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... To efficiently process a large number of images needed for high-resolution 3-D reconstructions, we suggest a two-step CTF fitting process that starts with automated fitting [41][42][43][44][45][46] and then completes with interactive graphic screening/verification. The reason for the need of final manual screening in practice is due to the inevitable occasional failures of automated fitting methods [42] and lack of reliable detection of these failures by the automated methods. ...
... To efficiently process a large number of images needed for high-resolution 3-D reconstructions, we suggest a two-step CTF fitting process that starts with automated fitting [41][42][43][44][45][46] and then completes with interactive graphic screening/verification. The reason for the need of final manual screening in practice is due to the inevitable occasional failures of automated fitting methods [42] and lack of reliable detection of these failures by the automated methods. In our recent automated fitting method, we have integrated four different automated fitting algorithms which can cross-validate the fitting results and only prompt users with the inconsistent results among the methods for further manual screening [42]. ...
... The reason for the need of final manual screening in practice is due to the inevitable occasional failures of automated fitting methods [42] and lack of reliable detection of these failures by the automated methods. In our recent automated fitting method, we have integrated four different automated fitting algorithms which can cross-validate the fitting results and only prompt users with the inconsistent results among the methods for further manual screening [42]. Once CTF fitting is completed, the determined CTF parameters should be saved to the image headers that are needed for subsequent CTF correction and further refinement. ...
With fast progresses in instrumentation, image processing algorithms, and computational resources, single particle electron cryo-microscopy (cryo-EM) 3-D reconstruction of icosahedral viruses has now reached near-atomic resolutions (3-4 Å). With comparable resolutions and more predictable outcomes, cryo-EM is now considered a preferred method over X-ray crystallography for determination of atomic structure of icosahedral viruses. At near-atomic resolutions, all-atom models or backbone models can be reliably built that allow residue level understanding of viral assembly and conformational changes among different stages of viral life cycle. With the developments of asymmetric reconstruction, it is now possible to visualize the complete structure of a complex virus with not only its icosahedral shell but also its multiple non-icosahedral structural features. In this chapter, we will describe single particle cryo-EM experimental and computational procedures for both near-atomic resolution reconstruction of icosahedral viruses and asymmetric reconstruction of viruses with both icosahedral and non-icosahedral structure components. Procedures for rigorous validation of the reconstructions and resolution evaluations using truly independent de novo initial models and refinements are also introduced.
... There are several stand-alone programs for measuring the effects of the CTF (Mindell and Grigorieff, 2003;Mallick et al., 2005;Jiang et al., 2012;Yang et al., 2009;Sidorov, 2002) and a system for CTF estimation is included in every single particle processing package (Lander et al., 2009;Ludtke et al., 1999;Heymann and Belnap, 2007;Frank et al., 1996;Tang et al., 2007;Hohn et al., 2007;Van Heel et al., 2012;Sorzano et al., 2004). Tomographic approaches have recently been integrating CTF estimation and correction for their data (Voortman et al., 2011;Philippsen et al., 2007;Mariani et al., 2011;Xiong et al., 2009;Zanetti et al., 2009;Fernández et al., 2006;Winkler and Taylor, 2003). ...
... When using the squared spectral density, the CTF is approximately a sine function. The spherical aberration term goes as the fourth power of s and can be effectively ignored as higher order perturbations (Jiang et al., 2012). A power spectrum from 1/100 Å À1 (0.0001 Å À2 in s 2 terms) to 1/5 Å À1 (0.04 Å À2 in s 2 terms) is divided up into ten disjointed divisions each covering an s 2 space of approximately 0.004 Å À2 . ...
... Raw movie frames were aligned using a 9 × 7 patch in MotionCor2, and contrast transfer function (CTF) parameters were estimated using Gctf and CTF in JSPR (25). Only the micrographs with consistent CTF values, including defocus and astigmatism parameters, were retained for subsequent image processing in a hierarchical way (26). ...
Phage-antibiotic synergy (PAS) represents a superior treatment strategy for pathogen infections with less probability of resistance development. Here, we aim to understand the molecular mechanism by which PAS suppresses resistance in terms of population evolution. A novel hypervirulent Klebsiella pneumoniae (KP) phage H5 was genetically and structurally characterized. The combination of H5 and ceftazidime (CAZ) showed a robust synergistic effect in suppressing resistance emergence. Single-cell Raman analysis showed that the phage-CAZ combination suppressed bacterial metabolic activities, contrasting with the upregulation observed with phage alone. The altered population evolutionary trajectory was found to be responsible for the contrasting metabolic activities under different selective pressures, resulting in pleiotropic effects. A pre-existing wcaJ point mutation (wcaJG949A) was exclusively selected by H5, conferring a fitness advantage and up-regulated activity of carbohydrate metabolism, but also causing a trade-off between phage resistance and collateral sensitivity to CAZ. The wcaJ point mutation was counter-selected by H5-CAZ, inducing various mutations in galU that imposed evolutionary disadvantages with higher fitness costs, and suppressed carbohydrate metabolic activity. H5 and H5-CAZ treatments resulted in opposite effects on the transcriptional activity of the phosphotransferase system and the ascorbate and aldarate metabolism pathway, suggesting potential targets for phage resistance suppression. Our study reveals a novel mechanism of resistance suppression by PAS, highlighting how the complexity of bacterial adaptation to selective pressures drives treatment outcomes.
IMPORTANCE
Phage-antibiotic synergy (PAS) has been recently proposed as a superior strategy for the treatment of multidrug-resistant pathogens to effectively reduce bacterial load and slow down both phage and antibiotic resistance. However, the underlying mechanisms of resistance suppression by PAS have been poorly and rarely been studied. In this study, we tried to understand how PAS suppresses the emergence of resistance using a hypervirulent Klebsiella pneumoniae (KP) strain and a novel phage H5 in combination with ceftazidime (CAZ) as a model. Our study reveals a novel mechanism by which PAS drives altered evolutionary trajectory of bacterial populations, leading to suppressed emergence of resistance. The findings advance our understanding of how PAS suppresses the emergence of resistance, and are imperative for optimizing the efficacy of phage-antibiotic therapy to further improve clinical outcomes.
... The initial set of particles was extracted from the original clean micrograph, and the resultant dataset was used for final cleaning and reconstruction. Raw movie frames were aligned using Motion-Cor2 (55) with a 9 × 7 patch, and the contrast transfer function (CTF) parameters were estimated using Gctf and ctf in JSPR (56). Only micrographs with consistent CTF values, including defocus and astigmatism, were kept for further processing. ...
Kisspeptin receptor (KISS1R), belonging to the class A peptide-GPCR family, plays a key role in the regulation of reproductive physiology after stimulation by kisspeptin and is regarded as an attractive drug target for reproductive diseases. Here, we demonstrated that KISS1R can couple to the G i/o pathway besides the well-known G q/11 pathway. We further resolved the cryo–electron microscopy (cryo-EM) structure of KISS1R-G q and KISS1R-G i complexes bound to the synthetic agonist TAK448 and structure of KISS1R-G q complex bound to the endogenous agonist KP54. The high-resolution structures provided clear insights into mechanism of KISS1R recognition by its ligand and can facilitate the design of targeted drugs with high affinity to improve treatment effects. Moreover, the structural and functional analyses indicated that conformational differences in the extracellular loops (ECLs), intracellular loops (ICLs) of the receptor, and the “wavy hook” of the Gα subunit may account for the specificity of G protein coupling for KISS1R signaling.
... The general strategy in the image processing follows the method described previously 42,43 in a hierarchical way. Raw movie frames were aligned with MotionCor2 using a 9 × 7 patch, and the contrast transfer function (CTF) parameters were estimated using Gctf and CTF in JSPR 44 . Only the micrographs with consistent CTF values, including defocus and astigmatism parameter, were kept for the following image processing. ...
DltB, a model member of the Membrane-Bound O-AcylTransferase (MBOAT) superfamily, plays a crucial role in D-alanylation of the lipoteichoic acid (LTA), a significant component of the cell wall of gram-positive bacteria. This process stabilizes the cell wall structure, influences bacterial virulence, and modulates the host immune response. Despite its significance, the role of DltB is not well understood. Through biochemical analysis and cryo-EM imaging, we discover that Streptococcus thermophilus DltB forms a homo-tetramer on the cell membrane. We further visualize DltB in an apo form, in complex with DltC, and in complex with its inhibitor amsacrine (m-AMSA). Each tetramer features a central hole. The C-tunnel of each protomer faces the intratetramer interface and provides access to the periphery membrane. Each protomer binds a DltC without changing the tetrameric organization. A phosphatidylglycerol (PG) molecule in the substrate-binding site may serve as an LTA carrier. The inhibitor m-AMSA bound to the L-tunnel of each protomer blocks the active site. The tetrameric organization of DltB provides a scaffold for catalyzing D-alanyl transfer and regulating the channel opening and closing. Our findings unveil DltB’s dual function in the D-alanylation pathway, and provide insight for targeting DltB as a anti-virulence antibiotic.
... The particle after initial cleaning was subjected to extraction from the original clean micrograph and the resultant dataset was used for final cleaning and reconstruction. Raw movie frames were aligned with MotionCor2 53 using a 9 × 7 patch and the contrast transfer function (CTF) parameters were estimated using Gctf and ctf in JSPR 54 . Only the micrographs with consistent CTF values including defocus and astigmatism were kept for following image processing. ...
Hydroxycarboxylic acid receptor 2 (HCAR2) belongs to the family of class A G protein-coupled receptors with key roles in regulating lipolysis and free fatty acid formation in humans. It is deeply involved in many pathophysiological processes and serves as an attractive target for the treatment of cardiovascular, neoplastic, autoimmune, neurodegenerative, inflammatory, and metabolic diseases. Here, we report four cryo-EM structures of human HCAR2–Gi1 complexes with or without agonists, including the drugs niacin (2.69 Å) and acipimox (3.23 Å), the highly subtype-specific agonist MK-6892 (3.25 Å), and apo form (3.28 Å). Combined with molecular dynamics simulation and functional analysis, we have revealed the recognition mechanism of HCAR2 for different agonists and summarized the general pharmacophore features of HCAR2 agonists, which are based on three key residues R111 3.36 , S179 45.52 , and Y284 7.43 . Notably, the MK-6892–HCAR2 structure shows an extended binding pocket relative to other agonist-bound HCAR2 complexes. In addition, the key residues that determine the ligand selectivity between the HCAR2 and HCAR3 are also illuminated. Our findings provide structural insights into the ligand recognition, selectivity, activation, and G protein coupling mechanism of HCAR2, which shed light on the design of new HCAR2-targeting drugs for greater efficacy, higher selectivity, and fewer or no side effects.
... (82). For the '9087-a 2A AR-G oA -scFv16 complex, raw movie frames were aligned with MotionCor2 (83) using a 9 × 7 patch and the contrast transfer function (CTF) parameters were estimated using Gctf and ctf in JSPR (84). Micrographs with consistent CTF values including defocus and astigmatism parameter were kept for the following image processing, which kept 3768 micrographs from 4217 raw movies. ...
Because nonopioid analgesics are much sought after, we computationally docked more than 301 million virtual molecules against a validated pain target, the α2A-adrenergic receptor (α2AAR), seeking new α2AAR agonists chemotypes that lack the sedation conferred by known α2AAR drugs, such as dexmedetomidine. We identified 17 ligands with potencies as low as 12 nanomolar, many with partial agonism and preferential Gi and Go signaling. Experimental structures of α2AAR complexed with two of these agonists confirmed the docking predictions and templated further optimization. Several compounds, including the initial docking hit '9087 [mean effective concentration (EC50) of 52 nanomolar] and two analogs, '7075 and PS75 (EC50 4.1 and 4.8 nanomolar), exerted on-target analgesic activity in multiple in vivo pain models without sedation. These newly discovered agonists are interesting as therapeutic leads that lack the liabilities of opioids and the sedation of dexmedetomidine.
... The particle after initial cleaning was subjected to extraction from the original clean micrograph and the resultant dataset was used for final cleaning and reconstruction. Raw movie frames were aligned with MotionCor2 53 using a 9 × 7 patch and the contrast transfer function (CTF) parameters were estimated using Gctf and ctf in JSPR 54 . Only the micrographs with consistent CTF values including defocus and astigmatism were kept for following image processing. ...
GPR88 is an orphan class A G-protein-coupled receptor that is highly expressed in the striatum and regulates diverse brain and behavioral functions. Here we present cryo-EM structures of the human GPR88-Gi1 signaling complex with or without a synthetic agonist (1R, 2R)-2-PCCA. We show that (1R, 2R)-2-PCCA is an allosteric modulator binding to a herein identified pocket formed by the cytoplasmic ends of transmembrane segments 5, 6, and the extreme C terminus of the α5 helix of Gi1. We also identify an electron density in the extracellular orthosteric site that may represent a putative endogenous ligand of GPR88. These structures, together with mutagenesis studies and an inactive state model obtained from metadynamics simulations, reveal a unique activation mechanism for GPR88 with a set of distinctive structure features and a water-mediated polar network. Overall, our results provide a structural framework for understanding the ligand binding, activation and signaling mechanism of GPR88, and will facilitate the innovative drug discovery for neuropsychiatric disorders and for deorphanization of this receptor. GPR88 is an orphan GPCR and regulates diverse brain functions. Here, the authors report two structures of the human GPR88-Gi complex, showing an allosteric ligand directly involved in the interaction interface between the receptor and G-protein, and a density which may represent an endogenous ligand of GPR88.
... The particles after initial cleaning were subjected to particle extraction from the originally cleaned micrograph, and the resultant dataset was used for final cleaning and reconstruction. Specifically, raw movie frames were aligned with MotionCor2 using a 9 × 7 patch, and the contrast transfer function (CTF) parameters were estimated using Gctf and ctf in JSPR 48 . Only the micrographs with consistent CTF values, including defocus and astigmatism parameters, were kept for subsequent image processing. ...
Melatonin receptors (MT1 and MT2 in humans) are family A G protein–coupled receptors that respond to the neurohormone melatonin to regulate circadian rhythm and sleep. Numerous efforts have been made to develop drugs targeting melatonin receptors for the treatment of insomnia, circadian rhythm disorder, and cancer. However, designing subtype-selective melatonergic drugs remains challenging. Here, we report the cryo-EM structures of the MT1–Gi signaling complex with 2-iodomelatonin and ramelteon and the MT2–Gi signaling complex with ramelteon. These structures, together with the reported functional data, reveal that although MT1 and MT2 possess highly similar orthosteric ligand-binding pockets, they also display distinctive features that could be targeted to design subtype-selective drugs. The unique structural motifs in MT1 and MT2 mediate structural rearrangements with a particularly wide opening on the cytoplasmic side. Gi is engaged in the receptor core shared by MT1 and MT2 and presents a conformation deviating from those in other Gi complexes. Together, our results provide new clues for designing melatonergic drugs and further insights into understanding the G protein coupling mechanism.
... Graph theory [8] is a branch of applied mathematics, which originated from the Konigsberg problem. It takes graph as the research object. ...
... Images were recorded using an FEI Falcon I direct electron detection camera with the Leginon program (35) at a calibrated magnification of ×59,000, yielding a pixel size of 1.09 Å. Defocus values for the contrast transfer function were calculated using CTFFIND3 (36) and were in the range of 1.5 to 3.5 mm in the final data set. A total of 8800 micrographs were collected, 6800 of which were used for particle selection after excluding those micrographs that had either poor power spectra or thick ice, using the method described by Jiang et al. (37). Program EMAN was used to select ribosome-sized single-particle images and then manually examine the quality of these images, yielding a data set of 380,000 single-particle images. ...
... CTF fitting was automatically performed using the fitctf2.py program in jspr (47) and then visually adjusted using the EMAN1.9 ctfit program (48). ...
Importance:
Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. It is recognized that BPL inactivates viral infectivity through modification of viral nucleic acids. However, its effect on viral proteins remains largely unknown. Here, we present high-resolution cryo-EM structures of BPL-treated coxsackievirus A16 (CVA16) mature virion and procapsid, which reveals an expanded overall conformation and characteristic features that are typical for the 135S-like uncoating intermediate. We further show that BPL concentration affects the binding of inactivated CVA16 particles to its receptor/antibody. Thus, BPL treatment can alter overall structure and surface properties of viral capsids, which may lead to antigenic and immunogenic variations. Our findings provide important information for future development of BPL-inactivated vaccines.
... CTF parameters of the particles from each micrograph were estimated using fitctf2.py (44). ...
Significance
The recently identified rhinovirus C (RV-C) species of picornaviruses might be associated with more severe respiratory infections than other rhinoviruses in children. The RV-C have been linked to 50–85% of hospital-level childhood asthma exacerbations that can lead to significant adult respiratory problems. However, there are currently no effective antiviral treatments or vaccines available. Using cryoelectron microscopy, we have determined the atomic structures of the full virion and native empty particle (NEP) of a cell-adapted RV-C strain. The structures highlight novel immunogenic sites on the virus surface, a probable binding site for the RV-C receptor, molecular determinants of antiviral resistance, and the rationale of utilizing NEPs as vaccine candidates. These results provide the basis for designing antiviral therapeutics and vaccines.
... The particle images were resampled from 0.650 to 0.975 Å /pixel using the Fourier cropping method. The jspr program fitctf2.py (Jiang et al., 2012) was used to determine the initial CTF parameters from the extracted particles. The estimation and correction of astigmatism aberration was performed later during iterative refinement using the aligner refineAstigmatism in jspr (Guo and Jiang, 2014;Liu et al., 2016). ...
The affinity cryoelectron microscopy (cryo-EM) approach has been explored in recent years to simplify and/or improve the sample preparation for cryo-EM, which can bring previously challenging specimens such as those of low abundance and/or unpurified ones within reach of the cryo-EM technique. Despite the demonstrated successes for solving structures to low to intermediate resolutions, the lack of near-atomic structures using this approach has led to a common perception of affinity cryo-EM as a niche technique incapable of reaching high resolutions. Here, we report a ∼2.6-Å structure solved using the antibody-based affinity grid approach with low-concentration Tulane virus purified from a low-yield cell-culture system that has been challenging to standard cryo-EM grid preparation. Quantitative analyses of the structure indicate data and reconstruction quality comparable with the conventional grid preparation method using samples at high concentration.
... Among many of the parameters (defocus, B-factor, noises, etc.) of the contrast transfer function (CTF), the defocus value is the most critical parameter (Jiang et al. 2012). In addition to determining the CTF parameters, the Thon rings in power spectrum of micrographs can be used for evaluating the quality of micrographs. ...
Virus 3D atomic structures provide insight into our understanding of viral life cycles and the development of antiviral drugs. X-ray crystallography and cryo-EM have been used to determine the atomic structure of viruses. However, limited availability of biological samples, biosafety issues due to virus infection, and sometimes inherent characteristics of viruses, pose difficulties on combining both methods in determining viral structures. These have made solving the high resolution structure of some medically important viruses very challenging. Here, we describe our recently employed protocols for determining the high-resolution structure of the virus-like particle of hepatitis E virus (HEV), a pathogen of viral hepatitis in human. These protocols include utilizing recombinant baculovirus system to generate sufficient amount of virus particles, single-particle cryo-EM to get an intermediate resolution structure as a phasing model, and X-ray crystallography for final atomic structure determination. Our protocols have solved the hepatitis E virus structure to the resolution of 3.5 Å. The combined methodology is generally applicable to other human infectious viruses.
... Similar results were obtained for these two dose-weighting modes, and the mode 1 strategy has been used for all the datasets processed in this work. The contrast transfer function (CTF) parameters were determined with jspr program fitctf2.py (Jiang et al., 2012) using the extracted particles. The whole dataset was then split into two halves, even and odd subsets, and processed independently with jspr as described before Liu et al., 2016). ...
... CTF fitting was automatically performed using the fitctf2.py program in jspr package [46], then visually validated and adjusted using EMAN1.9 ctfit program [47]. ...
Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.
... The contrast transfer function (CTF) parameters were first automatically fitted using the fitctf2.py program (Jiang et al., 2012) and then verified using EMAN ctfit program (Ludtke et al., 1999). These fitted CTF parameters were used only as initial parameters that would be further refined in later highresolution refinements. ...
Single-particle cryoelectron microscopy typically discards close-packed particle images as unusable data. Here, we report an image processing strategy and case study of obtaining near-atomic resolution 3D reconstructions from close-packed particles. Multiple independent de novo initial models were constructed to determine and cross-validate the particle parameters. The particles with consistent views were further refined including not only Euler angles and center positions but also defocus, astigmatism, beam tilt, and overall and anisotropic magnification. We demonstrated this strategy with a 2.9 Å resolution reconstruction of a 1.67 MDa virus-like particle of a circovirus, PCV2, recorded on 86 photographic films. The map resolution was further validated with a phase-randomization test and local resolution assessment, and the atomic model was validated with MolProbity and EMRinger. Close-packed virus particles were thus shown not only to be useful for high-resolution 3D reconstructions but also to allow data collection at significantly improved throughput for near-atomic resolution reconstructions.
... Images were recorded using an FEI Falcon I direct electron detection camera with the Leginon program (35) at a calibrated magnification of ×59,000, yielding a pixel size of 1.09 Å. Defocus values for the contrast transfer function were calculated using CTFFIND3 (36) and were in the range of 1.5 to 3.5 mm in the final data set. A total of 8800 micrographs were collected, 6800 of which were used for particle selection after excluding those micrographs that had either poor power spectra or thick ice, using the method described by Jiang et al. (37). Program EMAN was used to select ribosome-sized single-particle images and then manually examine the quality of these images, yielding a data set of 380,000 single-particle images. ...
During protein synthesis, elongation of the polypeptide chain by each amino acid is followed by a translocation step in which mRNA and transfer RNA (tRNA) are advanced by one codon. This crucial step is catalyzed by elongation factor G (EF-G), a guanosine triphosphatase (GTPase), and accompanied by a rotation between the two ribosomal subunits. A mutant of EF-G, H91A, renders the factor impaired in guanosine triphosphate (GTP) hydrolysis and thereby stabilizes it on the ribosome. We use cryogenic electron microscopy (cryo-EM) at near-atomic resolution to investigate two complexes formed by EF-G H91A in its GTP state with the ribosome, distinguished by the presence or absence of the intersubunit rotation. Comparison of these two structures argues in favor of a direct role of the conserved histidine in the switch II loop of EF-G in GTPase activation, and explains why GTP hydrolysis cannot proceed with EF-G bound to the unrotated form of the ribosome.
... The TEM instrument contrast transfer function parameters were determined automatically using fitctf2.py (60,61) and were then visually validated using EMAN ctfit program. Having lost interaction with scaffolding proteins, capsid proteins undergo conformational changes that result in symmetric hexamers that are further stabilized by the newly formed intracapsomere, noncovalent, topological linking. ...
Significance
T7 phage has been used as a model system to study dsDNA virus capsid assembly and maturation. Yet, atomic capsid models and details of capsid transformations are not elucidated. From our cryo-EM study we have derived near-atomic resolution reconstructions of the DNA-free procapsid, a DNA packaging intermediate, and the DNA-packaged, mature phage capsid. From these structures, we have derived the first near-atomic-level model of T7 capsid maturation. The structural knowledge obtained from this study can serve as a platform for analysis of other dsDNA viruses as well as a platform for the development of molecular tools such as improved phage display systems.
... Briefly, after estimating the contrast transfer function (CTF) parameters using the fitctf2.py program (Jiang et al., 2012), individual datasets were split into two halves, whereby de novo initial 3D models were built separately using the "random model" method by assigning random orientations to subsets of randomly picked particles from individual half datasets and iteratively refining until convergence. The 2D alignment for each half dataset was performed independently with the "projection matching" method that compares the CTF-corrected 2D images with the projections of the models, and 3D models were built using the "direct Fourier inversion" approach. ...
Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged E. coli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.
... Particles were manually selected from micrograph images with the graphic program boxer in the EMAN software package (22). The contrast transfer function parameters were automatically fitted and verified with the EMAN ctfit program (23,45). The whole data set (21,291 particles) was divided into two half subsets (even (D) BHK and EGFR ϩ BHK cells were preincubated with 1 g IgG control or increasing amounts of EGFR blocking antibody for 30 min, and then viruses were equally loaded onto cells for plaque assay. ...
Rational design and directed evolution are powerful tools to generate and improve protein function; however, their uses are mostly limited in enzyme and antibody engineering. Here we describe a directed evolution strategy, named tandem selection and enrichment system (TSES), and its use in generating virus with exclusive specificity for particular cellular receptor. In TSES, evolving viruses are sequentially and iteratively transferred between two different host cells, one for selecting receptor specificity and the other for enriching the fittest virus. By combining rational design and TSES, we generate a human Epidermal Growth Factor Receptor (EGFR) Specific Virus 1 (ESV1). ESV1 has the backbone of Sindbis virus (SINV) and displays an EGF domain engrafted onto structural protein E2 after residue Pro192, together with eight amino acid changes stabilizing the E2-EGF chimera. ESV1 utilizes EGFR to initiate infection and has lost the capacity to interact with all known SINV receptors. A 12.2 Å cryoEM reconstruction of ESV1 reveals that the E2-EGF fusion adopts a fixed conformation with EGF sitting at the top of E2 spike; The EGFR binding interface faces outward and the EGF domain completely masks SINV receptor binding. The cryoEM structure of ESV1 explains the desirable properties of ESV1 and provides insights for its further modification. TSES expands the scope of directed evolution and can be easily extended to other targeting molecules and viral systems.
Cryo-electron microscopy is a powerful methodology in structural biology and has been broadly used in high-resolution structure determination for challenging samples, which are not readily available for traditional techniques. In particular, the strength of super macro-complexes and the lack of a need for crystals for cryo-EM make this technique feasible for the structural study of complexes involved in antiviral innate immunity. This chapter presents detailed information and experimental procedures of Cryo-EM for determining the structures of the complexes using STING as an example. The procedures included a sample quality check, high-resolution data acquisition, and image processing for Cryo-EM 3D structure determination.
The multidrug resistance‐associated protein (MRP) ABCC4 facilitates substrate transport across the cytoplasmic membrane, crucial for normal physiology and mediating multidrug resistance in tumor cells. Despite intensive studies on MRPs, ABCC4's transport mechanism remains incompletely understood. In this study, we unveiled an inward‐open conformation with an ATP bound to degenerate NBD1. Additionally, we captured the structure with both ATP and substrate co‐bound in the inward‐open state. Our findings uncover the asymmetric ATP binding in ABCC4 and provide insights into substrate binding and transport mechanisms. ATP binding to NBD1 is parallel to substrate binding to ABCC4, and is a prerequisite for ATP‐bound NBD2‐induced global conformational changes. Our findings shed new light on targeting ABCC4 in combination with anticancer therapy.
Hydroxycarboxylic acid receptor 2 (HCAR2) belongs to the family of class A G-protein-coupled receptors with key roles in regulating lipolysis and free fatty acid formation in humans. It is deeply involved in many pathophysiological processes and serves as an attractive target for the treatment of neoplastic, autoimmune, neurodegenerative, inflammatory, and metabolic diseases. Here, we report four cryo-EM structures of human HCAR2-Gi1 complexes with or without agonists, including the drugs niacin and acipimox, and the highly subtype-specific agonist MK-6892. Combined with molecular docking and functional analysis, we have revealed the recognition mechanism of HCAR2 for different agonists and summarized the general pharmacophore features of HCAR2 agonists, which are based on three key residues R111 3.36 , S179 45.52 , and Y284 7.43 . Notably, the MK-6892-HCAR2 structure shows an extended binding pocket relative to other agonist-bound HCAR2 complexes. In addition, the key residues that determine the ligand selectivity between the HCAR2 and HCAR3 are also illuminated. Our findings provide structural insights into the ligand recognition, selectivity, activation, and G protein coupling mechanism of HCAR2, which sheds light on the design of new HCAR2-targeting drugs for greater efficacy, higher selectivity, and fewer or no side effects.
In this paper the bag-of-words model is applied to image classification and improves the existing problems of the traditional bag-of-words method. We propose a method of combination of corner detection and graph theory for ROI region extraction and fuzzy membership degree. First using corner detection for images, then the ROI region is defined by the method of graph theory. Then the SIFT features of the ROI region are extracted and the visual dictionary is generated. The visual dictionary can be more accurate to describe the image features, which can reduce the influence of background information and other interference information. Secondly, the concept of fuzzy membership function and information of feature space is introduced to improve the image of the visual histogram. Finally, support vector machine classifier is used to classify. Through the experiment of the Caltech 100 database, the result shows that the method improves the accuracy of classification compared with the traditional method.
Marine bacteriophage TW1 belongs to the Siphoviridae family and infects Pseudoalteromonas phenolica. Mass spectrometry analysis has identified 16 different proteins in the TW1 virion. Functions of most of these proteins have been predicted by bioinformatic methods. A 3.6 Å resolution cryoelectron microscopy map of the icosahedrally averaged TW1 head showed the atomic structures of the major capsid protein, gp57∗, and the capsid-stabilizing protein, gp56. The gp57∗ structure is similar to that of the phage HK97 capsid protein. The gp56 protein has two domains, each having folds similar to that of the N-terminal part of phage λ gpD, indicating a common ancestry. The first gp56 domain clamps adjacent capsomers together, whereas the second domain is required for trimerization. A 6-fold-averaged reconstruction of the distal part of the tail showed that TW1 has six tail spikes, which are unusual for siphophages but are similar to the podophages P22 and Sf6, suggesting a common evolutionary origin of these spikes.
Enterovirus 71 (EV71) is the major causative agent of severe hand, foot, and mouth disease, which affects millions of young children in the Asia-Pacific region annually. In this study, we engineered a novel EV71 virus-like particle (VLP) that lacks VP4 (therefore designated VLPΔVP4) and investigated its structure, antigenicity, and vaccine potential. The cryo-electron microscopy (cryo-EM) structure of VLPΔVP4 was reconstructed to 3.71-Å resolution. Results from structural and biochemical analyses revealed that VLPΔVP4 resembles the end product of the viral uncoating process, the 80S empty capsid. VLPΔVP4 is able to elicit high-titer neutralizing antibodies and to fully protect mice against lethal viral challenge. Mechanistic studies showed that, at the cellular level, the anti-VLPΔVP4 sera exert neutralization effects at both preand postattachment stages by inhibiting both virus attachment and internalization, and at the molecular level, the antisera can block multiple interactions between EV71 and its key receptors. Our study gives a better understanding of EV71 capsid assembly and provides important information for the design and development of new-generation vaccines for EV71, and perhaps for other enteroviruses, as well.
div class="title">A Digital Micrograph Script for Detection of Astigmatism in TEM Images
- Volume 22 Issue S3 - Rui Yan, Wen Jiang
Significance
The pathogenic trypanosomatids— Trypanosoma cruzi , Trypanosoma brucei , and Leishmania spp.—are the causative agents of Chagas disease, African trypanosomiasis, and leishmaniasis, respectively. These diseases, with high morbidity and mortality rates, affect millions of people worldwide. Current treatments typically use drugs with high toxicity and marginal efficacy. Here we present, a 2.5-Å structure of the T. cruzi ribosome large subunit by single-particle cryo-EM. Our structure highlights distinctive trypanosome interactions and has allowed us to propose a tentative model for assembly of the 60S large ribosomal subunit. These atomic details highlighting trypanosome-specific interactions and the differences between T. cruzi and the human ribosome can be used directly for structure-based drug design of antitrypanosome drugs.
Minimization of the astigmatism of the objective lens is a critical daily instrument alignment task essential for high resolution TEM imaging. Fast and sensitive detection of astigmatism is needed to provide real-time feedback and adjust the stigmators to efficiently reduce astigmatism. Currently the method used by many microscopists is to visually examine the roundness of a diffractogram (Thon rings) and iteratively adjust the stigmators to make the Thon rings circular. This subjective method is limited by poor sensitivity and potentially biased by the astigmatism of human eyes. In this study, an s² power spectra based method, s²stigmator, was developed to allow fast and sensitive detection of the astigmatism in TEM live images. The “radar”-style display provides real-time feedback to guide the adjustment of the objective lens stigmators. Such unique capability allowed us to discover the mapping of the two stigmators to the astigmatism amplitude and angle, which led us to develop a single-pass tuning strategy capable of significantly quicker minimization of the objective lens astigmatism.
With the advance of new instruments and algorithms, and the accumulation of experience over decades, single-particle cryo-EM has become a pivotal part of structural biology. Recently, we determined the structure of a eukaryotic ribosome at 2.5 Å for the large subunit. The ribosome was derived from Trypanosoma cruzi, the protozoan pathogen of Chagas disease. The high-resolution density map allowed us to discern a large number of unprecedented details including rRNA modifications, water molecules, and ions such as Mg2+ and Zn2+. In this paper, we focus on the procedures for data collection, image processing, and modeling, with particular emphasis on factors that contributed to the attainment of high resolution. The methods described here are readily applicable to other macromolecules for high-resolution reconstruction by single-particle cryo-EM. This article is protected by copyright. All rights reserved.
Importance:
A capsid assembled from viral proteins protects the virus genome during transmission from one cell to another. However, when a virus enters a cell the virus genome has to be released from the capsid in order to initiate infection. This process is not well understood for non-enveloped viruses. We address this gap in our current knowledge by studying the genome release of human Saffold virus-3. Saffold viruses cause diseases ranging from gastrointestinal disorders to meningitis. We show that before the genome is released, the Saffold virus-3 particle expands and holes form in the previously compact capsid. These holes serve as channels for the release of the genome and small capsid proteins VP4 that in related enteroviruses facilitate subsequent transport of the virus genome into the cell cytoplasm.
Single-particle cryo-electron microscopy (cryo-EM), accompanied with 3D reconstruction, is a broadly applicable tool for the structural characterization of macromolecules and nanoparticles. Recently, the cryo-EM field has pushed the limits of this technique to higher resolutions and samples of smaller molecular mass, however, some samples still present hurdles to this technique. Hybrid particles with electron-dense components, which have been studied using single-particle cryo-EM yet with limited success in 3D reconstruction due to the interference caused by electron-dense elements, constitute one group of such challenging samples. To process such hybrid particles, a masking method is developed in this work to adaptively remove pixels arising from electron-dense portions in individual projection images while maintaining maximal biomass signals for subsequent 2D alignment, 3D reconstruction, and iterative refinements. As demonstrated by the success in 3D reconstruction of an octahedron DNA/gold hybrid particle, which has been previously published without a 3D reconstruction, the devised strategy that combines adaptive masking and standard single-particle 3D reconstruction approach has overcome the hurdle of electron-dense elements interference, and is generally applicable to cryo-EM structural characterization of most, if not all, hybrid nanomaterials with electron-dense components.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
High-resolution structures of viruses have made important contributions to modern structural biology. Bacteriophages, the most diverse and abundant organisms on earth, replicate and infect all bacteria and archaea, making them excellent potential alternatives to antibiotics and therapies for multidrug-resistant bacteria. Here, we improved upon our previous electron cryomicroscopy structure of Salmonella bacteriophage epsilon15, achieving a resolution sufficient to determine the tertiary structures of both gp7 and gp10 protein subunits that form the T = 7 icosahedral lattice. This study utilizes recently established best practice for near-atomic to high-resolution (3-5 Å) electron cryomicroscopy data evaluation. The resolution and reliability of the density map were cross-validated by multiple reconstructions from truly independent data sets, whereas the models of the individual protein subunits were validated adopting the best practices from X-ray crystallography. Some sidechain densities are clearly resolved and show the subunit-subunit interactions within and across the capsomeres that are required to stabilize the virus. The presence of the canonical phage and jellyroll viral protein folds, gp7 and gp10, respectively, in the same virus suggests that epsilon15 may have emerged more recently relative to other bacteriophages.
Motor-driven packaging of a dsDNA genome into a preformed protein capsid through a unique portal vertex is essential in the life cycle of a large number of dsDNA viruses. We have used single-particle electron cryomicroscopy to study the multilayer structure of the portal vertex of the bacteriophage T7 procapsid, the recipient of T7 DNA in packaging. A focused asymmetric reconstruction method was developed and applied to selectively resolve neighboring pairs of symmetry-mismatched layers of the portal vertex. However, structural features in all layers of the multilayer portal vertex could not be resolved simultaneously. Our results imply that layers with mismatched symmetries can join together in several different relative orientations, and that orientations at different interfaces assort independently to produce structural isomers, a process that we call combinatorial assembly isomerism. This isomerism explains rotational smearing in previously reported asymmetric reconstructions of the portal vertex of T7 and other bacteriophages. Combinatorial assembly isomerism may represent a new regime of structural biology in which globally varying structures assemble from a common set of components. Our reconstructions collectively validate previously proposed symmetries, compositions, and sequential order of T7 portal vertex layers, resolving in tandem the 5-fold gene product 10 (gp10) shell, 12-fold gp8 portal ring, and an internal core stack consisting of 12-fold gp14 adaptor ring, 8-fold bowl-shaped gp15, and 4-fold gp16 tip. We also found a small tilt of the core stack relative to the icosahedral fivefold axis and propose that this tilt assists DNA spooling without tangling during packaging.
Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit
portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes
viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this
study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat
proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα
backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at
3.8- and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry.
The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat
protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins
and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating
the capsid assembly at the portal vertex and propagating its growth on a T = 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational
changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated
capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation
into infectious virions.
This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www.emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.
This report presents the conclusions of the X-ray Validation Task Force of the worldwide Protein Data Bank (PDB). The PDB has expanded massively since current criteria for validation of deposited structures were adopted, allowing a much more sophisticated understanding of all the components of macromolecular crystals. The size of the PDB creates new opportunities to validate structures by comparison with the existing database, and the now-mandatory deposition of structure factors creates new opportunities to validate the underlying diffraction data. These developments highlighted the need for a new assessment of validation criteria. The Task Force recommends that a small set of validation data be presented in an easily understood format, relative to both the full PDB and the applicable resolution class, with greater detail available to interested users. Most importantly, we recommend that referees and editors judging the quality of structural experiments have access to a concise summary of well-established quality indicators.
1. Introduction 308
2. Electron microscopy 311
2.1 Specimen preparation 311
2.2 The electron microscope 311
2.3 Acceleration voltage, defocus, and the electron gun 312
2.4 Magnification and data collection 313
3. Digitisation and CTF correction 317
3.1 The patchwork densitometer 318
3.2 Particle selection 320
3.3 Position dependent CTF correction 321
3.4 Precision of CTF determination 321
4. Single particles and angular reconstitution 323
4.1 Preliminary filtering and centring of data 323
4.2 Alignments using correlation functions 324
4.3 Choice of first reference images 324
4.4 Multi-reference alignment of data 325
4.5 MSA eigenvector/eigenvalue data compression 328
4.6 MSA classification 330
4.7 Euler angle determination (‘angular reconstitution’) 332
4.8 Sinograms and sinogram correlation functions 332
4.9 Exploiting symmetry 335
4.10 Three-dimensional reconstruction 337
4.11 Euler angles using anchor sets 339
4.12 Iterative refinements 339
5. Computational hardware/software aspects 341
5.1 The ( IMAGIC ) image processing workstation 342
5.2 Operating systems and GUIs 342
5.3 Computational logistics 344
5.4 Shared memory machines 344
5.5 Farming on loosely coupled computers 346
5.6 Implementation using MPI protocol 347
5.7 Software is what it's all about 347
6. Interpretation of results 348
6.1 Assessing resolution: the Fourier Shell Correlation 348
6.2 Influence of filtering 351
6.3 Rendering 351
6.4 Searching for known sub-structures 352
6.5 Interpretation 353
7. Examples 353
7.1 Icosahedral symmetry: TBSV at 5·9 Å resolution 354
7.2 The D6 symmetrical worm hemoglobin at 13 Å resolution 356
7.3 Functional states of the 70S E. coli ribosome 357
7.4 The 50S E. coli ribosomal subunit at 7·5 Å resolution 359
8. Perspectives 361
9. Acknowledgements 364
10. References 364
In the past few years, electron microscopy (EM) has established itself as an important – still upcoming – technique for studying the structures of large biological macromolecules. EM is a very direct method of structure determination that complements the well-established techniques of X-ray crystallography and NMR spectroscopy. Electron micrographs record images of the object and not just their diffraction patterns and thus the classical ‘phase’ problem of X-ray crystallography does not exist in EM. Modern microscopes may reach resolution levels better than ∼ 1·5 Å, which is more than sufficient to elucidate the polypeptide backbone in proteins directly. X-ray structures at such resolution levels are considered ‘excellent’. The fundamental problem in biological EM is not so much the instrumental resolution of the microscopes, but rather the radiation sensitivity of the biological material one wants to investigate. Information about the specimen is collected in the photographic emulsion with the arrival of individual electrons that have (elastically) interacted with the specimen. However, many electrons will damage the specimen by non-elastic interactions. By the time enough electrons have passed through the object to produce a single good signal-to-noise (SNR) image, the biological sample will have been reduced to ashes. In contrast, stable inorganic specimens in material science often show interpretable details down to the highest possible instrumental resolution.
Several factors, including spatial and temporal coherence of the electron microscope, specimen movement, recording medium, and scanner optics, contribute to the decay of the measured Fourier amplitude in electron image intensities. We approximate the combination of these factors as a single Gaussian envelope function, the width of which is described by a single experimental B-factor. We present an improved method for estimating this B-factor from individual micrographs by combining the use of X-ray solution scattering and numerical fitting to the average power spectrum of particle images. A statistical estimation from over 200 micrographs of herpes simplex virus type-1 capsids was used to estimate the spread in the experimental B-factor of the data set. The B-factor is experimentally shown to be dependent on the objective lens defocus setting of the microscope. The average B-factor, the X-ray scattering intensity of the specimen, and the number of particles required to determine the structure at a lower resolution can be used to estimate the minimum fold increase in the number of particles that would be required to extend a single particle reconstruction to a specified higher resolution. We conclude that microscope and imaging improvements to reduce the experimental B-factor will be critical for obtaining an atomic resolution structure.
We have implemented a Web-based simulation program for the contrast transfer function and envelope functions in Java and JavaScript. The simulation provides interactive controls of all the parameters in those functions. In addition to the predefined functions, users can easily define new functions that use any of the parameters in the preset functions. The most useful feature of this new simulation program is the convenient, universal accessibility through Web browsers on any computer platform that supports Java, such as Netscape and Internet Explorer.
A half-century after the determination of the first three-dimensional crystal structure of a protein, more than 40,000 structures ranging from single polypeptides to large assemblies have been reported. The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-A resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (epsilon15) particle, by single-particle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity. However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle's stability. Beyond epsilon15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.
We have implemented a Web-based simulation program for the contrast transfer function and envelope functions in Java and JavaScript. The simulation provides interactive controls of all the parameters in those functions. In addition to the predefined functions, users can easily define new functions that use any of the parameters in the preset functions. The most useful feature of this new simulation program is the convenient, universal accessibility through Web browsers on any computer platform that supports Java, such as Netscape and Internet Explorer.
Molecular networks provide descriptions of the organization of various biological processes, including cellular signaling, metabolism, and genetic regulation. Knowledge on molecular networks is commonly used for systems level analysis of biological function; research and method development in this area has grown tremendously in the past few years. This book will provide a detailed review of existing knowledge on the functional characterization of biological networks. In 15 chapters authored by an international group of prolific systems biology and bioinformatics researchers, it will organize, conceptualize, and summarize the existing core of research results and computational methods on understanding biological function from a network perspective. © Springer Science+Business Media, LLC 2012. All rights reserved.
The effects of defocussing and spherical aberration in the electron microscope image are most simply and directly displayed in the Fourier transform of the image. We have investigated the process of image formation by determining the changes in the transform of the image of thin crystals of catalase, which has discrete diffraction maxima in the resolution range of 100 Å to 20 Å, as a function of defocussing. The experimentally measured changes in amplitude and phase of these diffraction maxima have been measured and compared with the predictions of a first order theory of image formation. The theory is generally confirmed, and the transfer function of the microscope is completely determined by finding the relative contributions from phase and amplitude contrast. A “true” maximum contrast image of the catalase molecule, compensated for the effects of defocussing, is reconstructed from the set of micrographs in the focal series. The relation of this compensated image to individual underfocussed micrographs, and the use of underfocus contrast enhancement in conventional electron microscopy, is discussed.
This approach and the experimental methods can be extended to high resolution in order to compensate for spherical aberration as well as defocussing, thereby offering the potential for extending the resolution of the microscope well beyond the presently attainable 5 Å.
In this paper, we present Google, a prototype of a large-scale search engine which makes heavy use of the structure present in hypertext. Google is designed to crawl and index the Web efficiently and produce much more satisfying search results than existing systems. The prototype with a full text and hyperlink database of at least 24 million pages is available at http://google.stanford.edu/ To engineer a search engine is a challenging task. Search engines index tens to hundreds of millions of web pages involving a comparable number of distinct terms. They answer tens of millions of queries every day. Despite the importance of large-scale search engines on the web, very little academic research has been done on them. Furthermore, due to rapid advance in technology and web proliferation, creating a web search engine today is very different from three years ago. This paper provides an in-depth description of our large-scale web search engine -- the first such detailed public description we know of to date. Apart from the problems of scaling traditional search techniques to data of this magnitude, there are new technical challenges involved with using the additional information present in hypertext to produce better search results. This paper addresses this question of how to build a practical largescale system which can exploit the additional information present in hypertext. Also we look at the problem of how to effectively deal with uncontrolled hypertext collections where anyone can publish anything they want. Keywords World Wide Web, Search Engines, Information Retrieval, PageRank, Google 1.
SciPy is a Python-based ecosystem of open-source software for mathematics, science, and engineering.
See http://www.scipy.org/ .
In this paper, we present Google, a prototype of a large-scale search engine which makes heavy use of the structure present in hypertext. Google is designed to crawl and index the Web efficiently and produce much more satisfying search results than existing systems. The prototype with a full text and hyperlink database of at least 24 million pages is available at http://google.stanford.edu/ To engineer a search engine is a challenging task. Search engines index tens to hundreds of millions of web pages involving a comparable number of distinct terms. They answer tens of millions of queries every day. Despite the importance of large-scale search engines on the web, very little academic research has been done on them. Furthermore, due to rapid advance in technology and web proliferation, creating a web search engine today is very different from 3 years ago. This paper provides an in-depth description of our large-scale web search engine - the first such detailed public description we know of to date. Apart from the problems of scaling traditional search techniques to data of this magnitude, there are new technical challenges involved with using the additional information present in hypertext to produce better search results. This paper addresses this question of how to build a practical large-scale system which can exploit the additional information present in hypertext. Also we look at the problem of how to effectively deal with uncontrolled hypertext collections, where anyone can publish anything they want.
Nine different near-atomic resolution structures of icosahedral viruses, determined by electron cryo-microscopy and published between early 2008 and late 2010, fulfil predictions made 15 years ago that single-particle cryo-EM techniques could visualize molecular detail at 3-4Å resolution. This review summarizes technical developments, both in instrumentation and in computation, that have led to the new structures, which advance our understanding of virus assembly and cell entry.
To achieve cell entry, many nonenveloped viruses must transform from a dormant to a primed state. In contrast to the membrane fusion mechanism of enveloped viruses (e.g., influenza virus), this membrane penetration mechanism is poorly understood. Here, using single-particle cryo-electron microscopy, we report a 3.3 A structure of the primed, infectious subvirion particle of aquareovirus. The density map reveals side-chain densities of all types of amino acids (except glycine), enabling construction of a full-atom model of the viral particle. Our structure and biochemical results show that priming involves autocleavage of the membrane penetration protein and suggest that Lys84 and Glu76 may facilitate this autocleavage in a nucleophilic attack. We observe a myristoyl group, covalently linked to the N terminus of the penetration protein and embedded in a hydrophobic pocket. These results suggest a well-orchestrated process of nonenveloped virus entry involving autocleavage of the penetration protein prior to exposure of its membrane-insertion finger.
The three-dimensional reconstruction of macromolecules from two-dimensional single-particle electron images requires determination and correction of the contrast transfer function (CTF) and envelope function. A computational algorithm based on constrained non-linear optimization is developed to estimate the essential parameters in the CTF and envelope function model simultaneously and automatically. The application of this estimation method is demonstrated with focal series images of amorphous carbon film as well as images of ice-embedded icosahedral virus particles suspended across holes.
The random-conical reconstruction method has been highly successful in three-dimensional imaging of macromolecules under low-dose conditions. This article summarizes the different steps of this technique as applied to molecules prepared with negative staining or vitreous ice, and sketches out the current directions of development. We anticipate that by using new instrumental developments, transfer function correction and computational refinement techniques, a resolution in the range of 7-10 A could ultimately be achieved.
A study of the surface flatness of carbon films on copper grids used for cryo-electron microscopy has been carried out using a Hitachi S-900 low-voltage SEM. Dramatic changes in flatness were observed after cooling from room temperature to -170 degrees C. The changes were similar both for carbon films that had been floated from a mica surface and for those initially deposited on the surface of plastic films. Results demonstrate that films prepared on copper grids that appear flat at room temperature become extensively, but reversibly, puckered at -170 degrees C. The linear thermal expansion coefficient (alpha) for copper is 16.2 x 10(-6)/degrees C and the puckering can be explained by assuming that the coefficient for amorphous carbon is substantially less. Measurements on grids made of titanium, molybdenum and tungsten (coefficients 8.5, 5 and 4.5 x 10(-6)/degrees C, respectively) showed significantly less puckering.
We present EMAN (Electron Micrograph ANalysis), a software package for performing semiautomated single-particle reconstructions from transmission electron micrographs. The goal of this project is to provide software capable of performing single-particle reconstructions beyond 10 A as such high-resolution data become available. A complete single-particle reconstruction algorithm is implemented. Options are available to generate an initial model for particles with no symmetry, a single axis of rotational symmetry, or icosahedral symmetry. Model refinement is an iterative process, which utilizes classification by model-based projection matching. CTF (contrast transfer function) parameters are determined using a new paradigm in which data from multiple micrographs are fit simultaneously. Amplitude and phase CTF correction is then performed automatically as part of the refinement loop. A graphical user interface is provided, so even those with little image processing experience will be able to begin performing reconstructions. Advanced users can directly use the lower level shell commands and even expand the package utilizing EMAN's extensive image-processing library. The package was written from scratch in C++ and is provided free of charge on our Web site. We present an overview of the package as well as several conformance tests with simulated data.
In this work, a powerful parametric spectral estimation technique, 2D-auto regressive moving average modeling (ARMA), has been applied to contrast transfer function (CTF) detection in electron microscopy. Parametric techniques such as auto regressive (AR) and ARMA models allow a more exact determination of the CTF than traditional methods based only on the Fourier transform of the complete image or parts of it and performing some average (periodogram averaging). Previous works revealed that AR models can be used to improve CTF estimation and the detection of its zeros. ARMA models reduce the model order and the computing time, and more interestingly, achieve increased accuracy. ARMA models are generated from electron microscopy (EM) images, and then a stepwise search algorithm is used to fit all the parameters of a theoretical CTF model in the ARMA model previously calculated. Furthermore, this adjustment is truly two-dimensional, allowing astigmatic images to be properly treated. Finally, an individual CTF can be assigned to every point of the micrograph, by means of an interpolation at the functional level, provided that a CTF has been estimated in each one of a set of local areas. The user need only know a few a priori parameters of the experimental conditions of his micrographs, for turning this technique into an automatic and very powerful tool for CTF determination, prior to CTF correction in 3D-EM. The programs developed for the above tasks have been integrated into the X-Windows-based Microscopy Image Processing Package (Xmipp) software package, and are fully accessible at www.biocomp.cnb.uam.es.
Three-dimensional electron cryomicroscopy of randomly oriented single particles is a method that is suitable for the determination of three-dimensional structures of macromolecular complexes at molecular resolution. However, the electron-microscopical projection images are modulated by a contrast transfer function (CTF) that prevents the calculation of three-dimensional reconstructions of biological complexes at high resolution from uncorrected images. We describe here an automated method for the accurate determination and correction of the CTF parameters defocus, twofold astigmatism and amplitude-contrast proportion from single-particle images. At the same time, the method allows the frequency-dependent signal decrease (B factor) and the non-convoluted background signal to be estimated. The method involves the classification of the power spectra of single-particle images into groups with similar CTF parameters; this is done by multivariate statistical analysis (MSA) and hierarchically ascending classification (HAC). Averaging over several power spectra generates class averages with enhanced signal-to-noise ratios. The correct CTF parameters can be deduced from these class averages by applying an iterative correlation procedure with theoretical CTF functions; they are then used to correct the raw images. Furthermore, the method enables the tilt axis of the sample holder to be determined and allows the elimination of individual poor-quality images that show high drift or charging effects.
Accurate knowledge of defocus and tilt parameters is essential for the determination of three-dimensional protein structures at high resolution using electron microscopy. We present two computer programs, CTFFIND3 and CTFTILT, which determine defocus parameters from images of untilted specimens, as well as defocus and tilt parameters from images of tilted specimens, respectively. Both programs use a simple algorithm that fits the amplitude modulations visible in a power spectrum with a calculated contrast transfer function (CTF). The background present in the power spectrum is calculated using a low-pass filter. The background is then subtracted from the original power spectrum, allowing the fitting of only the oscillatory component of the CTF. CTFTILT determines specimen tilt parameters by measuring the defocus at a series of locations on the image while constraining them to a single plane. We tested the algorithm on images of two-dimensional crystals by comparing the results with those obtained using crystallographic methods. The images also contained contrast from carbon support film that added to the visibility of the CTF oscillations. The tests suggest that the fitting procedure is able to determine the image defocus with an error of about 10nm, whereas tilt axis and tilt angle are determined with an error of about 2 degrees and 1 degrees, respectively. Further tests were performed on images of single protein particles embedded in ice that were recorded from untilted or slightly tilted specimens. The visibility of the CTF oscillations from these images was reduced due to the lack of a carbon support film. Nevertheless, the test results suggest that the fitting procedure is able to determine image defocus and tilt angle with errors of about 100 nm and 6 degrees, respectively.
The current theory of image formation in electron microscopy has been semi-quantitatively successful in describing data. The theory involves parameters due to the transfer function of the microscope (defocus, spherical aberration constant, and amplitude constant ratio) as well as parameters used to describe the background and attenuation of the signal. We present empirical evidence that at least one of the features of this model has not been well characterized. Namely the spectrum of the noise background is not accurately described by a Gaussian and associated "B-factor;" this becomes apparent when one studies high-quality far-from focus data. In order to have both our analysis and conclusions free from any innate bias, we have approached the questions by developing an automated fitting algorithm. The most important features of this routine, not currently found in the literature, are (i). a process for determining the cutoff for those frequencies below which observations and the currently adopted model are not in accord, (ii). a method for determining the resolution at which no more signal is expected to exist, and (iii). a parameter-with units of spatial frequency-that characterizes which frequencies mainly contribute to the signal. Whereas no general relation is seen to exist between either of these two quantities and the defocus, a simple empirical relationship approximately relates all three.
We present a completely automated algorithm for estimating the parameters of the contrast transfer function (CTF) of a transmission electron microscope. The primary contribution of this paper is the determination of the astigmatism prior to the estimation of the CTF parameters. The CTF parameter estimation is then reduced to a 1D problem using elliptical averaging. We have also implemented an automated method to calculate lower and upper cutoff frequencies to eliminate regions of the power spectrum which perturb the estimation of the CTF parameters. The algorithm comprises three optimization subproblems, two of which are proven to be convex. Results of the CTF estimation method are presented for images of carbon support films as well as for images of single particles embedded in ice and suspended over holes in the support film. A MATLAB implementation of the algorithm, called ACE, is freely available.
This article formulates the multidimensional nuclear Overhauser effect spectroscopy (NOESY) interpretation problem using graph theory and presents a novel, bottom-up, topology-constrained distance network analysis algorithm for NOESY cross peak interpretation using assigned resonances. AutoStructure is a software suite that implements this topology-constrained distance network analysis algorithm and iteratively generates structures using the three-dimensional (3D) protein structure calculation programs XPLOR/CNS or DYANA. The minimum input for AutoStructure includes the amino acid sequence, a list of resonance assignments, and lists of 2D, 3D, and/or 4D-NOESY cross peaks. AutoStructure can also analyze homodimeric proteins when X-filtered NOESY experiments are available. The quality of input data and final 3D structures is evaluated using recall, precision, and F-measure (RPF) scores, a statistical measure of goodness of fit with the input data. AutoStructure has been tested on three protein NMR data sets for which high-quality structures have previously been solved by an expert, and yields comparable high-quality distance constraint lists and 3D protein structures in hours. We also compare several protein structures determined using AutoStructure with corresponding homologous proteins determined with other independent methods. The program has been used in more than two dozen protein structure determinations, several of which have already been published.
EMAN is a scientific image processing package with a particular focus on single particle reconstruction from transmission electron microscopy (TEM) images. It was first released in 1999, and new versions have been released typically 2-3 times each year since that time. EMAN2 has been under development for the last two years, with a completely refactored image processing library, and a wide range of features to make it much more flexible and extensible than EMAN1. The user-level programs are better documented, more straightforward to use, and written in the Python scripting language, so advanced users can modify the programs' behavior without any recompilation. A completely rewritten 3D transformation class simplifies translation between Euler angle standards and symmetry conventions. The core C++ library has over 500 functions for image processing and associated tasks, and it is modular with introspection capabilities, so programmers can add new algorithms with minimal effort and programs can incorporate new capabilities automatically. Finally, a flexible new parallelism system has been designed to address the shortcomings in the rigid system in EMAN1.
Structural genomics projects are providing large quantities of new 3D structural data for proteins. To monitor the quality of these data, we have developed the protein structure validation software suite (PSVS), for assessment of protein structures generated by NMR or X-ray crystallographic methods. PSVS is broadly applicable for structure quality assessment in structural biology projects. The software integrates under a single interface analyses from several widely-used structure quality evaluation tools, including PROCHECK (Laskowski et al., J Appl Crystallog 1993;26:283-291), MolProbity (Lovell et al., Proteins 2003;50:437-450), Verify3D (Luthy et al., Nature 1992;356:83-85), ProsaII (Sippl, Proteins 1993;17: 355-362), the PDB validation software, and various structure-validation tools developed in our own laboratory. PSVS provides standard constraint analyses, statistics on goodness-of-fit between structures and experimental data, and knowledge-based structure quality scores in standardized format suitable for database integration. The analysis provides both global and site-specific measures of protein structure quality. Global quality measures are reported as Z scores, based on calibration with a set of high-resolution X-ray crystal structures. PSVS is particularly useful in assessing protein structures determined by NMR methods, but is also valuable for assessing X-ray crystal structures or homology models. Using these tools, we assessed protein structures generated by the Northeast Structural Genomics Consortium and other international structural genomics projects, over a 5-year period. Protein structures produced from structural genomics projects exhibit quality score distributions similar to those of structures produced in traditional structural biology projects during the same time period. However, while some NMR structures have structure quality scores similar to those seen in higher-resolution X-ray crystal structures, the majority of NMR structures have lower scores. Potential reasons for this "structure quality score gap" between NMR and X-ray crystal structures are discussed.
Transmission electron microscopy, as most imaging devices, introduces optical aberrations that in the case of thin specimens are usually modeled in Fourier space by the so-called contrast transfer function (CTF). Accurate determination of the CTF is crucial for its posterior correction. Furthermore, the CTF estimation must be fast and robust if high-throughput three-dimensional electron microscopy (3DEM) studies are to be carried out. In this paper we present a robust algorithm that fits a theoretical CTF model to the power spectrum density (PSD) measured on a specific micrograph or micrograph area. Our algorithm is capable of estimating the envelope of the CTF which is absolutely needed for the correction of the CTF amplitude changes.
Wikipedia: Total Least Squares
- Wikipedia
Wikipedia, 2012. Wikipedia: Total Least Squares. http://en.wikipedia.org/wiki/ Total_least_squares.
Phase contrast electron microscopy Single-particle electron cryo-microscopy: towards atomic resolution
- F M Thon
- B Gowen
- R Matadeen
- E V Orlova
- R Finn
Thon, F., 1971. Phase contrast electron microscopy. Academic Press, New York. van Heel, M., Gowen, B., Matadeen, R., Orlova, E.V., Finn, R., et al., 2000. Single-particle electron cryo-microscopy: towards atomic resolution. Q. Rev. Biophys. 33, 307–369.
The Fourier transform of an electron micrograph: effects of defocussing and aberrations, and implications for the use of underfocus contrast enhancement
- Erikson
Erikson HP, Klug A. The Fourier transform of an electron micrograph: effects of defocussing and
aberrations, and implications for the use of underfocus contrast enhancement. Berichte der Bunsen-Gesellschaft. 1970; 74:1129-1137.
Single-particle electron cryomicroscopy: towards atomic resolution
- F Thon
- M Van Heel
- B Gowen
- R Matadeen
- E V Orlova
- R Finn
Thon, F. Phase contrast electron microscopy. Academic Press; New York: 1971.
van Heel M, Gowen B, Matadeen R, Orlova EV, Finn R, et al. Single-particle electron cryomicroscopy: towards atomic resolution. Quarterly reviews of biophysics. 2000; 33:307-369.
[PubMed: 11233408]