Instruct-ERIC Webinar Series: Structure Meets Function - Webinar #3
Instruct-ERIC webinar series continues on October 13 and will highlight some of the latest developments in structural biology, demonstrating how integrative methods are enabling scientists to decipher the mechanisms that underpin health and disease. Talks will be given by Bert Janssen, Markus Weingarth and Meindert Lamers.
Instruct-ERIC Best Practices in Cryo-EM Workshop
University od Leeds, United Kingdom, is organising an online training on the best practises in Cryo-EM. The workshop will be held on 12-13 October 2020.
Sessions will include presentations on best practice in sample preparation, imaging and data handling/processing, operational models in light of COVID-19, as well as round table discussions focused on specific topics.
A Celebration of Instruct-ERIC: Achievements, Impact and Expanding Ambitions
It is our great pleasure to invite you to a virtual event celebrating the achievements of Instruct-ERIC in the time since achieving European Research Infrastructure Consortium (ERIC) status in 2017. The event will celebrate the achievements of Instruct-ERIC, recognising the hard work and commitment of the consortium, staff, supporters and users, in the wider context of the successful European Research Infrastructure landscape.
Emerging Topics in Biomolecular Magnetic Resonance – Vipin Agarwal (TIFR Hyderabad) & Ruan Ke (University of Science & Technology of China)
The series Emerging topics in Biomolecular Magnetic Resonance will continue on September 10th at 16:00 CEST with the following lecturers and topics:
- Vipin Agarwal: Proton detected experiments for structural characterization of biomolecules and a-synuclin fibril polymorphs from different aggregation intermediates
- Ruan Ke: Fragment to lead evolution guided by paramagnetic NMR approaches
Josef Dadok National NMR Centre installed a nitrogen liquification unit for its 850 MHz NMR spectrometer
The liquification unit called BSNL (Bruker Smart Nitrogen Liquefier) uses an excess cooling capacity of the cryogenic probe system to liquify and recycle the nitrogen evaporating from the magnet. The nitrogen refill interval is thus extended from 2-3 weeks to more than 6 months.
Research scientist for the Centre of Molecular Structure - Biophysical methods facility
Biophysical Methods facility of BIOCEV is looking for a research scientist specialised in biophysical techniques and/or spectroscopic techniques.
Highlights of Coronavirus Structural Studies
Growth, detection, quantification, and inactivation of SARS-CoV-2 (Virology)
Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is the agent responsible for the coronavirus disease 2019 (COVID-19) global pandemic. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2003 SARS outbreak. Although numerous reagents were developed to study SARS-CoV infections, few have been applicable to evaluating SARS-CoV-2 infection and immunity. Current limitations in studying SARS-CoV-2 include few validated assays with fully replication-competent wild-type virus. M.S. Diamond et. al. have developed protocols to propagate, quantify, and work with infectious SARS-CoV-2. Here, we describe: (1) virus stock generation, (2) RT-qPCR quantification of SARS-CoV-2 RNA; (3) detection of SARS-CoV-2 antigen by flow cytometry, (4) quantification of infectious SARS-CoV-2 by focus-forming and plaque assays; and (5) validated protocols for virus inactivation. Collectively, these methods can be adapted to a variety of experimental designs, which should accelerate our understanding of SARS-CoV-2 biology and the development of effective countermeasures against COVID-19.
SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation (Nat. Struct. Mol. Biol.)
The SARS-CoV-2 non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, suppresses host innate immune functions. By combining cryo-electron microscopy and biochemistry, O. Mühlemann, N. Ban et.al. show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes, including the 43S pre-initiation complex and the non-translating 80S ribosome. The protein inserts its C-terminal domain into the mRNA channel, where it interferes with mRNA binding. They observe translation inhibition in the presence of Nsp1 in an in vitro translation system and in human cells. Based on the high-resolution structure of the 40S– Nsp1 complex, they identify residues of Nsp1 crucial for mediating translation inhibition. They further show that the full-length 5′ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines global inhibition of translation by Nsp1 with efficient translation of the viral mRNA to allow expression of viral genes.
Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient (Nat. Struct Mol. Biol.)
The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. Stuart D., Huang, K.Y.A. et al. have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (K(D)of 2 nM), and a 2.6-angstrom-resolution crystal structure of an RBD-EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.
Reader's Corner Archive
Conformational Ensembles of an Intrinsically Disordered Protein Consistent with NMR, SAXS, and Single-Molecule FRET (J.Amer.Chem.Soc.)
Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes their structural characterization challenging. Although challenging, characterization of the conformational ensembles of IDPs is of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture. G-N.W. Gomes and C. Gradinaru et.al. used integrative modeling and validation to apply conformational restraints and assess agreement with the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Förster Resonance Energy Transfer (smFRET). Agreement with such a diverse set of experimental data suggests that details of the generated ensembles can now be examined with a high degree of confidence. Using the disordered N-terminal region of the Sic1 protein as a test case, they examined relationships between average global polymeric descriptions and higher-moments of their distributions. To resolve apparent discrepancies between smFRET and SAXS inferences, they integrated SAXS data with NMR data and reserved the smFRET data for independent validation. Consistency with smFRET, which was not guaranteed a priori, indicates that, globally, the perturbative effects of NMR or smFRET labels on the Sic1 ensemble are minimal. Analysis of the ensembles revealed distinguishing features of Sic1, such as overall compactness and large end-to-end distance fluctuations, which are consistent with biophysical models of Sic1’s ultrasensitive binding to its partner Cdc4. Their results underscore the importance of integrative modeling and validation in generating and drawing conclusions from IDP conformational ensembles.
In-cell architecture of an actively transcribing-translating expressome (Science)
Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. J. Rappsilber et. al. combined whole-cell cross-linking mass spectrometry, cellular cryo–electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at subnanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. They pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome and propose that it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell.
Half a century of amyloids: past, present and future (Chem. Rev.)
Amyloid diseases are global epidemics with profound health, social and economic implications and yet remain without a cure. This dire situation calls for research into the origin and pathological manifestations of amyloidosis to stimulate continued development of new therapeutics. In basic science and engineering, the cross-beta architecture has been a constant thread underlying the structural characteristics of pathological and functional amyloids, and realizing that amyloid structures can be both pathological and functional in nature has fueled innovations in artificial amyloids, whose use today ranges from water purification to 3D printing. At the conclusion of a half century since Eanes and Glenner's seminal study of amyloids in humans, this review commemorates the occasion by documenting the major milestones in amyloid research to date, from the perspectives of structural biology, biophysics, medicine, microbiology, engineering and nanotechnology. We also discuss new challenges and opportunities to drive this interdisciplinary field moving forward.
NMR Methods for Structural Characterization of Protein-Protein Complexes (Front. Mol. Biosci.)
Protein-protein interactions and the complexes thus formed are critical elements in a wide variety of cellular events that require an atomic-level description to understand them in detail. Such complexes typically constitute challenging systems to characterize and drive the development of innovative biophysical methods. NMR spectroscopy techniques can be applied to extract atomic resolution information on the binding interfaces, intermolecular affinity, and binding-induced conformational changes in protein-protein complexes formed in solution, in the cell membrane, and in large macromolecular assemblies. Here Venditti V. et al. discuss experimental techniques for the characterization of protein-protein complexes in both solution NMR and solid-state NMR spectroscopy. The approaches include solvent paramagnetic relaxation enhancement and chemical shift perturbations (CSPs) for the identification of binding interfaces, and the application of intermolecular nuclear Overhauser effect spectroscopy and residual dipolar couplings to obtain structural constraints of protein-protein complexes in solution. Complementary methods in solid-state NMR are described, with emphasis on the versatility provided by heteronuclear dipolar recoupling to extract intermolecular constraints in differentially labeled protein complexes. The methods described are of particular relevance to the analysis of membrane proteins, such as those involved in signal transduction pathways, since they can potentially be characterized by both solution and solid-state NMR techniques, and thus outline key developments in this frontier of structural biology.
Structure of human GABA(B) receptor in an inactive state (Nature)
The human GABA(B) receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction. A unique GPCR that is known to require heterodimerization for function, the GABA(B) receptor has two subunits, GABA(B1) and GABA(B2), that are structurally homologous but perform distinct and complementary functions. GABA(B1) recognizes orthosteric ligands, while GABA(B2) couples with G proteins. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail. Although the VFT heterodimer structure has been resolved, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here O.B. Clarke, J. Frank, Q.R. Fan et al. present a near full-length structure of the GABA(B) receptor at atomic resolution, captured in an inactive state by cryo-electron microscopy. Their structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. They also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity. The structure of the GABA(B) receptor in an inactive state reveals, amongst other features, a latch between the two subunits that locks the transmembrane domain interface, and the presence of large phospholipids that may modulate receptor function.
Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy (Nat. Commun.)
Microtubules are important components of the eukaryotic cytoskeleton. Their structural organization is regulated by nucleotide binding and many microtubule-associated proteins (MAPs). While cryo-EM and X-ray crystallography have provided detailed views of inter- actions between MAPs with the microtubule lattice, little is known about how MAPs and their intrinsically disordered regions interact with the dynamic microtubule surface. NMR carries the potential to directly probe such interactions but so far has been precluded by the low tubulin yield. M. Baldus et. al. present a protocol to produce [13C, 15N]-labeled, functional microtubules (MTs) from human cells for solid-state NMR studies. This approach allowed them to demonstrate that MAPs can differently modulate the fast time-scale dynamics of C-terminal tubulin tails, suggesting distinct interaction modes. Their results pave the way for in-depth NMR studies of protein dynamics involved in MT assembly and their interactions with other cellular components.
Forced phage uncorking: viral DNA ejection triggered by a mechanically sensitive switch (Nanoscale)
The foremost event of bacteriophage infection is the ejection of genomic material into the host bacterium after virus binding to surface receptor sites. How ejection is triggered is yet unknown. Here M. S. Z. Kellermayer et.al. show, in single mature T7 phage particles, that tapping the capsid wall with an oscillating atomic-force-micro- scope cantilever triggers rapid DNA ejection via the tail complex. The triggering rate increases exponentially as a function of force, following transition-state theory, across an activation barrier of 23 kcal mol−1 at 1.2 nm along the reaction coordinate. The conformation of the ejected DNA molecule revealed that it had been exposed to a propulsive force. This force, arising from intra-capsid pressure, assists in initiating the ejection process and the transfer of DNA across spatial dimensions beyond that of the virion. Chemical immobilization of the tail fibers also resulted in enhanced DNA ejection, suggesting that the triggering process might involve a conformational switch that can be mechanically activated either by external forces or via the tail-fiber complex.
Structural insights into probe-dependent positive allosterism of the GLP-1 receptor (Nat. Chem. Biol.)
Drugs that promote the association of protein complexes are an emerging therapeutic strategy. K. Kobilka, K.W.Slopp et al. report discovery of a G protein-coupled receptor (GPCR) ligand that stabilizes an active state conformation by cooperatively binding both the receptor and orthosteric ligand, thereby acting as a ‘molecular glue’. LSN3160440 is a positive allosteric modulator of the GLP-1R optimized to increase the affinity and efficacy of GLP-1(9-36), a proteolytic product of GLP-1(7-36). The compound enhances insulin secretion in a glucose-, ligand- and GLP-1R-dependent manner. Cryo-electron microscopy determined the structure of the GLP-1R bound to LSN3160440 in complex with GLP-1 and heterotrimeric Gs. The modulator binds high in the helical bundle at an interface between TM1 and TM2, allowing access to the peptide ligand. Pharmacological characterization showed strong probe dependence of LSN3160440 for GLP-1(9-36) versus oxyntomodulin that is driven by a single residue. Their findings expand protein–protein modulation drug discovery to uncompetitive, active state stabilizers for peptide hormone receptors.