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.
PostEra Call: Help us Fight Coronavirus - Contribute your expertise to design inhibitors of the SARS-CoV-2 main protease
An international group of scientists from academia and industry is seeking opportunities to contribute to combatting COVID-19. They welcome contributions of many forms including scientific expertise, experimental capabilities, and indeed donations to make this possible.
Highlights of Coronavirus Structural Studies
Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2
Two nanobodies that bind SARS-CoV-2 spike RBD are shown to block interaction with receptor ACE2 and thus neutralize the virus, and have an additive effect with antibody CR3022. The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, Raymond Owens, James Naismith et al. have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (K(D)of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.
Stabilizing the Closed SARS-CoV-2 Spike Trimer
The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, P.M. Langedijk et al. report the structure-based design of soluble S trimers, with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. They identify two regions in the S-protein critical for the protein’s stability: the heptad repeat region 1 of the S2 subunit and subunit domain 1 at the interface with S2. We combined a minimal selection of mostly inter-protomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct while no longer containing a heterologous trimerization domain. The cryo-EM structure reveals a correctly folded, predominantly closed pre-fusion conformation. Highly stable and well producing S protein and the increased understanding of S protein structure will support vaccine development and serological diagnostics.
Molecular architecture of the SARS-CoV-2 virus
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins and the complexes of the spike (S) proteins with the cellular receptor ACE2 or neutralizing antibodies, detailed architecture of the intact virus remains to be unveiled. Here, Li et al. from Zhejiang University School of Medicine, Hangzhou, China, report the molecular assembly of the authentic SARS-CoV-2 virus using cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in both pre- and post-fusion conformations were determined to average resolutions of 9-11 Å. Compositions of the N-linked glycans from the native spikes were analyzed by mass spectrometry, which revealed highly similar overall processing states of the native glycans to that of the recombinant glycoprotein glycans. The in-situ architecture of the ribonucleoproteins (RNP) and its higher-order assemblies were revealed. These characterizations have revealed the architecture of the SARS-CoV-2 virus to an unprecedented resolution, and shed lights on how the virus packs its ~30 Kb long single-segmented RNA in the ~80 nm diameter lumen. Overall, the results unveiled the molecular architecture and assembly of the SARS-CoV-2 in native context.
Reader's Corner Archive
Structural biology of cell surface receptor-ligand interactions
Plants have evolved unique membrane receptors that interpret native and foreign cues to coordinate plant life and adaptation. This large family of receptor proteins have evolved very diverse ectodomains, acquiring the capacity to sense ligands of very different chemical nature. A mechanistic understanding on how these signaling systems work will help to comprehend and unveil key cell biology questions. The review by Steven Moussou and Julia Santiago in Current Opinion in Plant Biology aims to focus on the latest receptor-ligands interactions and regulatory mechanism that have been structurally characterized, as well as new receptor folds.
Challenges and perspectives for structural biology of lncRNAs-the example of the Xist lncRNA A-repeats
Following the discovery of numerous long non-coding RNA (lncRNA) transcripts in the human genome, their important roles in biology and human disease are emerging. Recent progress in experimental methods has enabled the identification of structural features of lncRNAs. However, determining high-resolution structures is challenging as lncRNAs are expected to be dynamic and adopt multiple conformations, which may be modulated by interaction with protein binding partners. The X-inactive specific transcript (Xist) is necessary for X inactivation during dosage compensation in female placental mammals and one of the best-studied lncRNAs. Recent progress has provided new insights into the domain organization, molecular features, and RNA binding proteins that interact with distinct regions of Xist. The A-repeats located at the 5' end of the transcript are of particular interest as they are essential for mediating silencing of the inactive X chromosome. In this review, recent progress with elucidating structural features of the Xist lncRNA, focusing on the A-repeats is discussed. The experimental and computational approaches employed that have led to distinct structural models, likely reflecting the intrinsic dynamics of this RNA are overviewed. The presence of multiple dynamic conformations may also play an important role in the formation of the associated RNPs, thus influencing the molecular mechanism underlying the biological function of the Xist A-repeats. Alisha Jones and Michael Sattler in Journal of Molecular Cell Biology propose that integrative approaches that combine biochemical experiments and high-resolution structural biology in vitro with chemical probing and functional studies in vivo are required to unravel the molecular mechanisms of lncRNAs.
PEGylated surfaces for the study of DNA–protein interactions by atomic force microscopy
DNA–protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers the ability to visualize DNA–protein interactions at nanometer resolution in near-physiological buffers, but it requires that the DNA be adhered to the surface of a solid substrate. This presents a problem when working in biologically relevant protein concentrations, where proteins may be present in large excess in solution; much of the biophysically relevant information can therefore be occluded by non-specific protein binding to the underlying substrate. Here we explore the use of PLLx-b-PEGy block copolymers to achieve selective adsorption of DNA on a mica surface for AFM studies. Through varying both the number of lysine and ethylene glycol residues in the block copolymers, Bart Hoogenboom, Alice Pyne et al. show selective adsorption of DNA on mica that is functionalized with a PLL10-b-PEG113/PLL1000–2000 mixture as viewed by AFM imaging in a solution containing high concentrations of streptavidin. They demonstrate – through the use of biotinylated DNA and streptavidin – that this selective adsorption extends to DNA–protein complexes and that DNA-bound streptavidin can be unambiguously distinguished in spite of an excess of unbound streptavidin in solution. Finally, they apply this to the nuclear enzyme PARP1, resolving the binding of individual PARP1 molecules to DNA by in-liquid AFM.
More than Proton Detection—New Avenues for NMR Spectroscopy of RNA
This minireview written by Harald Schwalbe, Boris Fürtig and coworkers reports on the development of NMR methods that utilize detection on low-g nuclei (heteronuclei like 13C or 15N with lower gyromagnetic ratio than 1H) to obtain unique structural and dynamic information for large RNA molecules in solution. Experiments involve through-bond correlations of nucleobases and the phosphodiester backbone of RNA for chemical shift assignment and make information on hydrogen bonding uniquely accessible. Previously unobservable NMR resonances of amino groups in RNA nucleobases are now detected in experiments involving conformational exchange-resistant double-quantum 1H coherences, detected by 13C NMR spectroscopy. Furthermore, 13C and 15N chemical shifts provide valuable information on conformations. All the covered aspects point to the advantages of low-g nuclei detection experiments in RNA.
Toward high-resolution in situ structural biology with cryo-electron tomography and subtomogram averaging
Cryo-electron tomography (cryo-ET) provides unprecedented insights into the molecular constituents of biological environments. In combination with an image processing method called subtomogram averaging (STA), detailed 3D structures of biological molecules can be obtained in large, irregular macromolecular assemblies or in situ, without the need for purification. The contextual meta-information these methods also provide, such as a protein’s location within its native environment, can then be combined with functional data. This allows the derivation of a detailed view on the physiological or pathological roles of proteins from the molecular to cellular level. Despite their tremendous potential in in situ structural biology, cryo-ET and STA have been restricted by methodological limitations, such as the low obtainable resolution. Exciting progress now allows one to reach unprecedented resolutions in situ, ranging in optimal cases beyond the nanometer barrier. Here, Florian Schur reviews current frontiers and future challenges in routinely determining high-resolution structures in in situ environments using cryo-ET and STA.
Chemical cross-linking with mass spectrometry: a tool for systems structural biology
Biological processes supporting life are orchestrated by a highly dynamic array of protein structures and interactions comprising the interactome. Defining the interactome, visualizing how structures and interactions change and function to support life is essential to improved understanding of fundamental molecular processes, but represents a challenge unmet by any single analytical technique. Chemical cross-linking with mass spectrometry provides identification of proximal amino acid residues within proteins and protein complexes, yielding low resolution structural information. This approach has predominantly been employed to provide structural insight on isolated protein complexes, and has been particularly useful for molecules that are recalcitrant to conventional structural biology studies. In this review, Juan D. Chavez and James E. Bruce discuss recent developments in cross-linking and mass spectrometry technologies that are providing large-scale or systems-level interactome data with successful applications to isolated organelles, cell lysates, virus particles, intact bacterial and mammalian cultured cells and tissue samples.
In‐Cell EPR: Progress towards Structural Studies Inside Cells
Exploring the structure and dynamics of biomolecules in the context of their intracellular environment has become the ultimate challenge for structural biology. As the cellular environment is barely reproducible in vitro, investigation of biomolecules directly inside cells has attracted a growing interest. Among magnetic resonance approaches, site‐directed spin labeling (SDSL) coupled to electron paramagnetic resonance (EPR) spectroscopy provides competitive and advantageous features to capture protein structure and dynamics inside cells. To date, several in‐cell EPR approaches have been successfully applied to both bacterial and eukaryotic cells. In this minireview, the major advances of in‐cell EPR spectroscopy are summarized, as well as the challenges this approach still poses.
A standardized citation metrics author database annotated for scientific field
Citation metrics are widely used and misused. John P. A. Ioannidis et. al. created a publicly available data-base of 100,000 top scientists that provides standardized information on citations, h-index, co-authorship-adjusted hm-index, citations to papers in different authorship positions, and a composite indicator. Separate data are shown for career-long and single-year impact. Metrics with and without self-citations and ratio of citations to citing papers are given. Scientists are classified into 22 scientific fields and 176 subfields. Field- and subfield-specific percentiles are also provided for all scientists who have published at least five papers. Career-long data are updated to end of 2017 and to end of 2018 for comparison.