Talk of Dr. Alcicek on Zero- and Ultralow-field NMR
We are pleased to announce that the third Kiel Imaging Seminar (KIS) will take place next Monday, Jan. 16th.
We are pleased to announce that the third Kiel Imaging Seminar (KIS) will take place next Monday, Jan. 16th.
Instruct allocates funds to support small pilot research and development projects in any area of structural biology up to a maximum of €15,000 per project.
The EMBO workshop "Visualising the complex dynamics of biological membranes” is on March 13-16, at the Tel Aviv University.
Early career researchers, please submit abstracts for short talks. The program features a 'Dinner with speakers' event, with a chance for students and postdocs looking for a next career step, as well as PIs interested in new team members to discuss more informally. There will also be a tour to Jerusalem or hiking around the Dead Sea on the 15th for a full day.
The meeting will highlight the latest discoveries in structural and membrane biology along with a view to the future of how to tackle questions of higher complexity. The program will combine recent progress in single particle analysis, tomography, advances in molecular simulations, and high-speed atomic force microscopy.
On May 2 and 3, 2023 the Nuclear Magnetic Resonance Symposium will take place at the Institute of Science and Technology Austria (ISTA). The days after, May 4 and 5, 2023, a NMR workshop will be held for a select group of PhD students.
We are very pleased to announce that the 13th "NMR, a tool for Biology" conference will be held at the Institut Pasteur in Paris from May 15th to 17th, 2023.
Since the beginning of the COVID-19 pandemic, many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have emerged that are resistant to varying extents to neutralizing antibody responses induced by current vaccines and natural infection, especially the recent Omicron variants. Neutralizing potency and breadth for an antibody are often somewhat mutually exclusive. Here, we delineate the molecular interaction between a therapeutic antibody (ADG20) and SARS-CoV-2 receptor-binding domain (RBD) by X-ray crystallography and characterize its binding epitope. We show that this site is targeted by a few rare antibodies that have both potency and breadth. These findings provide insights into the design of more universal vaccines and broad therapeutic antibodies, which are pressingly needed.
Population antibody response is thought to be important in selection of virus variants. We report that SARS-CoV-2 infection elicits a population immune response that is mediated by a lineage of VH1-69 germline antibodies. A representative antibody R1-32 from this lineage was isolated. By cryo-EM, we show that it targets a semi-cryptic epitope in the spike receptor-binding domain. Binding to this non-ACE2 competing epitope results in spike destruction, thereby inhibiting virus entry. On the basis of epitope location, neutralization mechanism and analysis of antibody binding to spike variants, we propose that recurrent substitutions at 452 and 490 are associated with immune evasion of the identified population antibody response. These substitutions, including L452R (present in the Delta variant), disrupt interactions mediated by the VH1-69-specific hydrophobic HCDR2 to impair antibody-antigen association, enabling variants to escape. The first Omicron variants were sensitive to antibody R1-32 but subvariants that harbour L452R quickly emerged and spread. Our results provide insights into how SARS-CoV-2 variants emerge and evade host immune responses.
SARS-CoV-2 spike protein, which forms the basis for high pathogenicity and transmissibility of the virus, is a prime target for the development of both diagnostics and vaccines for the debilitating disease caused by the virus. We present a full model of spike methodically crafted and used to study its atomic-level dynamics by multiple microsecond simulations. The results shed light on the impact of posttranslational modifications on the pathogenicity of the virus. We show how glycan–glycan and glycan–lipid interactions broaden the protein’s dynamical range and thereby, its effective interaction with the surface receptors on the host cell. Palmitoylation of the spike membrane domain, however, results in a unique deformation pattern that might prime the membrane for fusion.
The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm, providing the highest-resolution structural information to date. Our subtomogram averages reveal mammalian sperm-specific protein complexes within the microtubules, the radial spokes and nexin–dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm-specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.
The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of β-tubulin using human prefoldin and TRiC. We find unstructured β-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded β-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin’s core and middle domains and GTP-binding. Thus, TRiC’s chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.
RNA modifications are widespread in biology and abundant in ribosomal RNA. However, the importance of these modifications is not well understood. We show that methylation of a single nucleotide, in the catalytic center of the large subunit, gates ribosome assembly. Massively parallel mutational scanning of the essential nuclear GTPase Nog2 identified important interactions with rRNA, particularly with the 2′-O-methylated A-site base Gm2922. We found that methylation of G2922 is needed for assembly and efficient nuclear export of the large subunit. Critically, we identified single amino acid changes in Nog2 that completely bypass dependence on G2922 methylation and used cryoelectron microscopy to directly visualize how methylation flips Gm2922 into the active site channel of Nog2. This work demonstrates that a single RNA modification is a critical checkpoint in ribosome biogenesis, suggesting that such modifications can play an important role in regulation and assembly of macromolecular machines.
Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure–activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein–nanobody complex are associated with tighter binding.To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.
Leucine-rich repeat kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinson’s disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an association enhanced by PD mutations. We report a cryo-EM structure of the catalytic half of LRRK2, containing its kinase, in a closed conformation, and GTPase domains, bound to microtubules. We also report a structure of the catalytic half of LRRK1, which is closely related to LRRK2 but is not linked to PD. Although LRRK1’s structure is similar to that of LRRK2, we find that LRRK1 does not interact with microtubules. Guided by these structures, we identify amino acids in LRRK2’s GTPase that mediate microtubule binding; mutating them disrupts microtubule binding in vitro and in cells, without affecting LRRK2’s kinase activity. Our results have implications for the design of therapeutic LRRK2 kinase inhibitors.
Drug-drug interaction of the antiviral sofosbuvir and the antiarrhythmics amiodarone has been reported to cause fatal heartbeat slowing. Sofosbuvir and its analog, MNI-1, were reported to potentiate the inhibition of cardiomyocyte calcium handling by amiodarone, which functions as a multi-channel antagonist, and implicate its inhibitory effect on L-type Cav channels, but the molecular mechanism has remained unclear. Here we present systematic cryo-EM structural analysis of Cav1.1 and Cav1.3 treated with amiodarone or sofosbuvir alone, or sofosbuvir/MNI-1 combined with amiodarone. Whereas amiodarone alone occupies the dihydropyridine binding site, sofosbuvir is not found in the channel when applied on its own. In the presence of amiodarone, sofosbuvir/MNI-1 is anchored in the central cavity of the pore domain through specific interaction with amiodarone and directly obstructs the ion permeation path. Our study reveals the molecular basis for the physical, pharmacodynamic interaction of two drugs on the scaffold of Cavchannels.
Cryogenic electron tomography (cryo-ET) is the application of tomographic principles of data acquisition and reconstruction to frozen-hydrated biological specimens. It combines a close-to-life preservation of cellular structures with the power of high-resolution three-dimensional imaging, which allows us to study the molecular architecture of cells, or their molecular sociology, in unprecedented detail. The journey of cryo-ET to the inner space of cells has been a long and tedious one. In the paper, some of the limitations of the method will be discussed along with prospects to overcome them.
Single-particle cryogenic electron microscopy (cryo-EM) has emerged as a powerful technique to visualize the structural landscape sampled by a protein complex. However, algorithmic and computational bottlenecks in analyzing heterogeneous cryo-EM datasets have prevented the full realization of this potential. CryoDRGN is a machine learning system for heterogeneous cryo-EM reconstruction of proteins and protein complexes from single-particle cryo-EM data. Central to this approach is a deep generative model for heterogeneous cryo-EM density maps, which we empirically find is effective in modeling both discrete and continuous forms of structural variability. Once trained, cryoDRGN is capable of generating an arbitrary number of 3D density maps, and thus interpreting the resulting ensemble is a challenge. Here, we showcase interactive and automated processing approaches for analyzing cryoDRGN results. Specifically, we detail a step-by-step protocol for the analysis of an existing assembling 50S ribosome dataset, including preparation of inputs, network training and visualization of the resulting ensemble of density maps. Additionally, we describe and implement methods to comprehensively analyze and interpret the distribution of volumes with the assistance of an associated atomic model. This protocol is appropriate for structural biologists familiar with processing single-particle cryo-EM datasets and with moderate experience navigating Python and Jupyter notebooks. It requires 3–4 days to complete. CryoDRGN is open source software that is freely available.