Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we ...report atomic-resolution crystal structures of Ca(2+)- and Mg(2+)-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca(2+)-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca(2+)-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca(2+)-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca(2+) triggering, moves en bloc as Ca(2+) influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. ...Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.
Significance The extremely short and bright X-ray pulses produced by X-ray free-electron lasers unlock new opportunities in crystallography-based structural biology research. Efficient methods to ...deliver crystalline material are necessary due to damage or destruction of the crystal by the X-ray pulse. Crystals for the first experiments were 5 µm or smaller in size, delivered by a liquid injector. We describe a highly automated goniometer-based approach, compatible with crystals of larger and varied sizes, and accessible at cryogenic or ambient temperatures. These methods, coupled with improvements in data-processing algorithms, have resulted in high-resolution structures, unadulterated by the effects of radiation exposure, from only 100 to 1,000 diffraction images.
The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-áÅ resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β ₂- adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.
X‐ray free‐electron lasers (XFELs) have the ability to produce ultra‐bright femtosecond X‐ray pulses for coherent diffraction imaging of biomolecules. While the development of methods and algorithms ...for macromolecular crystallography is now mature, XFEL experiments involving aerosolized or solvated biomolecular samples offer new challenges in terms of both experimental design and data processing. Skopi is a simulation package that can generate single‐hit diffraction images for reconstruction algorithms, multi‐hit diffraction images of aggregated particles for training machine learning classifiers using labeled data, diffraction images of randomly distributed particles for fluctuation X‐ray scattering algorithms, and diffraction images of reference and target particles for holographic reconstruction algorithms. Skopi is a resource to aid feasibility studies and advance the development of algorithms for noncrystalline experiments at XFEL facilities.
The Skopi software package provides tools to generate realistic simulations of coherent X‐ray diffractive imaging of noncrystalline biological samples, which in turn will aid algorithm development for a range of experiments at X‐ray free‐electron laser sources.
X‐ray free‐electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, ...more efficient sample‐delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre‐existing slurry grown off‐chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash‐cooling. Proof‐of‐principle tests with a model system (hen egg‐white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single‐crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.
X-ray free electron lasers (XFELs) reduce the effects of radiation damage on macromolecular diffraction data and thereby extend the limiting resolution. Previously, we adapted classical ...post-refinement techniques to XFEL diffraction data to produce accurate diffraction data sets from a limited number of diffraction images (Uervirojnangkoorn et al., 2015), and went on to use these techniques to obtain a complete data set from crystals of the synaptotagmin-1 / SNARE complex and to determine the structure at 3.5 Å resolution (Zhou et al., 2015). Here, we describe new advances in our methods and present a reprocessed XFEL data set of the synaptotagmin-1 / SNARE complex. The reprocessing produced small improvements in electron density maps and the refined atomic model. The maps also contained more information than those of a lower resolution (4.1 Å) synchrotron data set. Processing a set of simulated XFEL diffraction images revealed that our methods yield accurate data and atomic models.
The crystal structure of the trans-acyltransferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for the direct ...delivery of the sample into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes for polyketide synthase reaction dynamics are discussed.
Massive Scale Data Analytics at LCLS-II Thayer, Jana; Chen, Zhantao; Claus, Richard ...
EPJ Web of Conferences,
2024, Letnik:
295
Journal Article, Conference Proceeding
Recenzirano
Odprti dostop
The increasing volumes of data produced at light sources such as the Linac Coherent Light Source (LCLS) enable the direct observation of materials and molecular assemblies at the length and ...timescales of molecular and atomic motion. This exponential increase in the scale and speed of data production is prohibitive to traditional analysis workflows that rely on scientists tuning parameters during live experiments to adapt data collection and analysis. User facilities will increasingly rely on the automated delivery of actionable information in real time for rapid experiment adaptation which presents a considerable challenge for data acquisition, data processing, data management, and workflow orchestration. In addition, the desire from researchers to accelerate science requires rapid analysis, dynamic integration of experiment and theory, the ability to visualize results in near real-time, and the introduction of ML and AI techniques. We present the LCLS-II Data System architecture which is designed to address these challenges via an adaptable data reduction pipeline (DRP) to reduce data volume on-thefly, online monitoring analysis software for real-time data visualization and experiment feedback, and the ability to scale to computing needs by utilizing local and remote compute resources, such as the ASCR Leadership Class Facilities, to enable quasi-real-time data analysis in minutes. We discuss the overall challenges facing LCLS, our ongoing work to develop a system responsive to these challenges, and our vision for future developments.
Microcrystals present a significant impediment to the determination of macromolecular structures by X‐ray diffraction methods. Although microfocus synchrotron beamlines and X‐ray free‐electron lasers ...(XFELs) can enable the collection of interpretable diffraction data from microcrystals, there is a need for efficient methods of harvesting small volumes (<2 µl) of microcrystals grown under common laboratory formats and delivering them to an X‐ray beam source under native growth conditions. One approach that shows promise in overcoming the challenges intrinsic to microcrystal analysis is to pair so‐called `fixed‐target' sample‐delivery devices with microbeam‐based X‐ray diffraction methods. However, to record weak diffraction patterns it is necessary to fabricate devices from X‐ray‐transparent materials that minimize background scattering. Presented here is the design of a new micro‐diffraction device consisting of three layers fabricated from silicon nitride, photoresist and polyimide film. The chip features low X‐ray scattering and X‐ray absorption properties, and uses a customizable blend of hydrophobic and hydrophilic surface patterns to help localize microcrystals to defined regions. Microcrystals in their native growth conditions can be loaded into the chips with a standard pipette, allowing data collection at room temperature. Diffraction data collected from hen egg‐white lysozyme microcrystals (10–15 µm) loaded into the chips yielded a complete, high‐resolution (<1.6 Å) data set sufficient to determine a high‐quality structure by molecular replacement. The features of the chip allow the rapid and user‐friendly analysis of microcrystals grown under virtually any laboratory format at microfocus synchrotron beamlines and XFELs.
Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme ...cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.
PDF
