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29 Aug 2023

23rd Central European NMR Users Meeting

Welcome to the 23rd Central European Users Meeting (13-15 September 2023, Faculty of Architecture - Prague, Czech Republic), a scientific event dedicated to NMR users and enthusiasts. This year's event promises to be engaging, informative, and productive, with a range of presentations, discussions, and networking opportunities.

The agenda for the event is now available on the website, where you can also find all the information you need to attend, including the registration form. Take a look!

9 Aug 2023

Save the date (Instruct-ERIC Biennial Structural Biology Conference 2024)

The Instruct-ERIC Biennial Structural Biology Conference 2024 (IBSBC2024) will be taking place 22-24 May 2024.

The conference showcases the latest in integrative structural biology from leading scientists across the world, and will be taking place in the sunny seaside town of Cascais, Portugal.

This is the 6th Biennial Conference, and will explore the latest cutting-edge developments in structural biology, including imaging technologies, artificial intelligence and more, as well as insight into the role structural biology plays in the wider global context.

Biennial registration is open now.

3 Aug 2023

Register now to the IMAGINE project introduction and match-making event

Registration is open to join the first IMAGINE meeting open to all Instruct Centres on the 15th of September 2023 from 14:00 to 17:00 CEST. This meeting will highlight the new services (imaging instrumentation, tools and methods...) developed in the project and initiate collaborations with Instruct Centres external to IMAGINE. Indeed, Instruct Centres external to IMAGINE might take part in the validation and testing of the new services developed.

 

18 Jul 2023

Instruct-ERIC R&D Pilot Project Call Awardees 2023

Congratulations to the latest R&D Pilot Project Call Awardees Alexandre Ourjoumtsev, IBMC, France, Steffen Klein, EMBL Heidelberg, Germany, Francesca Paoletti, University of Trieste, Italy, and Heikki Saari, Finnish Red Cross Blood Service, Finland. All have received funding for preliminary research through Instruct-ERIC. The call was for small scale research projects in integrated structural biology, helping researchers develop external funding for projects. The selected researchers will each receive 15 000 EUR and will utilise several key technologies in the Instruct-ERIC service catalogue. R&D Projects will take place over the next 12 months and will pave the way for innovative advances in integrated structural biology.

10 Jul 2023

[3dem] iNext-Discovery Sample Preparation Course 2023

eBIC, Diamond Light Source, are pleased to announce the iNext-Discovery Sample Preparation Course 2023. This course is aimed at newcomers to the field of single particle analysis (SPA) using cryo-EM for their research.

The course will run at Diamond, Oxfordshire, UK on 16-19 Oct 2023. Delegates can expect theoretical lectures from expert & active scientists in the field including, Becky Thompson (TFS), Pete Harrison (eBIC) and Miriam Weckener (SPT Labtech), vitrification demonstration and hands on time using vitrobots or GP2, screening and data collection practical(s) on a Titan Krios, as well as demonstration of next generation freezing technologies i.e. SPT Labtech Chameleon.

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Highlights of Coronavirus Structural Studies

10 Nov 2021

Molecular basis of immune evasion by the Delta and Kappa SARS-CoV-2 variants (Science)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission leads to the emergence of variants, including the B.1.617.2 (Delta) variant of concern which is causing a new wave of infections and has become globally dominant. D. Veesler et.al. show that these variants dampen the in vitro potency of vaccine-elicited serum neutralizing antibodies and provide a structural framework for describing their immune evasion. Mutations in the B.1.617.1 (Kappa) and B.1.617.2 (Delta) spike glycoproteins abrogate recognition by several monoclonal antibodies via alteration of key antigenic sites, including remodeling of the B.1.617.2 (Delta) N-terminal domain. They determined electron cryo-microscopy (cryo-EM) structures of the B.1.617.1 and B.1.617.2 S glycoproteins to gain insights into their reduced antibody sensitivity and provide a structural framework to understand the role of shared and distinct mutations.  The ACE2 binding affinities of the B.1.617.1 (Kappa) and B.1.617.2 (Delta) receptor-binding domains are comparable to the Wuhan-Hu-1 isolate whereas B.1.617.2+ (Delta+) exhibits markedly reduced affinity.

8 Nov 2021

Crystal structure of SARS-CoV-2 main protease in complex with protease inhibitor PF-07321332 (Protein & Cell)

The crystal structure of SARS-CoV-2 Mpro in complex with PF-07321332 at 1.6 Å resolution was determined. PF-07321332 is a reversible covalent inhibitor carrying a nitrile warhead that targets SARS-CoV-2 Mpro. The overall structure of SARS-CoV-2 Mpro is a functional dimer, consistent with previous findings. The 1.6-Å crystal structure of SARS-CoV-2 Mpro in complex with the protease inhibitor PF-07321332 reveals critical insight for pharmaceutical development. With the emergence of SARS-CoV-2 variants with immune evasion, therapeutics targeting a conserved but essential viral target among the variants may synergize the COVID-19 vaccines. The improved oral bioavailability would enhance the clinical utility of PF-07321332 as a therapeutic to prevent progression among early-stage non-hospitalized patients or as a prophylaxis in the pre- and post-exposure setting. Further optimization of such covalent peptidomimetic inhibitors would generate potent drugs to contain the current COVID-19 pandemic and increase public health preparedness for potential future pandemic.

8 Nov 2021

NMR Spectroscopy of the Main Protease of SARS-CoV-2 and Fragment-Based Screening Identify Three Protein Hotspots and an Antiviral Fragment (Angewandte Chemie Int. Ed)

The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, J. Charton, X. Hanoulle et. al. studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. They performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.

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Newsletters

Reader's Corner Archive

9 Feb 2023

Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing (Nature Communications)

During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIPN-term) and DNA binding domain (PIPDBD). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIPN-term is released from PCNA and only PIPDBDis required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.

9 Feb 2023

Mechanism of IFT-A polymerization into trains for ciliary transport (Cell)

Intraflagellar transport (IFT) is the highly conserved process by which proteins are transported along ciliary microtubules by a train-like polymeric assembly of IFT-A and IFT-B complexes. IFT-A is sandwiched between IFT-B and the ciliary membrane, consistent with its putative role in transporting transmembrane and membrane-associated cargoes. Here, we have used single-particle analysis electron cryomicroscopy (cryo-EM) to determine structures of native IFT-A complexes. We show that subcomplex rearrangements enable IFT-A to polymerize laterally on anterograde IFT trains, revealing a cooperative assembly mechanism. Surprisingly, we discover that binding of IFT-A to IFT-B shields the preferred lipid-binding interface from the ciliary membrane but orients an interconnected network of β-propeller domains with the capacity to accommodate diverse cargoes toward the ciliary membrane. This work provides a mechanistic basis for understanding IFT-train assembly and cargo interactions.

9 Feb 2023

IFT-A structure reveals carriages for membrane protein transport into cilia (Cell)

Intraflagellar transport (IFT) trains are massive molecular machines that traffic proteins between cilia and the cell body. Each IFT train is a dynamic polymer of two large complexes (IFT-A and -B) and motor proteins, posing a formidable challenge to mechanistic understanding. Here, we reconstituted the complete human IFT-A complex and obtained its structure using cryo-EM. Combined with AlphaFold prediction and genome-editing studies, our results illuminate how IFT-A polymerizes, interacts with IFT-B, and uses an array of β-propeller and TPR domains to create “carriages” of the IFT train that engage TULP adaptor proteins. We show that IFT-A⋅TULP carriages are essential for cilia localization of diverse membrane proteins, as well as ICK—the key kinase regulating IFT train turnaround. These data establish a structural link between IFT-A’s distinct functions, provide a blueprint for IFT-A in the train, and shed light on how IFT evolved from a proto-coatomer ancestor.

9 Feb 2023

Cryo-EM structure of the human adenosine A2B receptor-Gs signaling complex (Science Advances)

The human adenosine A2B receptor (A2BR) is a class A G protein-coupled receptor that is involved in several major physiological and pathological processes throughout the body. A2BR recognizes its ligands adenosine and NECA with relatively low affinity, but the detailed mechanism for its ligand recognition and signaling is still elusive. Here, we present two structures determined by cryo-electron microscopy of A2BR bound to its ag-onists NECA and BAY60-6583, each coupled to an engineered Gs protein. The structures reveal conserved orthos-teric binding pockets with subtle differences, whereas the selectivity or specificity can mainly be attributed to regions extended from the orthosteric pocket. We also found that BAY60-6583 occupies a secondary pocket, where residues V2506.51 and N2737.36 were two key determinants for its selectivity against A2BR. This study offers a better understanding of ligand selectivity for the adenosine receptor family and provides a structural template for further development of A2BR ligands for related diseases.

9 Feb 2023

Structural basis for the severe adverse interaction of sofosbuvir and amiodarone on L-type Cav channels (Cell)

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.

24 Jan 2023

In situ cryo-electron tomography reveals the asymmetric architecture of mammalian sperm axonemes (Nat. Mol. Struct. Biol.)

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.

24 Jan 2023

Structural visualization of the tubulin folding pathway directed by human chaperonin TRiC/CCT (Cell)

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.

24 Jan 2023

A single 2′-O-methylation of ribosomal RNA gates assembly of a functional ribosome (Nat. Mol. Struct. Biol.)

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.

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