New timsTOF Pro Mass Spectrometer installed at CEITEC.
New timsTOF Pro Mass Spectrometer (Bruker) has been installed at the Proteomics Core Facility, CEITEC MU. The new instrument brings another separation dimension (according collisional cross sections) in qualitative and quantitative characterization of complex protein samples as it is equipped by trapped ion mobility spectrometry (TIMS) module.
Emerging Topics in Biomolecular Magnetic Resonance – A new on-line lecture series
A new on-line lecture series on Emerging Topics in Biomolecular Magnetic Resonance, organized by Loren Andreas, Stefan Glöggler, Christian Griesinger, Mei Hong, Oscar Millet, Art Palmer, and Markus Zweckstetter, starts on Thursday, June 4, 2020.
Cryo-EM reaches yet another milestone – Comments on two recent cryo-EM atomic resolution studies
Two studies published during last two weeks have reported that single particle cryo-EM data can be now resolved to 1.20Å or 1.25Å, respectively. The groups of Sjors H.W. Scheres (LMB-MRC, Cambridge) and Holger Stark (MPI Göttingen) have for the first time shown that atomic resolution is attainable by single particle cryo-EM.
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Single-particle cryo-EM at atomic resolution
A. Radu Aricescu & Sjors H.W. Scheres et al. show in the bioRxiv paper that using a new electron source, energy filter and camera, a 1.7 Å resolution cryo-EM reconstruction for a prototypical human membrane protein, the β3 GABAA receptor homo-pentamer, can be obtained.
Breaking the next Cryo-EM resolution barrier – Atomic resolution determination of proteins!
In a new bioRxiv paper, Holger Stark et a. report a 1.25 Å resolution structure of apoferritin obtained by cryo-EM with a newly developed electron microscope providing unprecedented structural details. Their apoferritin structure has almost twice the 3D information content of the current world record reconstruction (at 1.54 Å resolution). For the first time in cryo-EM they can visualize individual atoms in a protein, see density for hydrogen atoms and single atom chemical modifications.
Highlights of Coronavirus Structural Studies
Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease
COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, Nir London, Frank von Delft, Martin A. Walsh et.al. performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. The crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments was progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.
Controlling the SARS-CoV-2 Spike Glycoprotein Conformation
The coronavirus (CoV) viral host cell fusion spike (S) protein is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The highly flexible S-protein, with its mobile domains, presents a moving target to the immune system. In the bioRcin paper, to better understand S-protein mobility, a structure-based vector analysis of available β-CoV S-protein structures was implemented. Rory Henderson, Priyamvada Acharya et.al. from Duke Human Vaccine Institute, Durham, USA found that despite overall similarity in domain organization, different β-CoV strains display distinct S-protein configurations. Based on this analysis, we developed two soluble ectodomain constructs in which the highly immunogenic and mobile receptor binding domain (RBD) is locked in either the all-RBDs ‘down’ position or is induced to display a previously unobserved in SARS-CoV-2 2-RBDs ‘up’ configuration. These results demonstrate that the conformation of the S-protein can be controlled via rational design and provide a framework for the development of engineered coronavirus spike proteins for vaccine applications.
Distinct conformational states of SARS-CoV-2 spike protein
The ongoing SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic has created urgent needs for intervention strategies to control the crisis. The spike (S) protein of the virus forms a trimer and catalyzes fusion between viral and target cell membranes - the first key step of viral infection. Bing Chen et. al. report two cryo-EM structures, both derived from a single preparation of the full-length S protein, representing the prefusion (3.1Å resolution) and postfusion (3.3Å resolution) conformations, respectively. The spontaneous structural transition to the postfusion state under mild conditions is independent of target cells. The prefusion trimer forms a tightly packed structure with three receptor-binding domains clamped down by a segment adjacent to the fusion peptide, significantly different from recently published structures of a stabilized S ectodomain trimer. The postfusion conformation is a rigid tower-like trimer, but decorated by N-linked glycans along its long axis with almost even spacing, suggesting possible involvement in a mechanism protecting the virus from host immune responses and harsh external conditions. These findings advance understanding of how SARS-CoV-2 enters a host cell and may guide development of vaccines and therapeutics.
Reader's Corner Archive
Mapping Structural Dynamics of Proteins with Femtosecond Stimulated Raman Spectroscopy
The structure–function relationships of biomolecules have captured the interest and imagination of the scientific community and general public since the field of structural biology emerged to enable the molecular understanding of life processes. Proteins that play numerous functional roles in cellular processes have remained in the forefront of research, inspiring new characterization techniques. In this review in Annual Review of Physical Chemistry, Chong Fang and Longteng Tang present key theoretical concepts and recent experimental strategies using femtosecond stimulated Raman spectroscopy (FSRS) to map the structural dynamics of proteins, highlighting the flexible chromophores on ultrafast timescales. In particular, wavelength-tunable FSRS exploits dynamic resonance conditions to track transient-species-dependent vibrational motions, enabling rational design to alter functions. Various ways of capturing excited-state chromophore structural snapshots in the time and/or frequency domains are discussed. Continuous development of experimental methodologies, synergistic correlation with theoretical modeling, and the expansion to other nonequilibrium, photo-switchable, and controllable protein systems will greatly advance the chemical, physical, and biological sciences.
The regulation and functions of DNA and RNA G-quadruplexes
DNA and RNA can adopt various secondary structures. Four-stranded G-quadruplex (G4) structures form through self-recognition of guanines into stacked tetrads, and considerable biophysical and structural evidence exists for G4 formation in vitro. Computational studies and sequencing methods have revealed the prevalence of G4 sequence motifs at gene regulatory regions in various genomes, including in humans. Experiments using chemical, molecular and cell biology methods have demonstrated that G4s exist in chromatin DNA and in RNA, and have linked G4 formation with key biological processes ranging from transcription and translation to genome instability and cancer. In the paper published in Nature Reviews Molecular Cell Biology, Balasubramanian, S. et al. first discuss the identification of G4s and evidence for their formation in cells using chemical biology, imaging and genomic technologies. They then discuss possible functions of DNA G4s and their interacting proteins, particularly in transcription, telomere biology and genome instability. Roles of RNA G4s in RNA biology, especially in translation, are also discussed. Furthermore, they consider the emerging relationships of G4s with chromatin and with RNA modifications. Finally, they discuss the connection between G4 formation and synthetic lethality in cancer cells, and recent progress towards considering G4s as therapeutic targets in human diseases.
The architecture of the Gram-positive bacterial cell wall
The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics. Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor. In Gram-positive bacteria, peptidoglycan is tens of nanometers thick, generally portrayed as a homogeneous structure that provides mechanical strength. S. J. Foster & J. K. Hobbs et.al. applied atomic force microscopy to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The paper published in Nature shows that the mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface, providing information complementary to traditional structural biology approaches.
Emerging solution NMR methods to illuminate the structural and dynamic properties of proteins
The first recognition of protein breathing was more than 50 years ago. Today, we are able to detect the multitude of interaction modes, structural polymorphisms, and binding-induced changes in protein structure that direct function. Solution-state NMR spectroscopy has proved to be a powerful technique, not only to obtain high-resolution structures of proteins, but also to provide unique insights into the functional dynamics of proteins. Hari Arthanari, Gerhard Wager et. al. in the Current Opinion in Structural Biology review summarize recent technical landmarks in solution NMR that have enabled characterization of key biological macromolecular systems. These methods have been fundamental to atomic resolution structure determination and quantitative analysis of dynamics over a wide range of time scales by NMR. The ability of NMR to detect lowly populated protein conformations and transiently formed complexes plays a critical role in its ability to elucidate functionally important structural features of proteins and their dynamics.
Fragment-based drug discovery using cryo-EM
Recent advances in electron cryo-microscopy (cryo-EM) structure determination have pushed the resolutions obtainable by the method into the range widely considered to be of utility for drug discovery. Harren Jhoti et. al. in Drug Discovery Today review the use of cryo-EM in fragment-based drug discovery (FBDD) based on in-house method development. They demonstrate not only that cryo-EM can reveal details of the molecular interactions between fragments and a protein, but also that the current reproducibility, quality, and throughput are compatible with FBDD. In addition, they exemplify this using the test system β-galactosidase (Bgal) and the oncology target pyruvate kinase 2 (PKM2).
Integrating cryo-EM and NMR data
Single-particle cryo-electron microscopy (cryo-EM) is increasingly used as a technique to determine the atomic structure of challenging biological systems. Recent advances in microscope engineering, electron detection, and image processing have allowed the structural determination of bigger and more flexible targets than possible with the complementary techniques X-ray crystallography and NMR spectroscopy. However, there exist many biological targets for which atomic resolution cannot be currently achieved with cryo-EM, making unambiguous determination of the protein structure impossible. Although determining the structure of large biological systems using solely NMR is often difficult, highly complementary experimental atomic-level data for each molecule can be derived from the spectra, and used in combination with cryo-EM data. Gunnar F. Schröder et.al. review in Current Opinion in Structural Biology strategies with which both techniques can be synergistically combined, in order to reach detail and understanding unattainable by each technique acting alone; and the types of biological systems for which such an approach would be desirable.
Integrated multidisciplinarity in the natural sciences
The integration of multiple perspectives in both the arts and natural sciences is tremendously powerful and arguably necessary for capturing relevant features of complex phenomena. Individual methods and models comprise abstractions from and idealizations of nature, and only the integration of multiple models, methods, and representations provides a means to reach more accurate results than relying on any single approach. In the Mildred Cohn Award Lecture at the 2019 ASBMB meeting, and the subsequent Journal of Biological Chemistry award article, A. Gronenborn illustrates the power of such multidisciplinary work by highlighting the successful integration of data and multiple views afforded by NMR spectroscopy, cryo-electron micros- copy, cryo-electron tomography, X-ray crystallography, computation, and functional assays made possible through collaborative efforts by members of the Pittsburgh Center for HIV Protein Interactions. This approach permitted her to generate the first all-atom model of a native HIV-1 capsid core.
Cryo-electron microscopy for the study of virus assembly
Although viruses are extremely diverse in shape and size, evolution has led to a limited number of viral classes or lineages, which is probably linked to the assembly constraints of a viable capsid. Viral assembly mechanisms are restricted to two general pathways, (i) co-assembly of capsid proteins and single-stranded nucleic acids and (ii) a sequential mechanism in which scaffolding-mediated capsid precursor assembly is followed by genome packaging. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), which are revolutionizing structural biology, are central to determining the high-resolution structures of many viral assemblies as well as those of assembly intermediates. This wealth of cryo-EM data has also led to the development and redesign of virus-based platforms for biomedical and biotechnological applications. In the Nature Chemical Biology review, D. Luque and J. R. Castón discuss recent viral assembly analyses by cryo-EM and cryo-ET showing how natural assembly mechanisms are used to encapsulate heterologous cargos including chemicals, enzymes, and/or nucleic acids for a variety of nanotechnological applications.