LEAPS is the League of European Accelerator-based Photon Sources, a strategic consortium initiated by the Directors of the Synchrotron Radiation and Free Electron Laser (FEL) user facilities in Europe. Its primary goal is to actively and constructively ensure and promote the quality and impact of the fundamental, applied and industrial research carried out at their respective facility to the greater benefit of European science and society.
In this context LEAPS will organize 14-19 May, 2023 the second LEAPS Conference aiming at bringing together the latest achievements from the Life Sciences user community with those from synchrotron radiation source development and instrumentation.
The focus of the second LEAPS Conference will be the opportunities emerging from the Life Sciences R&D. This conference will showcase where today and future synchrotron radiation contributes to the advancements in Life Sciences.
The conference has limited places. There is a pre-registration where an abstract needs to be submitted. Non-presenting participants should explain in a short statement why the meeting is relevant to them. In a second step, selected participants will be informed and asked/prompted to pay the registration fee.
The conference will be held at the Hotel Hermitage, La Biodola Bay, Elba Island, Italy from the 14 (arrival date) to the 19 (departure date) of May 2023.
Metagenomics is a technique that enables us to look at parts of the biosphere that were masked to us. With present estimates suggesting that ~98% of the microorganisms are not amenable to growth under lab conditions, metagenomics is helping us to understand the role of microorganisms in different environments.
Two metagenomic discoveries of light harvesting by microorganisms will be discussed. The first is the finding of light-driven rhodopsin proton pumps in marine microorganisms. These rhodopsins are now believed to be present in 70% of marine bacteria and archaea in the photic zone. The second is the discovery of cyanophages (viruses infecting cyanobacteria) that carry in their genomes photosynthetic genes coding for both photosystem-I and II proteins. It was suggested that the horizontal transfer of these genes might be involved in increasing viral fitness.
Furthermore, the discoveries of strange rhodopsins, the bestrhodopsin ion-channel complexes and the potassium channelrhodopsins, to be used as potential tools for optogenetics, will also be described.
The implications of metagenomics on basic and applied science as a whole will be elaborated.
Since many years imaging methods on all length scales and their development are dominating the progress in life sciences. Atomic structure and dynamics link mechanism, phenotype and diseases to a mechanistic chemical explanation. Only if you reach this level, rational intervention, like needed in drug discovery, is possible. There is an ever increasing need for correlative multi-scale analysis in biology and medicine to capture the intrinsic complexity of life sciences on all time and length scales. There is not a single biophysical method that can address all the needs of life sciences. Only a highly complementary environment can support modern mechanistic understanding of biology or drug discovery. A large facility site that is well integrated with academic research can optimally provide this challenging interdisciplinary culture. Traditionally, X-ray crystallography was dominating the interaction of large facilities with life sciences. Today, there is an evolving need for more imaging methods and for biophysics that cannot be addressed with crystallisation and crystallography. Intrinsically disordered protein mesophases and their regulation are at the center of cellular nano organisation that guide development and cellular dynamics. Biophysical methods that can address cell-cell interactions and study complex cellular assemblies in tissues are the most needed tools for the future understanding of living organisms. Genomics, proteomics and structural biology will play an important role for a long time to come, but they need to be developed further. Atomic resolution in cell structural biology has been demonstrated in recent electron tomography case studies and this development will accelerate. Electron microscopy and X-ray crystallography addressing dynamics on Ångström to nanometer resolution are developing fast. Life sciences are inherently interdisciplinary and their progress depends on novel methods development. Large facilities can play a pivotal role in the future of life sciences because they can provide excellent project stability and they are intrinsically interdisciplinary. Life sciences at large facilities can only be relevant if life science research is closely integrated into the strategy of the large facility. If this is the case, life sciences can be a major driver of the future of large facilities in Europe and worldwide.
The femtosecond-duration pulses of X-ray free-electron lasers overcome the limitations of exposures of biological materials by outrunning radiation damage. In this way, it has become possible to obtain meaningful data from samples too small for conventional analyses, at the expense of measuring only a single exposure from a single object. Diffraction data collected in a serial fashion from a stream of randomly oriented crystals or particles can be merged into a 3D dataset by discovering the latent parameters of orientation, crystal twin component, or structural evolution. This measurement paradigm enables 3D images to be obtained from ensembles of structures, even in the limit of extremely low signal per object. Current developments continue to improve serial diffraction experiments with the goal to image even smaller and weaker scatterers.
G. Fiorini, S. Marshall, W. Figg, M. Kibria, F. Arif, P. Rabe, M. A. McDonough, C. J. Schofield
In humans, three HIF prolyl-hydroxylases (PHD1-3) play key roles in hypoxia sensing. The PHDs are 2-oxoglutarate (2OG)-dependent dioxygenases that catalyse trans-4-prolyl hydroxylation of the hypoxia-inducible factors (HIFs). Prolyl-hydroxylation enables the proteasomal degradation of HIF, causing the suppression of the hypoxic response. The aim of this work is to use time-resolved crystallography and spectroscopical techniques to elucidate the mechanism of the PHDs, by characterising the reaction intermediates. High-resolution structures of an anoxic PHD2.Fe.2OG.HIF2αCODD complex were obtained and exposure to O2, validated the possibility of monitoring the reaction in crystallo leading to the first high-resolution structures of a PHD2:product complex. One of PHDs peculiarities is their slow reaction with O2. It is unclear how O2 diffuses towards the active site, but of particular interest is residue Thr387. PHD2.substrate crystal structures show Thr387 to be involved in hydrogen bond networks with water molecules in the first and second coordination sphere of the active site. Mutations of the Thr387 to less polar residues cause an increase in enzyme activity. Crystal structures of these mutants showed a lack of water molecules near the active site when compared with the wild-type PHD2, providing a possible explanation for the increased turnover. Additionally, it was possible to spectroscopically identify a potential “ferryl” intermediate not yet reported for PHDs. Overall, there results elucidate key binding interactions between PHD2, product and cofactor, confirm the stereoselectivity of the hydroxylation, and identify a gate keeper residue in PHD2.
Advances in computer science, particularly in the field of AI, is having a significant impact on biomolecular research. AI methods such as deep neural networks have shown great promise in predicting protein structures as demonstrated by the AlphaFold project . Here, I will outline recent achievements as well as present a case study that benchmarks AlphaFold’s performance for G protein-coupled receptors (GPCRs) - a large and diverse family of membrane proteins that are involved in many physiological processes. For this, we carried out a comprehensive structural analysis and large-scale molecular simulations for hundreds of GPCRs from AlphaFold in different activation states (active, inactive). In a second case study, I will highlight the potential of atomistic computer simulations  to sample the dynamics of thousands of intramolecular contacts in a prototypical GPCR linked to relevant functional signaling outcomes. All in all, this lecture will shed light on the advancements but also existing challenges of AI and computer simulations to study complex biological systems such as GPCRs.
High Performance Computing, i.e. scientific computing where performance matters, has been somewhat intimidating for non-experts. With the recent surge of machine learning, HPC technologies have found massive adoption in the commercial software world, which in turn allows us to make better use of extreme-scale computing and data in scientific workflows. Alps is how we call the new supercomputing infrastructure at CSCS, on which we are providing a practical synthesis of cloud-native and HPC, as well as AI technologies for science. We will discuss our plans, opportunities to better deal with large-scale scientific data and its analysis, and what we believe are the main investment hat domain sincere communities should be making now.
X-ray free electron laser (XFEL) is an exciting new technology that could significantly extend our structural knowledge of biological systems. One of the experimental approaches currently pursued is “single particle analysis,” in which intense laser light from XFEL is used to observe single molecular complexes. Since it does not require crystallization, various systems could be studied under different physiological conditions. However, applications to biological systems are still challenging due to their low diffraction power and require further developments of experimental as well as computational analysis techniques.
Therefore, we have been developing programs to perform three-dimensional reconstruction from a data set of diffraction patterns by adopting algorithms used in cryo-EM 3D reconstruction to handle XFEL diffraction patterns. This approach was successfully tested with X-ray tomography experimental data. Using synthetic data, we have estimated the experimental conditions necessary to achieve sub-nanometer resolutions for molecules such as the ribosome. In addition, we have investigated the molecular size dependence on the achievable resolution. Finally, after processing nanoparticles' experimental diffraction patterns to reduce background noise, we obtained a 3D shape consistent with scanning electron microscope images.
While cryo-electron microscopy (cryo-EM) is rapidly gaining in popularity, its time resolution is currently insufficient to directly observe proteins in action, leaving our understanding of these nanoscale machines fundamentally incomplete. We have recently introduced a novel approach to time-resolved cryo-EM that affords microsecond time resolution and thus promises to overcome these limitations. Our method involves melting a cryo sample with a laser beam, which allows dynamics of the embedded particles to occur in liquid once a suitable stimulus is provided. While the dynamics occur, the heating laser is switched off at a well-defined point in time, causing the sample to rapidly cool and revitrify. The particles are thus trapped in their transient configurations, in which they can subsequently be imaged. We demonstrate that our approach affords a time resolution of 5 µs or better. Moreover, near-atomic resolution reconstructions can be obtained from revitrified samples, showing that the revitrification process leaves the protein structure intact. Finally, I will present a microsecond time-resolved pH jump experiment, in which we observe the dynamics of the capsid of CCMV, an icosahedral plant virus. These results highlight the potential of our method to fundamentally advance our understanding of protein function through direct observation of dynamics.
Fourth generation synchrotron sources create new opportunities for expanding the research in structural biology and in protein crystallography in particular. The ESRF Extremely Brilliant Source upgrade programme was completed with the construction of the new ID29, the first world beamline completely dedicated to room temperature experiment and time-resolved macromolecular serial crystallography. The beamline characteristics were designed in order to obtain diffraction data from micrometer sized crystals and achieve a microsecond time resolution. This needed the development of a new class of instrumentation which included a new double chopper timing system, that is able to produce X-ray pulses of 10 microseconds, a new diffractometer, the MD3upSSX, that presents a flexible sample environment, that accommodate fixed target, viscous injectors, microfluidics or tape drive. The experimental setup is completed with a Jungfrau 4M detector that has been integrated in the ESRF data acquisition pipeline and can be operated up to 1 khz data acquisition rate.
In this presentation we will report from the first ground-breaking experiments that took place in this initial year of operation of ID29, along with the beamline development roadmap and future plans.
A frontier challenge in bioscience is to obtain deep mechanistic understanding from atomic resolution, time-resolved structures of macromolecules engaged in function. To this end, we have developed sample-efficient delivery and reaction initiation strategies that use room temperature microcrystal slurries and serial crystallography methods for time-resolved studies. Our overriding hypothesis is that microcrystal enzyme-substrate complexes will form many times faster than the average enzyme catalytic rate, which is supported by increasing numbers of observations. However, interpreting electron density maps from reaction cycle intermediates can be challenging, especially when mixtures of species are often present in the data. Therefore, to help reduce ambiguity we have also pioneered strategies to simultaneously collect time-resolved serial crystallography (tr-SMX/SFX) diffraction data in the forward direction, and X-ray emission spectroscopy (tr-XES) data at ~ 90º, using either X-ray free electron laser (XFEL) sources, or synchrotron sources such as Diamond Light Source in Oxfordshire UK. The resulting atomic and electronic structures are fully correlated and have been applied to a range of metalloenzymes. For instance, isopenicillin N synthase (IPNS) uses nonheme iron to catalyse the O2-dependent conversion of its tripeptide substrate delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) into isopenicillin N (IPN, the precursor of all penicillin/cephalosporin beta-lactam antibiotics). The unique four electron oxidation reaction leading to the beta-lactam bicyclic ring proceeds via two high-valent iron species, an Fe(III)-superoxo and a high-spin Fe(IV)=O oxyferryl species. These enable two sequential C-H bond cleavage steps that each exhibit large kinetic isotope effects (KIE). Our recent tr-SFX and tr-XES studies have characterised the Fe(III)-superoxo species and revealed unexpected, correlated motions throughout the whole protein caused by O2 binding.
Mammalian rhodopsin is our light receptor for vision. It belongs to the highly druggable G protein-coupled receptor family. It hosts the retinal chromophore which, like a switch, isomerizes in less than 200 femtoseconds upon photon absorption. This triggers sequential intramolecular changes in rhodopsin, initiating the signalling cascade generating in milliseconds vision events to the brain via the optic nerve. However, the intramolecular events transforming the rhodopsin resting state[1-2] (dark) into the transducin-binding activated state[3-5] (Meta II) are not completely understood.
We experimentally determined ultrafast changes of native bovine rhodopsin at room temperature using time-resolved serial femtosecond crystallography, already successfully used for the proton pump bacteriorhodopsin[6-7], at SACLA and SwissFEL X-ray free electron lasers. Thousands of rhodopsin microcrystals grown in the dark are successively injected in the light of a pump laser and probed after various time-delays using an XFEL. After 1 picosecond, we observe a highly distorted all-trans retinal that has induced changes in its binding pocket while the excess energy of the absorbed 480nm-photon dissipates inside rhodopsin through an anisotropic protein breathing motion towards the extracellular domain. Interestingly, some amino acids known to be key elements later in the transduction of the signal are involved in the ultrafast changes.
Single molecule scattering experiments with femtosecond high-intensity free-electron laser (XFEL) pulses provide a new route to macromolecular structure determination. In these serial crystallography experiments, and despite the ultra-high brilliance of XFEL lasers, the signal to noise is expected to be in the extreme Poisson regime with only 10-100 recorded diffracting photons per image. Further, in each single scattering event, the orientation of the specimen molecule is random and unknown. As a further layer of complexity, many biomolecules show structural heterogeneity and conformational motions between different distinct structures; these structural dynamics are averaged out by existing refinement methods. To overcome these limitations, here we developed and tested a rigorous Bayesian approach and demonstrate that it should be possible to determine not only a single structure, but an entire structural ensemble to near-atomistic resolution from these experiments. We further show that as few as three photon per scattering image suffice for this purpose. Unexpectedly, much fewer images are required to determine an ensemble of n structures of m atoms each than a single structure of n × m atoms, i.e., of the same total number of degrees of freedom. These findings shows that X-ray scattering experiments using state-of-the-art free electron lasers should allow one to determine not only biomolecular structures, but whole structure ensembles and, ultimately, ‘molecular movies’.
The sarcomere is the smallest contractile unit in cardiac and skeletal muscle, where actin and myosin filaments slide past each other to generate tension. This molecular machinery is supported by a subset of highly organised cytoskeletal proteins that perform architectural, mechanical, and signalling functions. Sarcomere ultrastructure is highly organised and delimited by Z-disks, which play a critical role in mechanical stabilisation and force transmission.
In the Z-disks – the lateral boundaries of the sarcomere machinery – the protein α-actinin-2 cross-links antiparallel actin filaments from adjacent sarcomeres, and additionally serves as a binding platform for a number of other Z-disk proteins. In striated muscle cells, the Z-disk represents a highly organized three-dimensional assembly containing a large directory of proteins orchestrated in a multi-protein complex centred on its major component α-actinin, with still poorly understood hierarchy and three-dimensional interaction map. To investigate the molecular structural architecture of the Z-disk, the assembly hierarchy, and structure-function relationships, we are employing an integrative structural biology strategy that combines molecular biophysics, structural, and biochemical approaches.
FATZ proteins interact with α-actinin and five other core Z-disk proteins, contributing to the assembly and maintenance of myofibrils as a hub for protein interactions. I will present our studies on the interaction of the major Z-disk protein α-actinin-2 with FATZ-1 and Z-portion of titin, forming dynamic fuzzy complexes, and discuss findings in view of asymmetric sorting of α-actinin and sarcomeric Z-disk architecture and assembly.
Furthermore, our recent discover that FATZ-1 can phase-separate and form biomolecular condensates with α-actinin-2 and other three Z-disk proteins raises the intriguing question of whether FATZ proteins can create an interaction hub for Z-disk proteins during myofibrillogenesis via membrane-less compartmentalization.
Cytochrome c oxidase (CcO) catalyses the reduction of molecular oxygen to water while the energy released in this process is used to pump protons across a biological membrane. Even though many members of the CcO superfamily have been structurally characterized in detail, there is no detailed structural understanding of how unidirectional proton translocation takes place. A billion-fold jump in the peak X-ray brilliance delivered by X-ray free electron laser (XFEL) and the development of serial femtosecond crystallography (SFX) allowed the determination of protein structures at room temperature, opening up the opportunities for time-resolved (TR) experiments in measuring ultrafast reactions in proteins. In this project, we track structural changes at the active site of ba3-type cytochrome c oxidase upon photoinitiated release of oxygen molecule from cobal-based cage compound. Additionally, we use X-ray absorption spectroscopy (XAS) to investigate the detailed atomic structures of the metal co-factors in different redox states.
In this tutorial I will briefly review the principles of synchrotron radiation, introduce the basics of tomographic microscopy and then showcase some of the latest imaging results, with particular focus on time-resolved experiments. I will then further discuss on how some of the tools originally designed for machine diagnostics purposes have developed into powerful, potentially game-changing instruments with a clinical impact. The tutorial has been conceived to provide an “easy-entry” into the fascinating world of modern X-ray imaging.
The electron Bio-Imaging Centre (eBIC) is a UK national cryoEM facility located at the UK synchrotron Diamond Light Source. eBIC provides UK and international scientists with state-of-the-art experimental equipment, expertise, and training, in the field of cryo-electron microscopy, for single particle analysis, electron tomography, electron diffraction, and correlative microscopy. eBIC also develops cutting-edge technology with in-house research program, output of which further benefits our users and beyond. The location of eBIC enables scientists to combine their techniques with many of the other cutting-edge approaches that Diamond offers. In this presentation, I will demonstrate the power of emerging in situ structural biology and methods development towards high resolution structures of protein complexes in the most native cellular context. I will also showcase the integration of multi-scale imaging modalities encompassing cryo-electron tomography, soft x-ray imaging and cryoFIB/SEM volume imaging to investigate virus infections in cells.
Propagation-based X-ray phase-contrast tomography (XPCT) offers a unique potential to extend histology and pathohistology by a scalable, isotropic resolution without destructive slicing of the specimen and quantitative density-based contrast.
Here, we use virtual histology based on XPCT to image the human lung at synchrotron radiation endstations (P10, DESY) in different setups as well as by laboratory µCT instruments, and screen a broad range of sample preparation approaches. By comparing and optimizing setups and image parameters, we provide benchmarks of image quality and resolution on multiple scales down to the sub-cellular level. Using examples of several medically relevant pulmonary pathologies, we illustrate the advantages of obtaining three dimensional (3D) reconstructions rather than the two-dimensional images known from conventional histology. To complement manual evaluation of the data, we introduce quantitative morphometric parameters to characterize lung tissue and to compare between physiological and pathological states.
By optimizing imaging workflows and reconstruction routines, we can take advantage of the superior contrast and image quality provided by synchrotron radiation. Thus we can find a good compromise between resolution and field of view, providing ground truth for validation of laboratory data which is important for future standardization and translation of the method.
The 3D complexity of biological tissues and intricate structural-functional connections call for state-of-the-art X-ray imaging approaches. Unlike other imaging techniques, X-ray phase-contrast tomography (XPCT) offers a highly sensitive 3D imaging approach to investigate different disease-relevant networks at levels ranging from the single cell through to the whole organ. We present here a concomitant study of the evolution of tissue damage and inflammation in different organs affected by the disease in the murine model for multiple sclerosis, a demyelination autoimmune disorder of the central nervous system. XPCT identifies and monitors structural and cellular alterations throughout the central nervous system, but also in the gut, and eye, of mice induced to develop multiple sclerosis-like disease and sacrificed at pre-symptomatic and symptomatic time points. This approach rests on a multiscale analysis to detect early appearance of imaging indicators potentially acting as biomarkers that can predict the disease. The longitudinal data obtained permit an original evaluation of the sequential evolution of multi-organ damages in the murine model showing the disease development and progression, of relevance for the human case.
Malaria is one of the most deadly diseases to affect humanity and it is caused by a parasite which has co-evolved with humans for millennia. It has proved hugely challenging to develop a vaccine to target this adaptable and changeable parasite. I will talk about how we use structural approaches to design improved vaccine immunogens, showing how we combine structural biology methods to understand the key host-parasite interactions in malaria and to determine the epitopes of the most effective growth-neutralising antibodies. Finally I will discuss how we use structure-guided protein design to make novel vaccine immunogens focused to induce only the most effective antibody responses.
High-resolution ribosome structures determined by cryo X-ray crystallography have provided important insights into the mechanism of translation. Such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. We use serial femtosecond X-ray crystallography (SFX) with an X-ray free-electron laser (XFEL) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature. Small 30S ribosomal subunit microcrystals programmed with decoding complexes and bound to either antibiotic compounds or their next-generation derivatives diffracted to high resolution. Our results demonstrate the feasibility of using SFX to better understand the structural mechanisms underpinning the interactions between ribosomes and other substrates such as antibiotics and decoding complexes. We have determined the structure of a large (50S) ribosomal subunit in a record-short time by using the record-low amount of sample during an XFEL beamtime. This structure is the largest one solved to date by any FEL source to near-atomic resolution (3 MDa). We expect that these results will enable routine structural studies, at near-physiological temperatures, of the large ribosomal subunit bound to clinically relevant classes of antibiotics targeting it, e.g. macrolides and ketolides, also with the goal of aiding the development of the next generation of these classes of antibiotics. Overall, the ability to collect diffraction data at near-physiological temperatures promises to provide new fundamental insights into the structural dynamics of the ribosome and other medically important drug targets with their functional and inhibitor complexes.
Imaging the structure of neuronal networks is of fundamental importance for the understanding of information processing in the healthy and diseased brain. But neuronal networks in the brain span several millimeters in each dimension and the smallest neurites and synaptic connections are on the order of a few tens of nanometers in size. To date, only volume electron microscopy (vEM) provides sufficient resolution and sufficient field of view for the dense reconstruction of synaptic-resolution neuronal circuits. Using vEM, mm3-sized samples can be acquired at synaptic resolution within a couple of months. However, these vEM techniques are all based on cutting tens of thousands of ultrathin (30 – 50 nm) sample sections, which is a delicate process and inherently prone to failure. Sectioning artifacts and the limited resolution in Z are the major source of errors of state-of-the-art automated neuron segmentation algorithms. Therefore, synchrotron X-ray nanotomography is a promising, nondestructive alternative for ultrastructural imaging of large tissue samples such as whole brains.
Neurodegenerative diseases are characterised by the accumulation of amyloids in human brain. Cryo-electron microscopy (cryo-EM) of amyloid filaments from post mortem brains has recently shown that distinct amyloid conformations define different neurodegenerative diseases. We describe the structural characterisation of in vitro model systems for amyloid formation that aim to replicate the same structures as observed in diseased brains, with a focus on tau. We show that by using N- and C-terminally truncated tau constructs, under specific conditions can lead to structures like those observed in Alzheimer’s Disease (AD) and Chronic Traumatic Encephalopathy (CTE). We show that NMR and high through-put cryo-EM approaches can be used to study the molecular mechanisms of amyloid assembly. We show that amyloid-prone regions within the tau sequence can form dimers in solution which assemble into protofibril structures. These protofibrils are intermediate filament structures which can rearrange into mature filaments, like those observed in disease.
The IBS hosts a number of state of the art structural biology platforms that are accessible via the national FRISBI and the European Instruct programs providing technician-supported user access to instrumentation including cell imaging, cryo electron microscopy and NMR. Furthermore, the IBS participates in the operation of the ESRF CM01 electron microscopy facility equipped with a Titan Krios cryo-electron microscope (cryoEM) for single particle experiments. Construction of a second cryo electron microscopy platform at ESRF, CM02 is currently underway, which will be equipped with a Titan Krios electron microscope for single particle and cryo electron tomography experiments. CM02 will be operated as a French CRG beam line by IBS starting at the end of 2023. In addition, IBS operates the CRG crystallography beam line FIP-2 on the Bending Magnet section 07 (BM07) of the ESRF for multiwavelength anomalous diffraction experiments and in situ (in plate) crystal screening and data collection. Together with the ESRF, IBS runs the icOS Lab dedicated to optical spectroscopy experiments, such as UV/vis absorption, fluorescence (soon available on MASSIF-3 and FIP-2), and Raman spectra (soon on ID30B) of protein crystals.
Here I will present two examples of integrative structural biology approaches applied to enveloped virus host interaction. First, I will discuss vaccine development against SARS CoV-2. Although the current epidemic of SARS CoV-2 seems to slow down, there is a high need for vaccines that can provide broad protection against current or newly arising variants of concern (VOCs). Secondly, I will present structural biology approaches to understand the cellular machinery called ESCRT that is recruited by many enveloped viruses to catalyze the release of newly formed enveloped viruses from host cells.
Structural biology had a major impact on the delivery of effective responses to the COVID-19 pandemic. I will give some illustrations of these from our experiences, however I will attempt to embed them in a consideration of the broader developments.
Large infrastructures allowed the high resolution structural analysis of virus proteins and particles via X-ray crystallography. For small proteins this has been honed into ultra-high throughput methods accelerating antiviral drug discovery. Circa nine years ago the field of virus structure was opened up by the resolution revolution in cryo-EM. This made complex analysis easier and revealed features that broke icosahedral symmetry. Top-end microscopes were, and remain, expensive and to democratise access we integrated them into a synchrotron facility (Diamond) and a pan-European research infrastructure (Instruct-ERIC). Technology developments have accelerated the method roughly an order of magnitude since the revolution began. Now lower voltage microscopes might take this a step further by making machines affordable by many more labs. Meanwhile the next step in structural biology, in situ analysis using electron tomography, is a reality. It remains unclear what key technology innovations will increase the reach of this method, but it is likely that the next generation or two of instruments will be increasingly expensive, and probably most should be incorporated into large facilities. Alongside these developments there are open questions about the long term impact on biology of electron ptychography and liquid phase electron microscopy. However electrons are fundamentally limited in their ability to penetrate cells and tissue, and so it is likely that imaging with light, electrons and X-rays will need to be combined to address the most challenging questions, e.g. of virus pathogenesis. There are real opportunities for large scale science infrastructures to accelerate technical innovation and markedly improve pandemic preparedness.
The study presents the first observation of critical steps in protein synthesis at temperatures close to those in the human body using ultrafast and ultrabright X-ray free electron laser (XFEL) pulses. Over half of known antibiotics target prokaryotic ribosomes, the site of protein synthesis, and the large ribosomal subunit (50S) is specifically targeted by antibiotics such as macrolides and ketolides. The study determined the structure of 50S ribosomal subunit at ambient temperature, providing insight into how antibiotics interact with ribosomes and how peptide bonds are formed. This is a significant milestone as it's one of the largest structures determined using XFEL, with a record short beam time of 47 minutes. The use of serial femtosecond X-ray crystallography (SFX) will further advance our understanding of ribosome structure and function, leading to the development of new antibiotics.
Time-resolved serial crystallography has emerged as a method to study protein dynamics at atomic resolution. In this method, reactions need to be initiated synchronously. Many experiments have used light as a trigger system to study endogenously photoactive proteins; however, only a small fraction of proteins are naturally photoactive. Therefore, work needs to be done to ensure this method can be applied to a wider range of pharmacologically relevant proteins. Synthetic photoswitches have been developed through the field of photopharmacology that convert between binding and nonbinding conformations. Here we present the characterisation of eight synthetic photoswitches, derived from an approved treatment against Parkinson’s disease. We observed that photoisomerisation properties can differ in crystallo from in solution, indicating that not all photoswitches are suitable for time-resolved crystallography. We applied synthetic photoswitches to observe the process of ligand dissociation from the human A2A receptor, a G protein-coupled receptor. An understanding of this process can help guide the development of drugs with increased residence times. Using data collected at SLS, MaxIV and the SwissFEL, we follow how the binding pocket adapts to accommodate ligands and movement in the hydrogen-bonded lid of the binding pocket during ligand dissociation.
Macromolecular crystallography is a well-established method in structural biology and after focusing on static structures, the method is now developing towards the investigation of structural dynamics, e.g. by looking at protein-ligand or enzyme-substrate interactions. In time-resolved serial crystallography and room-temperature data collection, the reaction is triggered within the crystals – either optically or chemically. For the latter, the use of micron-sized crystals is necessary to ensure short diffusion times and quick saturation within each crystal. However, certain crystal morphologies e.g. small solvent channels can prevent sufficient ligand diffusion. Presented here is a method combining protein crystallization and data collection in a novel one-step-process to overcome the aforementioned challenges. We successfully performed corresponding experiments as a proof-of-principle using hen egg-white lysozyme with crystallization times of a few seconds. This method called JINXED (Just in time crystallization for easy structure determination) promises to result in high-quality data due to the avoidance of crystal handling and could enable time-resolved experiments regardless of crystal morphology by adding potential ligands to the crystallization buffer, simulating co-crystallization approaches. In combination with upcoming automated data processing, this method may offer the possibility to combine high-throughput ligand screenings and detailed dynamical investigations with a high level of automation.
Advances in structure determination and computational methods facilitate the discovery and optimization of pharmaceutical active compounds in the majority of all projects. Still, integral membrane proteins are drug targets for more than 60% of all approved drugs, but are underexplored because of their challenges to be expressed, purified and get high resolution structures or enable biophysical methods to investigate target engagement and ligand binding kinetics.
The presentation includes recent advances in technologies and their application to relevant drug targets such as the cryo-EM structure of the human TRPV4 ion-channel with bound small molecule agonist. The agonist binding activates the channel opening with a significant structural change enabling direct observation of agonist pharmacology by high resolution cryo-EM analysis. Next example is LPTDE, a clinically validated antibiotics drug target. Due to limited size of 120 kDa and the monomeric -sheet transmembrane architecture, the leadXpro proprietary tool of Pro-Macrobodies was essential for the successful EM structure at 2.9 A resolution. The cryo-EM structure of the ion-channel Kv3.1 at 2.6 Å resolution using full length wild type protein apo and with the small molecule positive modulator Lu AG00563 reveals a novel ligand binding site for the Kv class of ion channels located between the voltage sensory domain and the channel pore. Recently, leadXpro disclosed the first structure of a proton sensing GPCR. Structure-based optimization of GPR65 activity modulators will open novel therapeutic options for life-threatening diseases such as inflammatory bowel disease and cancer.
Construct engineering, application of in meso in situ serial X-ray crystallography (IMISX) is exemplified with the GPCR structure of CCR2 in complex with an antagonist ligand. This is combined with detailed binding characterization using grating-coupled interferometry (GCI, Creoptix) to facilitate drug design with binding kinetic, affinity.
The outlook at future perspectives includes further advances in cryo-EM and the application of serial X-ray crystallography at synchrotron and femtosecond pulsed Free Electron Lasers (FEL). Here, determination of room temperature structures and observation of structural dynamic of ligand binding and associated conformational changes. All new developments in structural biology will further enhance the impact to the design of candidate drug compounds.
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Cheng, R.K.Y., Towards an optimal sample delivery method for serial crystallography at XFEL, Crystals, 2020, 10, 215;
Apel, A-C., Crystal structure of CC chemokine receptor 2A in complex with an orthosteric antagonist provides insights for the design of selective antagonists, Structure 27, (2019)
AIDS caused by infection of human immunodeficiency virus (HIV) is one of the big issues in the world. Antiretroviral therapy (ART) using multiple drugs has developed, and suppression of HIV in a body is possible. However, latent HIV reservoirs can be found in body, and HIV can hide for years inside reservoirs. This reservoir is resistant to ART and leads to viral rebound once the treatment is stopped. To remove the reservoir and cure AIDS is the biggest goal. To achieve this goal, Otsuka group have recently developed a compound inositol hexaphosphate (IP6) named L-HIPPO targeted to suppress membrane localization of Gag and induce apoptosis of the host cell containing the un-budded viruses. The L-HIPPO was designed based on the fact that the MA domain of Gag mediates membrane binding through its interaction with inositol phospholipid PIP2 in the membrane.The main structural component of HIV-1 is the Gag polyprotein, which has five domains. By that, there is no structure of Gag that includes all of its five domains. We aim to first use home source XRD then use time-resolved Serial Femtosecond X-ray crystallography (SFX) to understand dynamic structure of HIV-1 Gag itselfs and with IP6 /L-HIPPO (or PIP2) complexes.
Azobenzene photoisomerization can be chemically implemented in protein ligands to actuate on biological receptors and to manipulate their activity with light. Azobenzene small molecule photoswitches can be designed and synthesized to serve for real-time regulation of receptors with high spatiotemporal accuracy using specific illumination patterns. The basis for this is a different interaction mode of the ligand isomers with the biological receptor. Therefore, light can be used for a precise control of physiology, on/off drug activation and targeting localized organs in free behaving animals. Strikingly, the photomolecular isomerization can also be employed in structural studies. Photoswitchable ligands co-crystallized with biomolecules can be used for triggering molecular actions in the crystal upon illumination. The bound ligand can be very fast photoisomerized, sharply generating a new state, which induces a receptor rearrangement that can be experimentally measured. This light switch in the crystallized receptor, which is reminiscent of some photon activated endogenous receptor systems, opens unprecedented possibilities to measure structural changes at atomic resolution and at very short-time scale. This approach will involve cooperative work of chemists, biologists, physicists and engineers, and may open a new perspective in dynamic studies of biological processes that can change our understanding of life and applied to invent radically new therapeutic approaches.
Photopharmacology offers a powerful approach to alter ligand affinity and biological activity of small molecule drugs using light as a trigger. However, understanding the molecular mechanisms underlying this process has been challenging due to the inability of conventional structural biology to resolve the relevant transitions. In this presentation, I will outline how we employed time-resolved serial crystallography at the Swiss Light Source (SLS) and the Swiss X-ray Free Electron Laser (SwissFEL) to capture the release of azo-combretastatin A4, an anti-cancer compound, and the resulting conformational changes in tubulin. We obtained a series of structural snapshots logarithmically spaced in time from 100 fs to 100 ms, which, along with computer simulations and time-resolved spectroscopy, provide direct molecular insight into how the cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of microtubule-destabilizing drugs.
 M. Wranik et al., Watching the release of a photopharmacological drug from tubulin using time-resolved serial crystallography. Nat Commun 14, 903 (2023).
We are studying drug discovery targeting membrane proteins. In particular, GPCRs are our main targets, and we are investigating the complex structures of receptors and compounds to elucidate action mechanisms of these compounds and lead to novel drug discovery.
In the first part of my talk, we will discuss the X-ray structure of the complex of orexin 2 receptor and a dual orexin receptor antagonist lemborexant. We will discuss how both high affinity and fast koff are achieved for the compound.
In the second part of this talk, we will discuss recent progress in our time-resolved crystallography at SACLA. Dynamic structures of drug targets have the potential to facilitate new drug discovery that is difficult to achieve by studying static 3D structures. In this talk, we will focus on the dynamic structures of microbial rhodopsins, which are related to GPCRs. We would like to present dynamic structures of a chloride-pump rhodopsin and our recent instrumental developments at SACLA.
Around 50% of the current antibiotic arsenal targets the ribosome, thus resistance to ribosome-targeting antibiotics poses severe challenges to antimicrobial treatments. Here, we characterize a 12-nucleotide deletion in the rplF gene encoding the uL6 ribosomal protein, which was identified in a tobramycin-resistant strain of Pseudomonas aeruginosa isolated from a cystic fibrosis patient. To understand this resistance, we determined multiple structures of wild-type and mutant ribosomes characterizing their conformational landscape. Our analysis reveals how detuning of the ribosome dynamics alters spontaneous rotational movement circumventing inhibition. The mutation compromises the 50S assembly, triggering structural instability and inducing a different rotational dynamic of the 70S. We found 3 new binding sites of tobramycin, and a fourth one exclusive of the mutant, which acts as an allosteric activator to skip inhibition. Our data also illustrate why the mutation enhances sensitivity to chloramphenicol, providing evidence of a molecular mechanism leading to collateral sensitivity. Our work reveals the complex scenario faced to combat ribosome antibiotic resistance, as minor changes generated in regions far away from the antibiotic docking site can derail translation inhibition. This information may be used for the development of new antibiotics that target the effects of the mutations restoring the antimicrobial effect.
Synchrotron radiation and free electron laser facilities are by nature large-scale multidisciplinary research infrastructures, as exemplified by LEAPS partners which offer a comprehensive and unique portfolio of instruments, methods, and techniques, to support a wide and diverse range of applications, from physics, chemistry, engineering to biology and medicine.
In this context, the Life Sciences community, notably structural biology, has always played a central role in the development of such research infrastructures. As a matter of fact, the size of this community and its scientific productivity have been at the origin of many synchrotron and FEL projects. Following the outstanding achievements of the structural biology community, many other emerging techniques, notably X-ray imaging, are also becoming key tools to underpin a more integrative approach. More complex and challenging questions, aiming at linking structural information to biological function in a cellular context and ultimately to ecosystems, require multi-modal and multi-scale research strategies which fit well with new capabilities of modern photon science infrastructures.
However, as illustrated by the dramatic development of cryo-electron microscopy and the advent of new computational methods (e.g., Alphafold), the landscape is changing very rapidly and photon source facilities need to go beyond their initial remit of provider of infrastructure for data collection and to adapt in many aspects (instrument performance, access modes, support labs, service for non-expert users, …).
This presentation aims at laying the grounds for a general discussion on this upcoming necessary evolution in a rapidly changing context. Current trends, bottlenecks, and challenges, as well as new opportunities in the development of life sciences at synchrotron and free electron laser facilities in Europe, will be discussed and illustrated by examples.
The capabilities of light source user facilities continually expand. High brightness and high time resolution are two technical advances that are popular globally. In the U.S., investments are being made across many facilities, in these and other areas, to support the need for new imaging and dynamic studies. I will provide a view of the strategy behind, and implementation of, these advances.
Synchrotrons have a foundational imaging role in the bioscience area. Hundreds of laboratories across the US depend on synchrotrons for their research. An exemplary, outstanding and under-appreciated outcome from the past decade is the machine learning (ML) based protein structure prediction. Training the ML relied on the imaging of thousands of structures generated by a large crystallography community at synchrotrons. The synchrotron community can follow this strategy to provide unique large data sets for ML training and further major insights and breakthroughs in biology. As in the case of crystallography, no single lab can produce the diverse biological material that is needed to train ML and solve a general problem. Synchrotrons have well developed user programs and provide sufficient access to foster large communities. User communities in scattering, footprinting, and spectroscopy are particularly well positioned to generate such data sets and outcomes if properly supported. Further, synchrotrons are uniquely situated to combine information from these techniques and provide multi-modal and multi-scale information that together provides greater insight than the techniques applied individually. I’ll provide examples from the Advanced Light Source of efforts to create collection infrastructure at scales sufficient to make use of ML and examples of multi-scale imaging. Recent examples stem from cross-synchrotron and neutron studies that addressed COVID19.
The Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory has partnered with the U.S. National Institute of General Medical Sciences (NIGMS) to create the Center for Structural Dynamics in Biology (SDB). The center serves the biomedical research community by developing tools and technologies for studying protein dynamics at LCLS and the Stanford Synchrotron Radiation Lightsource (SSRL). Beyond developing technologies and methods for enabling cutting-edge bioscience experiments at LCLS, the Center aims to standardize bioscience experiments at LCLS and SSRL, together with other light sources worldwide.
The tools and technologies developed by the Center boost the synergy between LCLS and SSRL, expand the spectroscopic capabilities across beamlines, and push towards the automation of the structural biology beamtime at LCLS, where reasonable. The Center furthermore develops improved sample preparation, delivery, and activation tools for serial crystallography experiments. The biomedical research community is actively involved in the Center’s operations through collaboration (supporting nine Driving Biomedical Projects), user training, and outreach activities. The overall work of the Center will enable all scientists to use the capabilities of X-ray free electron lasers and synchrotron light sources for studying the structure and motion of life’s molecules.
Respiratory complexes located in the internal membranes of our mitochondria are true macromolecular batteries: they couple the flow of electrons through clusters of metals and cofactors with a transfer of protons to create a gradient that provides the energy necessary for the ATP production and therefore to the nourishment of essential life processes. The first complex in the respiratory chain, named Complex I (CI), is one of the largest membrane proteins, made up of 45 subunits. The processes of its assembly and its sophisticated regulation are still poorly understood, although it is known that their disruption leads to neurodegenerative diseases such as Alzheimer's.
While exploring the molecular basis for protein recognition in the Mitochondrial CI Assembly (MCIA) complex, using a combination of biochemical, biophysical and structural techniques, we discovered that the assembly of the MCIA complex juggles between two incompatible activities: fatty acid oxidation and CI assembly. Cryo-EM and crystal structures of the partners in complex and alone allowed us further understanding into how they switch from one function to another. Furthermore, our recent mitochondrial analyses in amyloidogenic cells provide insights into the relationship of CI assembly in neurological dysfunction, suggesting whether MCIA components could detect early AD pre-symptomatic stages.
Modern methods in Structural Biology aim to provide detailed insights into how proteins are able to catalyse and control chemical reactions. This requires understanding the role of the conformational dynamics of proteins in the regulation of enzyme catalysis, for example by allosteric phenomena. In recent years, time-resolved (TR) X-ray crystallography and structural enzymology have been revived by the advent of serial crystallography at advanced X-ray sources and by new methods for sample delivery, reaction initiation and data processing, making TR studies increasingly accessible. However, electron density maps resulting from TR studies typically correspond to mixtures of states: their interpretation thus presents a serious challenge, that we are addressing by supplementing TR structural data with ultra-high resolution data from macrocrystals of highly homogenous mechanistically trapped states. Routinely achieving ultra-high resolution requires optimised sample quality, top-hat beams of adjustable size and shape, high-energy data collection with CdTe detectors, and optimised data collection work- flows. In this talk we will present some of our recent work, highlighting what is now possible for Structural Enzymology using X-ray crystallography, yet currently inaccessible by cryo-EM and Artificial Intelligence methods.
Time-resolved X-ray crystallography allows time-dependent structural changes to be visualized as they evolve within protein crystals and can yield unique structural insights into the course of a biochemical reaction (1). Serial crystallography (2) is now routinely used for time-resolved X-ray diffraction studies of macromolecules at X-ray free electron laser and synchrotron radiation facilities (1). As the field grows, biological reactions that are not naturally light sensitive will be increasingly studied using time-resolved serial crystallography. In this work, a laser-flash was used to release photocaged oxygen and thereby initiate the reduction of oxygen to water in microcrystals of cytochrome c oxidase (3). Our structural results suggest how this reaction may be coupled to gating the uptake of protons from the cytoplasm and to the release of protons to the periplasm. Similar interdisciplinary experiments will allow other biological reactions to become accessible to time-resolved diffraction.
G. Branden, R. Neutze, Advances and challenges in time-resolved macromolecular crystallography. Science 373, eaba0954 (2021).
H. N. Chapman et al., Femtosecond X-ray protein nanocrystallography. Nature 470, 73 (2011).
R. Andersson et al., Serial femtosecond crystallography structure of cytochrome c oxidase at room temperature. Sci Rep 7, 4518 (2017).