technology. SodB showed the greatest differential protein expression, a greater than 40 fold increase in response to antibiotic challenge. SodB is an enzyme that protects bacteria from oxidative stress by dismutating highly reactive superoxide radicals. While the superoxide dismutase family has been extensively studied, little is known about A. baumannii SodB. We expressed SodB recombinantly from E. coli and purified it to >95% homogeneity. We evaluated the activity of SodB using a tetrazolium salt assay and established it is an iron dependent enzyme using inductively coupled plasma – optical emission spectrometry. We also determined the structure of SodB with iron bound to 1.45 angstroms resolution. The crystal belonged to the C121 monoclinic space group with unit cell sides (angstroms) and angles (degrees) 97.28, 40.74, 76.63 and 90, 123, 90, respectively. In total, these data establish the importance of protection from oxidative stress in antibiotic resistant A. baumannii and provide the first steps for a structure‐based drug design approach to target inhibition of SodB as a therapeutic strategy.Support or Funding InformationThis work was funded by National Science Foundation HRD1547757, National Institutes of Health R25MD010396 and R01HD090061, and Office of Medical Research, Department of Veterans Affairs IK2BX001701.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cryo-EM Imaging of Kv1.2 Channels with Membrane Potential Applied

We use cryo-EM methods to image Kv1.2 potassium channels reconstituted into small (40-50 nm) liposomes. A zero-potential structure at 7.5 angstrom resolution agrees well with the crystal structure of the detergent-solubilized “native” channel complex (S. Long, E. Campbell and R. MacKinnon, 2005). Flux assay results confirm that a negative membrane potential can be established in this system, and we now report results from our imaging these channels in what should be their resting state.

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately tento twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Probing Light Atoms at Subnanometer Resolution: Realization of Scanning Transmission Electron Microscope Holography.

Atomic resolution imaging of light elements in electron-transparent materials has long been a challenge. Biomolecular materials, for example, are rapidly altered by incident electrons. We demonstrate a scanning transmission electron microscopy (STEM) technique, called STEM holography, capable of efficient structural analysis of beam-sensitive nanomaterials. STEM holography measures the absolute phase and amplitude of electrons passed through a specimen via interference with a vacuum reference wave. We use an amplitude-dividing nanofabricated grating to prepare multiple beams focused at the sample. We configure the postspecimen microscope imaging system to overlap the beams, forming an interference pattern. We record and analyze the pattern at each 2D-raster-scan-position, reconstructing the complex object wave. As a demonstration, we image gold nanoparticles on an amorphous carbon substrate at 2.4 Å resolution. STEM holography offers higher contrast of the carbon while maintaining gold atomic lattice resolution compared to high angle annular dark field STEM.

Electroviscous Retardation of the Squeeze Out of Nanoconfined Ionic Liquids

Although many works have confirmed the layering structure of nanoconfined ionic liquids (ILs), fundamental studies of their dynamic properties are less abundant. This work evaluates the resistance of molecularly thin ion layers to flow out of films confined between atomically flat surfaces. We measure the time-dependent drainage of six different ionic liquids from large surface separations down to ∼3 nm with angstrom resolution in dynamic force measurements with a surface forces apparatus and determine the effective viscosity of the films of thickness smaller than ∼10 nm by numerically solving the equation of motion. The latter requires appropriate models for the equilibrium surface forces (electrostatic, solvation, and van der Waals forces) and the Reynolds theory of lubrication to describe the hydrodynamic drag induced by the motion of the solid surface in the viscous liquid. We show that the effective viscosity of the six ILs becomes up to 2 orders of magnitude larger than the viscosity of the unconfin...

Real-space Demonstration of 0.4 Angstrom Resolution at 80 keV via Electron Ptychography with a High Dynamic Range Pixel Array Detector

Real-space Demonstration of 0.4 Angstrom Resolution at 80 keV via Electron Ptychography with a High Dynamic Range Pixel Array Detector

A Method for Generation of Synthetic 2D and 3D Cryo-EM Images

Cryo-electron microscopy (cryo-EM) is widely used in structural biology for resolving 3D models of particles with Angstrom resolution. The most popular techniques for such high-resolution model reconstruction are single-particle cryo-EM and cryo-electron tomography (cryo-ET). The cornerstone of both techniques is the registration of images: 2D images in cryo-EM and 3D images in cryo-ET. There are several registration methods for 2D and 3D cryo-EM images; however, it is hard to evaluate these methods due to the lack of ground truth for real data. Moreover, evaluation of image registration methods on real data is complicated by a high level of noise. In this paper, we propose image synthesis methods for generating realistic 2D single-particle cryo-EM images and 3D cryo-ET subtomogram images. The proposed algorithms model the artifacts typical of the real EM image acquisition pipeline: EM-specific noise, missing wedge effect, 2D projection, and contrast transfer function. We also present some examples of the 2D and 3D synthetic images generated.

Identification and nanomechanical characterization of the fundamental single-strand protofilaments of amyloid α-synuclein fibrils

The formation and spreading of amyloid aggregates from the presynaptic protein α-synuclein in the brain play central roles in the pathogenesis of Parkinson's disease. Here, we use high-resolution atomic force microscopy to investigate the early oligomerization events of α-synuclein with single monomer angstrom resolution. We identify, visualize, and characterize directly the smallest elementary unit in the hierarchical assembly of amyloid fibrils, termed here single-strand protofilaments. We show that protofilaments form from the direct molecular assembly of unfolded monomeric α-synuclein polypeptide chains. To unravel protofilaments' internal structure and elastic properties, we manipulated nanomechanically these species by atomic force spectroscopy. The single-molecule scale identification and characterization of the fundamental unit of amyloid assemblies provide insights into early events underlying their formation and shed light on opportunities for therapeutic intervention at the early stages of aberrant protein self-assembly.

Thermometry with Subnanometer Resolution in the Electron Microscope Using the Principle of Detailed Balancing.

We measure phonon energy gain and loss down to 20 meV in a single nanostructure using an atom-wide monochromatic electron beam. We show that the bulk and surface, energy loss and energy gain processes obey the principle of detailed balancing in nanostructured systems at thermal equilibrium. By plotting the logarithm of the ratio of the loss and gain bulk/surface scattering as a function of the excitation energy, we find a linear behavior, expected from detailed balance arguments. Since that universal linearity scales with the inverse of the nanosystem temperature only, we can measure the temperature of the probed object with precision down to about 1 K without reference to the nanomaterial. We also show that subnanometer spatial resolution (down to ∼2 Å) can be obtained using highly localized acoustic phonon scattering. The surface phonon polariton signal can also be used to measure the temperature near the nanostructure surfaces, but with unavoidable averaging over several nanometers. Comparison between transmission and aloof probe configurations suggests that our method exhibits noninvasive characteristics. Our work demonstrates the validity of the principle of detailed balancing within nanoscale materials at thermal equilibrium, and it describes a transparent method to measure nanoscale temperature, thus representing an advance in the development of a noninvasive method for measurements with angstrom resolution.

Structural and functional analysis of Utp24, an endonuclease for processing 18S ribosomal RNA.

The precursor ribosomal RNA is processed by multiple steps of nucleolytic cleavage to generate mature rRNAs. Utp24 is a PIN domain endonuclease in the early 90S precursor of small ribosomal subunit and is proposed to cleave at sites A1 and A2 of pre-rRNA. Here we determine the crystal structure of Utp24 from Schizosaccharomyces pombe at 2.1 angstrom resolution. Utp24 structurally resembles the ribosome assembly factor Utp23 and both contain a Zn-finger motif. Functional analysis in Saccharomyces cerevisiae shows that depletion of Utp24 disturbs the assembly of 90S and abolishes cleavage at sites A0, A1 and A2. The 90S assembled with inactivated Utp24 is arrested at a post-A0-cleavage state and contains enriched nuclear exosome for degradation of 5' ETS. Despite of high sequence conservation, Utp24 from other organisms is unable to form an active 90S in S. cerevisiae, suggesting that Utp24 needs to be precisely positioned in 90S. Our study provides biochemical and structural insight into the role of Utp24 in 90S assembly and activity.