Femtosecond x-ray diffraction from an aerosolized beam of protein nanocrystals

We demonstrate near-atomic-resolution Bragg diffraction from aerosolized single granulovirus crystals using an x-ray free-electron laser. The form of the aerosol injector is nearly identical to conventional liquid-microjet nozzles, but the x-ray-scattering background is reduced by several orders of magnitude by the use of helium carrier gas rather than liquid. This approach provides a route to study the weak diffuse or lattice-transform signal arising from small crystals. The high speed of the particles is particularly well suited to upcoming MHz-repetition-rate x-ray free-electron lasers

Development of Container Free Sample Exposure for Synchrotron X-ray Footprinting.

The method of X-ray footprinting and mass spectrometry (XFMS) on large protein assemblies and membrane protein samples requires high flux density to overcome the hydroxyl radical scavenging reactions produced by the buffer constituents and the total protein content. Previously, we successfully developed microsecond XFMS using microfluidic capillary flow and a microfocused broadband X-ray source at the Advanced Light Source synchrotron beamlines, but the excessive radiation damage incurred when using capillaries prevented the full usage of a high-flux density beam. Here we present another significant advance for the XFMS method: the instrumentation of a liquid injection jet to deliver container free samples to the X-ray beam. Our preliminary experiments with a liquid jet at a bending magnet X-ray beamline demonstrate the feasibility of the approach and show a significant improvement in the effective dose for both the Alexa fluorescence assay and protein samples compared to conventional capillary flow methods. The combination of precisely controlled high dose delivery, shorter exposure times, and elimination of radiation damage due to capillary effects significantly increases the signal quality of the hydroxyl radical modification products and the dose-response data. This new approach is the first application of container free sample handling for XFMS and opens up the method for even further advances, such as high-quality microsecond time-resolved XFMS studies

Visualizing aerosol-particle injection for diffractive-imaging experiments

Delivering sub-micrometer particles to an intense x-ray focus is a crucial aspect of single-particle diffractive-imaging experiments at x-ray free-electron lasers. Enabling direct visualization of sub-micrometer aerosol particle streams without interfering with the operation of the particle injector can greatly improve the overall efficiency of single-particle imaging experiments by reducing the amount of time and sample consumed during measurements. We have developed in-situ non-destructive imaging diagnostics to aid real-time particle injector optimization and x-ray/particle-beam alignment, based on laser illumination schemes and fast imaging detectors. Our diagnostics are constructed to provide a non-invasive rapid feedback on injector performance during measurements, and have been demonstrated during diffraction measurements at the FLASH free-electron laser.Comment: 15 page

Methods and Instrumentation of Sample Injection for XFEL Experiments

abstract: ABSTRACT X-Ray crystallography and NMR are two major ways of achieving atomic resolution of structure determination for macro biomolecules such as proteins. Recently, new developments of hard X-ray pulsed free electron laser XFEL opened up new possibilities to break the dilemma of radiation dose and spatial resolution in diffraction imaging by outrunning radiation damage with ultra high brightness femtosecond X-ray pulses, which is so short in time that the pulse terminates before atomic motion starts. A variety of experimental techniques for structure determination of macro biomolecules is now available including imaging of protein nanocrystals, single particles such as viruses, pump-probe experiments for time-resolved nanocrystallography, and snapshot wide- angle x-ray scattering (WAXS) from molecules in solution. However, due to the nature of the "diffract-then-destroy" process, each protein crystal would be destroyed once probed. Hence a new sample delivery system is required to replenish the target crystal at a high rate. In this dissertation, the sample delivery systems for the application of XFELs to biomolecular imaging will be discussed and the severe challenges related to the delivering of macroscopic protein crystal in a stable controllable way with minimum waste of sample and maximum hit rate will be tackled with several different development of injector designs and approaches. New developments of the sample delivery system such as liquid mixing jet also opens up new experimental methods which gives opportunities to study of the chemical dynamics in biomolecules in a molecular structural level. The design and characterization of the system will be discussed along with future possible developments and applications. Finally, LCP injector will be discussed which is critical for the success in various applications.Dissertation/ThesisDoctoral Dissertation Physics 201

Highly focused supersonic microjets

The paper describes the production of thin, focused microjets with velocities up to 850 m/s by the rapid vaporization of a small mass of liquid in an open liquid-filled capillary. The vaporization is caused by the absorption of a low-energy laser pulse. A likely explanation of the observed phenomenon is based on the impingement of the shock wave caused by the nearly-instantaneous vaporization on the free surface of the liquid. An experimental study of the dependence of the jet velocity on several parameters is conducted, and a semi-empirical relation for its prediction is developed. The coherence of the jets, their high velocity and good reproducibility and controllability are unique features of the system described. A possible application is to the development of needle-free drug injection systems which are of great importance for global health care.Comment: 10 pages, 11figure

Highly focused supersonic microjets

The paper describes the production of thin, focused microjets with velocities up to 850 m/s by the rapid vaporization of a small mass of liquid in an open liquid-filled capillary. The vaporization is caused by the absorption of a low-energy laser pulse. A likely explanation of the observed phenomenon is based on the impingement of the shock wave caused by the nearly-instantaneous vaporization on the free surface of the liquid. An experimental study of the dependence of the jet velocity on several parameters is conducted, and a semi-empirical relation for its prediction is developed. The coherence of the jets, their high velocity and good reproducibility and controllability are unique features of the system described. A possible application is to the development of needle-free drug injection systems which are of great importance for global health care.Comment: 10 pages, 11figure

Injection Methods and Instrumentation for Serial X-ray Free Electron Laser Experiments

abstract: Scientists have used X-rays to study biological molecules for nearly a century. Now with the X-ray free electron laser (XFEL), new methods have been developed to advance structural biology. These new methods include serial femtosecond crystallography, single particle imaging, solution scattering, and time resolved techniques. The XFEL is characterized by high intensity pulses, which are only about 50 femtoseconds in duration. The intensity allows for scattering from microscopic particles, while the short pulses offer a way to outrun radiation damage. XFELs are powerful enough to obliterate most samples in a single pulse. While this allows for a “diffract and destroy” methodology, it also requires instrumentation that can position microscopic particles into the X-ray beam (which may also be microscopic), continuously renew the sample after each pulse, and maintain sample viability during data collection. Typically these experiments have used liquid microjets to continuously renew sample. The high flow rate associated with liquid microjets requires large amounts of sample, most of which runs to waste between pulses. An injector designed to stream a viscous gel-like material called lipidic cubic phase (LCP) was developed to address this problem. LCP, commonly used as a growth medium for membrane protein crystals, lends itself to low flow rate jetting and so reduces the amount of sample wasted significantly. This work discusses sample delivery and injection for XFEL experiments. It reviews the liquid microjet method extensively, and presents the LCP injector as a novel device for serial crystallography, including detailed protocols for the LCP injector and anti-settler operation.Dissertation/ThesisDoctoral Dissertation Physics 201

Studying protein dynamics with X-ray free-electron lasers: Opportunities & Limitations

Protein structure and function are intimately connected. To deduce the mechanisms underlying specific functions, it is therefore of high interest to investigate structural changes during a reaction. Recently, the development of serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) has attracted a great deal of attention by enabling time-resolved (TR) experiments at atomic spatial and femtosecond temporal resolution, thereby allowing unprecedented insight into protein dynamics. The high intensity of the XFEL pulse destroys any sample that has been exposed to the focused beam. A new protein crystal thus needs to be supplied for each pulse. This is typically achieved using a continuously flowing jet. For light-triggered reactions, an optical pulse starts the reaction in crystals of photosensitive proteins and the X-ray pulse then interrogates the system after a given time interval. For such experiments there are two main issues: First, appropriate conditions have to be found for triggering the reaction of interest. Second, the measurement of weak signals is severely limited by the low data collection rate (≤ 120 Hz) at first-generation XFELs. Moreover high sample consumption is an issue at these X-ray sources. The goals of this thesis were therefore twofold: In the first part, techniques were developed to enable studying the ultrafast isomerization following photon absorption by bacteriorhodopsin in a TR-SFX experiment. Extending these results, light-matter interactions changing the incident excitation intensity were quantified based on experiments and calculations. This allowed establishing guidelines how to generally determine appropriate excitation conditions in SFX employing light triggering. These findings are fundamental to avoid multiphoton artefacts arising from excessive excitation and are thus essential for studying biological reactions which take place almost exclusively in the single photon regime. In the second part of this thesis, opportunities and challenges of SFX experiments at next-generation XFELs were explored. These new machines generate X-ray pulses at MHz peak repetition rate and promise significantly higher throughput and more efficient sample usage. However, the short spacing between pulses introduces new challenges: it needs to be ensured that fresh sample is supplied sufficiently fast for each X-ray pulse. Moreover, it has been shown that the XFEL pulse launches shock waves in the sample carrying jet. These may damage sample probed by subsequent pulses. Here, first experiments at MHz peak repetition rate were conducted to investigate both issues. It was demonstrated that data collection of undamaged sample is indeed possible at 1.1 MHz repetition rate. At shorter pulse intervals (corresponding to 4.5 and 9.2 MHz), shock wave induced damage may lead to a significant loss in diffraction resolution of the crystal and even to structural changes in the protein. Together, the results of this thesis delineate the limitations of (TR-) SFX due to XFEL induced shock damage and pave the way towards exploiting the promising capabilities of MHz XFELs, in particular for studying biologically relevant light-triggered reactions in proteins.Proteinstruktur und –funktion sind eng miteinander verbunden. Um die zugrundeliegenden Mechanismen aufzuklären, ist es daher von hohem Interesse, strukturelle Änderungen während einer Reaktion zu verfolgen. Die Entwicklung serieller Femtosekunden-Kristallographie (SFX) an Freie-Elektronen-Lasern im Röntgenbereich (XFEL) hat folglich durch die einmalige Kombination von atomarer räumlicher und Femtosekunden zeitlicher Auflösung viel Aufmerksamkeit erregt, da sie beispiellose Einblicke in die Struktur und Dynamik von Proteinen erlaubt. XFEL Pulse besitzen eine solch hohe Intensität, dass die Probe letztendlich zerstört und für jeden Puls ein neuer Proteinkristall benötigt wird. Ein Flüssigkeitsstrahl (Jet) liefert daher kontinuierlich frisches Material. Mit diesem Ansatz lassen sich auch lichtgesteuerte Reaktionen beobachten, indem ein optischer Puls die Reaktion in einem Kristall aus photosensitiven Proteinen startet, und der Röntgenpuls nach einer festgelegten Zeit das System abfragt. Bei dieser Herangehensweise gibt es zwei grundlegende Probleme: Erstens müssen geeignete Bedingungen zum Starten der Reaktion gefunden werden. Zweitens ist an XFELs der ersten Generation die Messung schwacher Signale durch die geringe Repetitionsrate (≤ 120 Hz) limitiert, die zudem zu einem hohen Probenverbrauch führt. Diese Arbeit hat daher zwei Ziele: Im ersten Teil wurden Methoden entwickelt, die die Grundlage für das Verfolgen der ultraschnellen lichtinduzierten Isomerisierung in Bacteriorhodopsin mittels SFX bildet. Anknüpfend daran wurden die Anregungsintensität ändernde Licht-Materie-Wechselwirkungen mithilfe von Experimenten und Berechnungen quantifiziert, sodass ein allgemeiner Leitfaden für die Bestimmung passender Anregungsbedingungen aufgestellt werden konnte. Dies ist ein entscheidender Schritt für das Vermeiden biologisch irrelevanter Multiphotonen-Effekte. Im zweiten Teil der Arbeit wurden die Chancen und Herausforderungen von SFX an neuen XFELs untersucht, die Röntgenpulse mit bis zu MHz Wiederholrate produzieren können und dadurch versprechen, Durchsatz und Probeneffizienz zu erhöhen. Durch die kurzen Pulsabstände entstehen jedoch neue Probleme: einerseits muss die Zufuhr neuer Kristalle in den Strahl schnell genug geschehen. Andererseits wurde gezeigt, dass der XFEL Puls im Jet Schockwellen auslöst, die die Probe schädigen und so die Messung mit schnell aufeinanderfolgenden Pulsen beeinträchtigen könnte. In dieser Arbeit wurden erste Experimente bei MHz Wiederholrate durchgeführt und beide Problematiken untersucht. Messungen bei 1.1 MHz konnten erfolgreich ohne Beeinträchtigung durchgeführt werden. Es wurde aber auch gezeigt, dass bei kürzeren Pulsintervallen (entsprechend 4.5 und 9.2 MHz) die Schockwelle die Probe schädigen kann und dadurch zu einer reduzierten Auflösung der Kristalle, sowie zu Strukturänderungen im Protein führen können. Die Ergebnisse dieser Arbeit sind wegweisend für das Ausschöpfen der vielversprechenden Möglichkeiten von MHz XFELs, insbesondere für das Beobachten biologisch relevanter, ultraschneller, lichtinduzierter Reaktionen in Proteinen

Sample Delivery Enabled by 3D Printing for Reduced Sample Consumption and Mix-and-Inject Serial Crystallography at X-ray Free Electron Lasers

abstract: Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has enabled the determination of damage-free protein structures at ambient temperatures and of reaction intermediate species with time resolution on the order of hundreds of femtoseconds. However, currently available XFEL facility X-ray pulse structures waste the majority of continuously injected crystal sample, requiring a large quantity (up to grams) of crystal sample to solve a protein structure. Furthermore, mix-and-inject serial crystallography (MISC) at XFEL facilities requires fast mixing for short (millisecond) reaction time points ("), and current sample delivery methods have complex fabrication and assembly requirements. To reduce sample consumption during SFX, a 3D printed T-junction for generating segmented aqueous-in-oil droplets was developed. The device surface properties were characterized both with and without a surface coating for improved droplet generation stability. Additionally, the droplet generation frequency was characterized. The 3D printed device interfaced with gas dynamic virtual nozzles (GDVNs) at the Linac Coherent Light Source (LCLS), and a relationship between the aqueous phase volume and the resulting crystal hit rate was developed. Furthermore, at the European XFEL (EuXFEL) a similar quantity and quality of diffraction data was collected for segmented sample delivery using ~60% less sample volume than continuous injection, and a structure of 3-deoxy-D-manno- octulosonate 8-phosphate synthase (KDO8PS) delivered by segmented injection was solved that revealed new structural details to a resolution of 2.8 Å. For MISC, a 3D printed hydrodynamic focusing mixer for fast mixing by diffusion was developed to automate device fabrication and simplify device assembly. The mixer was characterized with numerical models and fluorescence microscopy. A variety of devices were developed to reach reaction intermediate time points, ", on the order of 100 – 103 ms. These devices include 3D printed mixers coupled to glass or 3D printed GDVNs and two designs of mixers with GDVNs integrated into the one device. A 3D printed mixer coupled to a glass GDVN was utilized at LCLS to study the oxidation of cytochrome c oxidase (CcO), and a structure of the CcO Pr intermediate was determined at " = 8 s.Dissertation/ThesisSupplementary Video D.1 - Droplet formation in a 3D printed droplet generatorDoctoral Dissertation Chemistry 201

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals.

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme