bims-strubi Biomed News
on Advances in structural biology
Issue of 2021–12–19
fourteen papers selected by
Alessandro Grinzato, European Synchrotron Radiation Facility
  1. The rapidly evolving role of cryo-EM in drug design.
  2. Cloud computing platforms to support cryo-EM structure determination.
  3. A cryo-electron microscopy support film formed by 2D crystals of hydrophobin HFBI.
  4. Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of Protein Complexes.
  5. A comprehensive approach to X-ray crystallography for drug discovery at a synchrotron facility - The example of Diamond Light Source.
  6. Microcrystal electron diffraction in macromolecular and pharmaceutical structure determination.
  7. Proteins, proteins everywhere.
  8. Cryo-shift: reducing domain shift in cryo-electron subtomograms with unsupervised domain adaptation and randomization.
  9. Progress in the Development of Structure-Based Vaccines.
  10. In a flash of light: X-ray free electron lasers meet native mass spectrometry.
  11. MorphOT: Transport-based interpolation between EM maps with UCSF ChimeraX.
  12. Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP.
  13. A drug discovery toolbox for Nuclear Magnetic Resonance (NMR) characterization of ligands and their targets.
  14. Transmission Electron Microscopic Methods for Plant Virology.
  1. Drug Discov Today Technol. 2020 Dec;pii: S1740-6749(20)30036-6. [Epub ahead of print]38 91-102
    The rapidly evolving role of cryo-EM in drug design.
    Christoph Wigge, Aleksandar Stefanovic, Mazdak Radjainia.
      Since the early 2010s, cryo-electron microscopy (cryo-EM) has evolved to a mainstream structural biology method in what has been dubbed the "resolution revolution". Pharma companies also began to use cryo-EM in drug discovery, evidenced by a growing number of industry publications. Hitherto limited in resolution, throughput and attainable molecular weight, cryo-EM is rapidly overcoming its main limitations for more widespread use through a new wave of technological advances. This review discusses how cryo-EM has already impacted drug discovery, and how the state-of-the-art is poised to further revolutionize its application to previously intractable proteins as well as new use cases.
    Keywords:  Cryo-EM SBDD; Drug discovery; Epitope mapping; Lead optimization; Protein structure
    DOI:  https://doi.org/10.1016/j.ddtec.2020.12.003
  2. Trends Biochem Sci. 2021 Dec 09. pii: S0968-0004(21)00244-9. [Epub ahead of print]
    Cloud computing platforms to support cryo-EM structure determination.
    Yilai Li, Michael A Cianfrocco.
      Leveraging the power of single-particle cryo-electron microscopy (cryo-EM) requires robust and accessible computational infrastructure. Here, we summarize the cloud computing landscape and picture the outlook of a hybrid cryo-EM computing workflow, and make suggestions to the community to facilitate a future for cryo-EM that integrates into cloud computing infrastructure.
    Keywords:  cloud computing; cryo-EM
    DOI:  https://doi.org/10.1016/j.tibs.2021.11.005
  3. Nat Commun. 2021 Dec 14. 12(1): 7257
    A cryo-electron microscopy support film formed by 2D crystals of hydrophobin HFBI.
    Hongcheng Fan, Bo Wang, Yan Zhang, Yun Zhu, Bo Song, Haijin Xu, Yujia Zhai, Mingqiang Qiao, Fei Sun.
      Cryo-electron microscopy (cryo-EM) has become a powerful tool to resolve high-resolution structures of biomacromolecules in solution. However, air-water interface induced preferred orientations, dissociation or denaturation of biomacromolecules during cryo-vitrification remains a limiting factor for many specimens. To solve this bottleneck, we developed a cryo-EM support film using 2D crystals of hydrophobin HFBI. The hydrophilic side of the HFBI film adsorbs protein particles via electrostatic interactions and sequesters them from the air-water interface, allowing the formation of sufficiently thin ice for high-quality data collection. The particle orientation distribution can be regulated by adjusting the buffer pH. Using this support, we determined the cryo-EM structures of catalase (2.29 Å) and influenza haemagglutinin trimer (2.56 Å), which exhibited strong preferred orientations using a conventional cryo-vitrification protocol. We further show that the HFBI film is suitable to obtain high-resolution structures of small proteins, including aldolase (150 kDa, 3.28 Å) and haemoglobin (64 kDa, 3.6 Å). Our work suggests that HFBI films may have broad future applications in increasing the success rate and efficiency of cryo-EM.
    DOI:  https://doi.org/10.1038/s41467-021-27596-8
  4. Methods Mol Biol. 2022 ;2420 217-232
    Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of Protein Complexes.
    Lucía Quintana-Gallardo, Moisés Maestro-López, Jaime Martín-Benito, Miguel Marcilla, Daniel Rutz, Johannes Buchner, José M Valpuesta, Jorge Cuéllar.
      Structural biology has recently witnessed the benefits of the combined use of two complementary techniques: electron microscopy (EM) and cross-linking mass spectrometry (XL-MS). EM (especially its cryogenic variant cryo-EM) has proven to be a very powerful tool for the structural determination of proteins and protein complexes, even at an atomic level. In a complementary way, XL-MS allows the precise characterization of particular interactions when residues are located in close proximity. When working from low-resolution, negative-staining images and less-defined regions of flexible domains (whose mapping is made possible by cryo-EM), XL-MS can provide critical information on specific amino acids, thus identifying interacting regions and helping to deduce the overall protein structure. The protocol described here is particularly well suited for the study of protein complexes whose intrinsically flexible or transient nature prevents their high-resolution characterization by any structural technique itself.
    Keywords:  Atomic structure; Chaperones; Chemical cross-linker; Cross-linking; Docking; Electron microscopy; Flexibility; Image processing; Mass spectrometry; Three-dimensional reconstruction
    DOI:  https://doi.org/10.1007/978-1-0716-1936-0_17
  5. Drug Discov Today Technol. 2020 Dec;pii: S1740-6749(20)30021-4. [Epub ahead of print]37 83-92
    A comprehensive approach to X-ray crystallography for drug discovery at a synchrotron facility - The example of Diamond Light Source.
    Marco Mazzorana, Elizabeth J Shotton, David R Hall.
      A detailed understanding of the interactions between drugs and their targets is crucial to develop the best possible therapeutic agents. Structure-based drug design relies on the availability of high-resolution structures obtained primarily through X-ray crystallography. Collecting and analysing quickly a large quantity of structural data is crucial to accelerate drug discovery pipelines. Researchers from academia and industry can access the highly automated macromolecular crystallography (MX) beamlines of Diamond Light Source, the UK national synchrotron, to rapidly collect diffraction data from large numbers of crystals. With seven beamlines dedicated to MX, Diamond offers bespoke solutions for a wide variety of user requirements. Working in synergy with state-of-the-art laboratories and other life science instruments to provide an integrated offering, the MX beamlines provide innovative and multidisciplinary approaches to the determination of structures of new pharmacological targets as well as the efficient study of protein-ligand complexes.
    DOI:  https://doi.org/10.1016/j.ddtec.2020.10.003
  6. Drug Discov Today Technol. 2020 Dec;pii: S1740-6749(20)30035-4. [Epub ahead of print]37 93-105
    Microcrystal electron diffraction in macromolecular and pharmaceutical structure determination.
    Max T B Clabbers, Hongyi Xu.
      Microcrystal electron diffraction (MicroED) has recently shown to be a promising technique for structure determination in structural biology and pharmaceutical chemistry. Here, we discuss the unique properties of electrons and motivate its use for diffraction experiments. We review the latest developments in MicroED, and illustrate its applications in macromolecular crystallography, fragment screening and structure guided drug discovery. We discuss the perspectives of MicroED in synthetic chemistry and pharmaceutical development. We anticipate that the rapid advances MicroED showcased here will promote further development of electron crystallography and open up new opportunities for drug discovery.
    Keywords:  3DED; Electron crystallography; Electron diffraction; Fragment screening; Ligand binding; MicroED; Pharmaceuticals; Structural biology
    DOI:  https://doi.org/10.1016/j.ddtec.2020.12.002
  7. Science. 2021 Dec 17. 374(6574): 1415
    Proteins, proteins everywhere.
    H Holden Thorp.
      The first protein structures were determined by x-ray crystallography in 1957 by John C. Kendrew and Max F. Perutz. As a bioinorganic chemist, I was delighted that the structures were myoglobin and hemoglobin, both heme proteins with big, beautiful iron atoms. It must have been an extraordinary experience to stare at a physical model of the structures and see something that had previously only been imagined. Not long afterward, Christian B. Anfinsen Jr. proposed that the structure of a protein was thermodynamically stable. It seemed possible that the three-dimensional structure of a protein could be predicted based on the sequence of its amino acids. This "protein-folding problem," as it came to be known, baffled scientists until this year, when the papers we've deemed the 2021 Breakthrough of the Year were published.
    DOI:  https://doi.org/10.1126/science.abn5795
  8. Bioinformatics. 2021 Nov 23. pii: btab794. [Epub ahead of print]
    Cryo-shift: reducing domain shift in cryo-electron subtomograms with unsupervised domain adaptation and randomization.
    Hmrishav Bandyopadhyay, Zihao Deng, Leiting Ding, Sinuo Liu, Mostofa Rafid Uddin, Xiangrui Zeng, Sima Behpour, Min Xu.
       MOTIVATION: Cryo-Electron Tomography (cryo-ET) is a 3D imaging technology that enables the visualization of subcellular structures in situ at near-atomic resolution. Cellular cryo-ET images help in resolving the structures of macromolecules and determining their spatial relationship in a single cell, which has broad significance in cell and structural biology. Subtomogram classification and recognition constitute a primary step in the systematic recovery of these macromolecular structures. Supervised deep learning methods have been proven to be highly accurate and efficient for subtomogram classification, but suffer from limited applicability due to scarcity of annotated data. While generating simulated data for training supervised models is a potential solution, a sizeable difference in the image intensity distribution in generated data as compared with real experimental data will cause the trained models to perform poorly in predicting classes on real subtomograms.
    RESULTS: In this work, we present Cryo-Shift, a fully unsupervised domain adaptation and randomization framework for deep learning-based cross-domain subtomogram classification. We use unsupervised multi-adversarial domain adaption to reduce the domain shift between features of simulated and experimental data. We develop a network-driven domain randomization procedure with 'warp' modules to alter the simulated data and help the classifier generalize better on experimental data. We do not use any labeled experimental data to train our model, whereas some of the existing alternative approaches require labeled experimental samples for cross-domain classification. Nevertheless, Cryo-Shift outperforms the existing alternative approaches in cross-domain subtomogram classification in extensive evaluation studies demonstrated herein using both simulated and experimental data.
    AVAILABILITYAND IMPLEMENTATION: https://github.com/xulabs/aitom.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btab794
  9. Methods Mol Biol. 2022 ;2412 15-33
    Progress in the Development of Structure-Based Vaccines.
    Sunil Thomas, Ann Abraham.
      The immune response elicited by vaccines against microorganisms makes it the most successful medical interventions against infectious diseases. Conventional vaccines have limitations in inducing immunity against many types of pathogenic microorganism. The genetic diversity of microorganisms, coupled with the high degree of sequence variability in antigenic proteins, presents a challenge to developing broadly effective conventional vaccines. Atomic-resolution structure determination is crucial for understanding antigenic protein function. Cryo-electron microscopy, nuclear magnetic resonance spectroscopy coupled with bioinformatics provide three-dimensional structure of the antigenic proteins and provide a wealth of information about the organization of individual atoms and their chemical makeup. The atomic detail information of proteins offers enormous potential to rationally engineer proteins to enhance their properties and act as effective immunogens to induce immunity. The observation that whole protein antigens are not necessarily essential for inducing immunity has led to the emergence "structural vaccinology." Structure-based vaccines are designed on the rationale that protective epitopes should be sufficient to induce immune responses and provide protection against pathogens. In 2013 we published a review on structure-based vaccines (Thomas and Luxon. Expert Rev Vaccines 12 1301-11, 2013). This review states the progress in development of structure-based vaccines since the first review.
    Keywords:  Antigen; Epitope; Structural biology; Structure-based vaccines
    DOI:  https://doi.org/10.1007/978-1-0716-1892-9_2
  10. Drug Discov Today Technol. 2021 Dec;pii: S1740-6749(21)00017-2. [Epub ahead of print]39 89-99
    In a flash of light: X-ray free electron lasers meet native mass spectrometry.
    for MS SPIDOC consortium
      During the last years, X-ray free electron lasers (XFELs) have emerged as X-ray sources of unparalleled brightness, delivering extreme amounts of photons in femtosecond pulses. As such, they have opened up completely new possibilities in drug discovery and structural biology, including studying high resolution biomolecular structures and their functioning in a time resolved manner, and diffractive imaging of single particles without the need for their crystallization. In this perspective, we briefly review the operation of XFELs, their immediate uses for drug discovery and focus on the potentially revolutionary single particle diffractive imaging technique and the challenges which remain to be overcome to fully realize its potential to provide high resolution structures without the need for crystallization, freezing or the need to keep proteins stable at extreme concentrations for long periods of time. As the issues have been to a large extent sample delivery related, we outline a way for native mass spectrometry to overcome these and enable so far impossible research with a potentially huge impact on structural biology and drug discovery, such as studying structures of transient intermediate species in viral life cycles or during functioning of molecular machines.
    DOI:  https://doi.org/10.1016/j.ddtec.2021.07.001
  11. Bioinformatics. 2020 Dec 16. pii: btaa1019. [Epub ahead of print]
    MorphOT: Transport-based interpolation between EM maps with UCSF ChimeraX.
    Arthur Ecoffet, Frédéric Poitevin, Khanh Dao Duc.
       MOTIVATION: Cryogenic Electron-Microscopy offers the unique potential to capture conformational heterogeneity, by solving multiple 3 D classes that co-exist within a single cryo-EM image dataset. To investigate the extent and implications of such heterogeneity, we propose to use an optimal-transport based metric to interpolate barycenters between EM maps and produce morphing trajectories.
    RESULTS: While standard linear interpolation mostly fails to produce realistic transitions, our method yields continuous trajectories that displace densities to morph one map into the other, instead of blending them.
    AVAILABILITY: Our method is implemented as a plug-in for ChimeraX called MorphOT, which allows the use of both CPU or GPU resources. The code is publicly available on GitHub (https://github.com/kdd-ubc/MorphOT.git), with documentation containing tutorial and datasets.
    SUPPLEMENTARY INFORMATION: User manual for MorphOT is available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btaa1019
  12. Nat Commun. 2021 Dec 13. 12(1): 7236
    Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP.
    Christine E Carbone, Anna B Loveland, Howard B Gamper, Ya-Ming Hou, Gabriel Demo, Andrei A Korostelev.
      During translation, a conserved GTPase elongation factor-EF-G in bacteria or eEF2 in eukaryotes-translocates tRNA and mRNA through the ribosome. EF-G has been proposed to act as a flexible motor that propels tRNA and mRNA movement, as a rigid pawl that biases unidirectional translocation resulting from ribosome rearrangements, or by various combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive structures of ribosome•EF-G intermediates at near-atomic resolution. Prior to translocation, EF-G binds near peptidyl-tRNA, while the rotated 30S subunit stabilizes the EF-G GTPase center. Reverse 30S rotation releases Pi and translocates peptidyl-tRNA and EF-G by ~20 Å. An additional 4-Å translocation initiates EF-G dissociation from a transient ribosome state with highly swiveled 30S head. The structures visualize how nearly rigid EF-G rectifies inherent and spontaneous ribosomal dynamics into tRNA-mRNA translocation, whereas GTP hydrolysis and Pi release drive EF-G dissociation.
    DOI:  https://doi.org/10.1038/s41467-021-27415-0
  13. Drug Discov Today Technol. 2020 Dec;pii: S1740-6749(20)30028-7. [Epub ahead of print]37 51-60
    A drug discovery toolbox for Nuclear Magnetic Resonance (NMR) characterization of ligands and their targets.
    Regan M LeBlanc, Michael F Mesleh.
      Information about the structure, dynamics, and ligand-binding properties of biomolecules can be derived from Nuclear Magnetic Resonance (NMR) spectroscopy and provides valuable information for drug discovery. A multitude of experimental approaches provides a wealth of information that can be tailored to the system of interest. Methods to study the behavior of ligands upon target binding enable the identification of weak binders in a robust manner that is critical for the identification of truly novel binding interactions. This is particularly important for challenging targets. Observing the solution behavior of biomolecules yields information about their structure, dynamics, and interactions. This review describes the breadth of approaches that are available, many of which are under-utilized in a drug-discovery environment, and focuses on recent advances that continue to emerge.
    Keywords:  Drug Discovery; Fragment-based discovery; Glycan structure and dynamics; Nuclear Magnetic Resonance (NMR); Nucleic acid structure and dynamics; Protein structure and dynamics
    DOI:  https://doi.org/10.1016/j.ddtec.2020.11.008
  14. Methods Mol Biol. 2022 ;2400 297-317
    Transmission Electron Microscopic Methods for Plant Virology.
    Li Xie, Jian Hong.
      Transmission electron microscopy (TEM) is an important tool for observing the ultrastructure of plant virions and their host cells. The two main applicable TEM technologies used in plant virology are negative staining and ultrathin section. Negative staining is mainly used to observe the high-resolution structure of virus particles under a transmission electron microscope. Sample preparation for negative staining is convenient and fast, making it suitable for studying the virions in crude sap or purified solution. A modification of negative staining, by combining immunological reaction, named as technique of immuno-negative staining, is used to enrich or identify viruses. Ultrathin section is used for ultrastructural cytopathological studies in the virus-infected host cells, including the morphology of virus particles, the structure of viral induced inclusion bodies, the subcellular distribution of virions and the structural alteration of the host cell induced by viral infection. Such information is valuable to analyze the behavior of virus in replication, assembly, and intercellular transportation, and thus to understand the viral infection cycle. The present chapter describes the operation details of negative staining and ultrathin section TEM.
    Keywords:  Cytopathology; Negative staining; Plant viruses; Transmission electron microscopy; Ultrathin section; Virion
    DOI:  https://doi.org/10.1007/978-1-0716-1835-6_28
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