ABSTRACT
Serial crystallography, born from groundbreaking experiments at the Linac Coherent Light Source in 2009, has evolved into a pivotal technique in structural biology. Initially pioneered at X-ray free-electron laser facilities, it has now expanded to synchrotron-radiation facilities globally, with dedicated experimental stations enhancing its accessibility. This review gives an overview of current developments in serial crystallography, emphasizing recent results in time-resolved crystallography, and discussing challenges and shortcomings.
Subject(s)
Synchrotrons , Crystallography, X-Ray/methods , Proteins/chemistry , Lasers , Models, Molecular , HumansABSTRACT
The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein's crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.
Subject(s)
Catalytic Domain , Coronavirus 3C Proteases , Cysteine , Disulfides , Oxidation-Reduction , SARS-CoV-2 , Disulfides/chemistry , Disulfides/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus 3C Proteases/chemistry , Cysteine/chemistry , Cysteine/metabolism , Crystallography, X-Ray , Humans , Models, Molecular , Protein Multimerization , COVID-19/virologyABSTRACT
The Lysinibacillus sphaericus proteins Tpp49Aa1 and Cry48Aa1 can together act as a toxin toward the mosquito Culex quinquefasciatus and have potential use in biocontrol. Given that proteins with sequence homology to the individual proteins can have activity alone against other insect species, the structure of Tpp49Aa1 was solved in order to understand this protein more fully and inform the design of improved biopesticides. Tpp49Aa1 is naturally expressed as a crystalline inclusion within the host bacterium, and MHz serial femtosecond crystallography using the novel nanofocus option at an X-ray free electron laser allowed rapid and high-quality data collection to determine the structure of Tpp49Aa1 at 1.62 Å resolution. This revealed the packing of Tpp49Aa1 within these natural nanocrystals as a homodimer with a large intermolecular interface. Complementary experiments conducted at varied pH also enabled investigation of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals-a crucial step in its mechanism of action. To better understand the cooperation between the two proteins, assays were performed on a range of different mosquito cell lines using both individual proteins and mixtures of the two. Finally, bioassays demonstrated Tpp49Aa1/Cry48Aa1 susceptibility of Anopheles stephensi, Aedes albopictus, and Culex tarsalis larvae-substantially increasing the potential use of this binary toxin in mosquito control.
Subject(s)
Bacillaceae , Bacillus , Culex , Pesticides , Animals , Bacillaceae/chemistry , Bacillaceae/metabolism , Mosquito Control , Larva/metabolismABSTRACT
Free-electron lasers (FEL) are revolutionizing X-ray-based structural biology methods. While protein crystallography is already routinely performed at FELs, Small Angle X-ray Scattering (SAXS) studies of biological macromolecules are not as prevalent. SAXS allows the study of the shape and overall structure of proteins and nucleic acids in solution, in a quasi-native environment. In solution, chemical and biophysical parameters that have an influence on the structure and dynamics of molecules can be varied and their effect on conformational changes can be monitored in time-resolved XFEL and SAXS experiments. We report here the collection of scattering form factors of proteins in solution using FEL X-rays. The form factors correspond to the scattering signal of the protein ensemble alone; the scattering contributions from the solvent and the instrument are separately measured and accurately subtracted. The experiment was done using a liquid jet for sample delivery. These results pave the way for time-resolved studies and measurements from dilute samples, capitalizing on the intense and short FEL X-ray pulses.
Subject(s)
Electrons , Proteins , Scattering, Small Angle , X-Rays , X-Ray Diffraction , Proteins/chemistry , LasersABSTRACT
With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its "off-time" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.
ABSTRACT
The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX.
Subject(s)
Electrons , Lasers , Crystallography, X-Ray , Radiography , X-RaysABSTRACT
Time-resolved studies of biomacromolecular crystals have been limited to systems involving only minute conformational changes within the same lattice. Ligand-induced changes greater than several angstroms, however, are likely to result in solid-solid phase transitions, which require a detailed understanding of the mechanistic interplay between conformational and lattice transitions. Here we report the synchronous behavior of the adenine riboswitch aptamer RNA in crystal during ligand-triggered isothermal phase transitions. Direct visualization using polarized video microscopy and atomic force microscopy shows that the RNA molecules undergo cooperative rearrangements that maintain lattice order, whose cell parameters change distinctly as a function of time. The bulk lattice order throughout the transition is further supported by time-resolved diffraction data from crystals using an X-ray free electron laser. The synchronous molecular rearrangements in crystal provide the physical basis for studying large conformational changes using time-resolved crystallography and micro/nanocrystals.
Subject(s)
Nucleic Acid Conformation , Phase Transition , RNA/chemistry , Riboswitch , Adenine/chemistry , Aptamers, Nucleotide/chemistry , Crystallography, X-Ray , Microscopy, Atomic Force/methods , Microscopy, Polarization/methods , Models, Molecular , Time-Lapse Imaging/methodsABSTRACT
Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.
Subject(s)
Crystallography/instrumentation , Electrons , Lab-On-A-Chip Devices , Lasers , Aldehyde-Lyases/ultrastructure , Escherichia coli Proteins/ultrastructure , HydrodynamicsABSTRACT
Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.
Subject(s)
Crystallography/methods , Proteins/chemistry , Microscopy, Electron, Scanning Transmission , Models, Molecular , Muramidase/chemistry , Muramidase/ultrastructure , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Occlusion Body Matrix Proteins/chemistry , Occlusion Body Matrix Proteins/ultrastructure , Particle Size , Protein Conformation , Proteins/ultrastructureABSTRACT
The new European X-ray Free-Electron Laser (European XFEL) is the first X-ray free-electron laser capable of delivering intense X-ray pulses with a megahertz interpulse spacing in a wavelength range suitable for atomic resolution structure determination. An outstanding but crucial question is whether the use of a pulse repetition rate nearly four orders of magnitude higher than previously possible results in unwanted structural changes due to either radiation damage or systematic effects on data quality. Here, separate structures from the first and subsequent pulses in the European XFEL pulse train were determined, showing that there is essentially no difference between structures determined from different pulses under currently available operating conditions at the European XFEL.
ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2 nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4 nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature.
Subject(s)
Electrons , Lasers , Microtubules/ultrastructure , Molecular Imaging/methods , Tubulin/ultrastructure , Algorithms , Crystallography, X-Ray/instrumentation , Crystallography, X-Ray/methods , Image Processing, Computer-Assisted , Molecular Imaging/instrumentation , Scattering, Radiation , Synchrotrons , X-RaysABSTRACT
X-ray free electron lasers (XFELs) provide ultrashort intense X-ray pulses suitable to probe electron dynamics but can also induce a multitude of nonlinear excitation processes. These affect spectroscopic measurements and interpretation, particularly for upcoming brighter XFELs. Here we identify and discuss the limits to observing classical spectroscopy, where only one photon is absorbed per atom for a Mn2+ in a light element (O, C, H) environment. X-ray emission spectroscopy (XES) with different incident photon energies, pulse intensities, and pulse durations is presented. A rate equation model based on sequential ionization and relaxation events is used to calculate populations of multiply ionized states during a single pulse and to explain the observed X-ray induced spectral lines shifts. This model provides easy estimation of spectral shifts, which is essential for experimental designs at XFELs and illustrates that shorter X-ray pulses will not overcome sequential ionization but can reduce electron cascade effects.
ABSTRACT
Better injectors resulting from careful iterative optimization used at high repetition XFELs in combination with better detectors and further developed algorithms might, in the not so distant future, result in a 'resolution revolution' in SPI, enabling the molecular and atomic imaging of the dynamics of biological macromolecules without the need to freeze or crystallize the sample.
ABSTRACT
The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a ß-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.
ABSTRACT
Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers (FELs) for serial femtosecond crystallography measurements. The high X-ray intensity in the focus initiates an explosion of the microjet and sample. With the advent of X-ray FELs with megahertz rates, the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse. This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using 4.3â nm radiation. The operation of gas-dynamic nozzles that produce liquid microjets with velocities greater than 80â mâ s-1 was demonstrated. Furthermore, this article provides optical images of X-ray-induced explosions together with Bragg diffraction from protein microcrystals exposed to trains of X-ray pulses repeating at rates of up to 4.5â MHz. The results indicate the feasibility for megahertz serial crystallography measurements with hard X-rays and give guidance for the design of such experiments.
ABSTRACT
High-resolution Bragg diffraction from aerosolized single granulovirus nanocrystals using an X-ray free-electron laser is demonstrated. The outer dimensions of the in-vacuum aerosol injector components are identical to conventional liquid-microjet nozzles used in serial diffraction experiments, which allows the injector to be utilized with standard mountings. As compared with liquid-jet injection, the X-ray scattering background is reduced by several orders of magnitude by the use of helium carrier gas rather than liquid. Such reduction is required for diffraction measurements of small macromolecular nanocrystals and single particles. High particle speeds are achieved, making the approach suitable for use at upcoming high-repetition-rate facilities.
ABSTRACT
The success of diffraction experiments from weakly scattering samples strongly depends on achieving an optimal signal-to-noise ratio. This is particularly important in single-particle imaging experiments where diffraction signals are typically very weak and the experiments are often accompanied by significant background scattering. A simple way to tremendously reduce background scattering by placing an aperture downstream of the sample has been developed and its application in a single-particle X-ray imaging experiment at FLASH is demonstrated. Using the concept of a post-sample aperture it was possible to reduce the background scattering levels by two orders of magnitude.
ABSTRACT
Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.
ABSTRACT
We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an agonist ergotamine, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2, the human smoothened receptor in complex with an antagonist cyclopamine, and finally the human angiotensin II type 1 receptor in complex with the selective antagonist ZD7155. All four datasets have been deposited, with minimal processing, in an HDF5-based file format, which can be used directly for crystallographic processing with CrystFEL or other software. We have provided processing scripts and supporting files for recent versions of CrystFEL, which can be used to validate the data.