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The timely volume describes recent discoveries and method developments that have revolutionized Structural Biology with the advent of X-ray Free Electron Lasers. It provides, for the first time, a comprehensive examination of this cutting-edge technology. It discusses of-the-moment topics such as growth and detection of nanocrystals, Sample Delivery Techniques for serial femtosecond crystallography, data collection methods at XFELs, and more. This book aims to provide the readers with an overview of the new methods that have been recently developed as well as a prospective on new methods under development. It highlights the most important and novel Structural Discoveries made recently with XFELS, contextualized with a big-picture discussion of future developments.



Chapter 1. X-Ray Free Electron Lasers and Their Applications

X-ray free electron lasers (FELs) represent the latest generation of X-ray sources, with unique properties and capabilities that present novel opportunities in the study of matter in unique forms as well as the study of interactions and dynamics on ultrafast timescales. For the purpose of this book focused on the use of X-ray FEL beams for the study of biological materials, the story begins with the availability of these novel sources to the scientific community as user facilities. Let us however take a quick step back and provide a brief historical background on what has led to the advent of X-ray FEL sources. This will be followed by a short description of the principles of operation of X-ray FELs and the breadth of their scientific use.
Sébastien Boutet, Makina Yabashi

Chapter 2. Serial Femtosecond Crystallography (SFX): An Overview

X-ray crystallography is a very powerful tool to determine structures of biological macromolecules at the atomic and molecular level and thereby enables insights into their function. Unfortunately, irradiating biological samples with X-rays is an inherently damaging and unavoidable process in conventional X-ray crystallography. The unique properties of X-ray free electron lasers (FELs), with pulse duration in the femtosecond range, allow data collection at time scales shorter than, or equivalent to, the time scales of the X-ray induced radiation damage pathways, offering a plausible way to diminish the ill effects of conventional radiation damage in biological structure determination. However, the high intensities of the X-ray FEL beam necessitated the development of novel techniques for sample preparation, characterization, introduction, data collection, and analysis. Serial femtosecond crystallography (SFX) represents a set of techniques developed to enable X-ray crystallography experiments at X-ray FELs, which encompasses multiple developments in sample introduction and data collection. This chapter summarizes the early experiments that demonstrated the SFX methods along with more recent developments and accomplishments that will be discussed in more detail in the following chapters of this book.
Mark S. Hunter, Petra Fromme

Chapter 3. Small Is Beautiful: Growth and Detection of Nanocrystals

With the advent of X-Ray free electron lasers (FELs), the field of serial femtosecond crystallography (SFX) was borne, allowing a stream of nanocrystals to be measured individually and diffraction data to be collected and merged to form a complete crystallographic data set. This allows submicron to micron crystals to be utilized in an experiment when they were once, at best, only an intermediate result towards larger, usable crystals. SFX and its variants have opened new possibilities in structural biology, including studies with increased temporal resolution, extending to systems with irreversible reactions, and minimizing artifacts related to local radiation damage. Perhaps the most profound aspect of this newly established field is that “molecular movies,” in which the dynamics and kinetics of biomolecules are studied as a function of time, are now an attainable commodity for a broad variety of systems, as discussed in Chaps. 11 and 12. However, one of the historic challenges in crystallography has always been crystallogenesis and this is no exception when preparing samples for serial crystallography methods. In the following chapter, we focus on some of the specific characteristics and considerations inherent in preparing a suitable sample for successful serial crystallographic approaches.
Jesse Coe, Alexandra Ros

Chapter 4. The Lipid Cubic Phase as a Medium for the Growth of Membrane Protein Microcrystals

The lipid-based bicontinuous cubic mesophase is a nanoporous membrane mimetic with applications in areas that include medicine, personal care products, foods, and the basic sciences. An application of particular note is as a medium in which to grow crystals of membrane proteins for structure determination by X-ray crystallography. At least two variations of the mesophase exist. One is the highly viscous cubic phase, known as the lipid cubic phase (LCP), which has well-developed long-range order. The other, the so-called sponge phase, is considerably more fluid and lacks long-range order. Both phase types have been shown to be amenable for growing microcrystals of membrane proteins and for use as a delivery medium to shuttle protein crystals to an X-ray free-electron laser beam for serial femtosecond crystallography. Here, we provide background on the physicochemical properties of these mesophases and how they function to grow microcrystals of membrane proteins. Protocols implemented for the generation and use of nanoliter volumes of mesophase of suitably high microcrystal density required for serial femtosecond crystallography are described. Prospects for future uses of lipid mesophases in the serial femtosecond crystallography arena are summarized.
Zina Al-Sahouri, Ming-Yue Lee, Dianfan Li, Wei Liu, Martin Caffrey

Chapter 5. Sample Delivery Techniques for Serial Crystallography

In serial femtosecond crystallography (SFX), protein microcrystals and nanocrystals are introduced into the focus of an X-ray free electron laser (FEL) beam ideally one-by-one in a serial fashion. The high photon density in each pulse is the double-edged sword that necessitates the serial nature of the experiments. The high photon count focused spatially and temporally leads to a diffraction-before-destruction snapshot, but this single snapshot is not enough for a high-resolution three-dimensional structural reconstruction. To recover the structure, more snapshots are required to sample all of reciprocal space from randomly oriented crystal diffraction, and in practice, some redundancy is necessary in these measurements. This chapter explores the different sample delivery techniques developed over the years to help enable serial crystallography experiments.
Raymond G. Sierra, Uwe Weierstall, Dominik Oberthuer, Michihiro Sugahara, Eriko Nango, So Iwata, Alke Meents

Chapter 6. When Diffraction Stops and Destruction Begins

It is now possible to solve protein structures with femtosecond X-ray free-electron laser (XFEL) pulses that were previously inaccessible to continuous synchrotron sources due to radiation damage. The key to this success is that diffraction probes the protein structure on femtosecond timescales, whereas nuclear motion takes tens to hundreds of femtoseconds to have a significant effect on the crystal structure. This is the essential idea behind the diffraction-before-destruction principle that underlies serial femtosecond crystallography (SFX) with XFELs. In practice, the principle works well enough to determine protein structures of comparable resolution to synchrotron protein crystallography, which has led to the many successes of XFEL crystallography to date.
Carl Caleman, Andrew V. Martin

Chapter 7. Climbing the Data Mountain: Processing of SFX Data

Serial femtosecond crystallography experiments produce mountains of data that require FEL facilities to provide many petabytes of storage space and large compute clusters for timely processing of user data. The route to reach the summit of the data mountain requires peak finding, indexing, integration, refinement, and phasing. Those who reach the summit get a crystal clear view of the “radiation damage-free” structure of a protein that is most consistent with the observed measurements. Data processing plays a critical role in the ability to measure accurate structure factor intensities from individual diffraction snapshots and combine them in three-dimensional space. Current developments in SFX aim to take into account the huge complexity of SFX experiments, modeling variations in the beam and crystals, uncertainties in geometry, partiality, mosaicity, and figures of merit that are unique to SFX.
Chun Hong Yoon, Thomas A. White

Chapter 8. Phasing Serial Crystallography Data

The development of serial femtosecond crystallography (SFX) at X-ray free electron lasers (X-ray FELs) allows for the use of tiny protein crystals down to just a few unit cells along an edge, measured at physiological temperatures, and with a time resolution far better than can be achieved with synchrotrons or electron microscopes. The unique properties of the X-ray FEL source has furthermore resulted in the appearance of entirely new ideas for solving the crystallographic phase problem. At the same time, in combination with work on phasing single-particle data (with one bioparticle per shot), SFX has stimulated research into new phasing methods for serial crystallography (SC) at synchrotrons, and protein crystallography in general. In the sense that these new phasing methods depend on the application of constraints, they might be considered developments of traditional “direct methods” such as density modification approaches.
Richard A. Kirian, Joe P. J. Chen, John C. H. Spence

Chapter 9. Structure Determination by Continuous Diffraction from Imperfect Crystals

The coherent diffraction pattern of a non-periodic finite object does not consist of Bragg peaks but is continuously and smoothly varying. Such patterns do not suffer from the well-known phase problem of crystallography. In this case, robust iterative algorithms exist to determine the electron density of the object from the diffraction pattern alone. Continuous diffraction is accessible from ensembles of aligned molecules, including disordered protein crystals. We discuss the application of the concepts of coherent diffractive imaging to such cases and describe the experimental considerations to adequately measure the weak continuous diffraction signals.
Kartik Ayyer, Oleksandr M. Yefanov, Henry N. Chapman

Chapter 10. Advances in Structure Determination of G Protein-Coupled Receptors by SFX

G protein-coupled receptors (GPCRs) constitute the largest superfamily of membrane proteins, members of which are involved in regulation of critical sensory and physiological processes in the human body. High-resolution GPCR structures are essential for the elucidation of the molecular mechanisms of signal transduction, and for the rational design of more effective therapeutics. GPCR structure determination is, however, hampered by challenges in their expression, stabilization, and crystallization. The recent emergence of X-ray free electron lasers (FELs), and establishment of serial femtosecond crystallography (SFX) have advanced the field of structural biology by enabling access to high-resolution structure and dynamics of challenging to crystallize and radiation damage-sensitive macromolecules. In this chapter we outline relevant SFX technology developments and its applications to structural studies of GPCRs, shedding light on ligand binding to antitumor and anti-addiction targets, uncovering molecular mechanisms behind distinct functions of angiotensin receptors, elucidating full-length structures of multidomain class B and Frizzled receptors, and revealing details of interactions between GPCRs and arrestins.
Benjamin Stauch, Linda Johansson, Andrii Ishchenko, Gye Won Han, Alexander Batyuk, Vadim Cherezov

Chapter 11. Time-Resolved Serial Femtosecond Crystallography, Towards Molecular Movies of Biomolecules in Action

Biological macromolecules, such as proteins, nucleic acids, and complexes thereof, are characterized by specific structural and dynamic features that are the basis of their respective biological activity, and define their dynamic personalities [29]. Understanding macromolecular activity thus requires studying structural changes over time and on various time-scales, such as equilibrium fluctuations and conformational changes orchestrating enzyme catalysis or enabling signal transduction. The first step in human vision, for instance, is the sub-picosecond time-scale photoisomerization of the retinal pigment in rhodopsin [73], which within microseconds leads to the conformational changes required for activation of transducin, the regulatory protein that initiates the signaling cascade beyond the macromolecular level.
Jacques-Philippe Colletier, Giorgio Schirò, Martin Weik

Chapter 12. Towards Molecular Movies of Enzymes

Macromolecular crystallography has been highly successful in the past 60 years as it has been the predominant method to solve macromolecular structures, with more than 100,000 protein structures determined and posted to structural databases [www.​rcsb.​org, (Berman et al., Nucleic Acids Res 28:235–242, 2000)]. Crystallography methods are capable of determining structures at high resolution (<1.5 Å) as demonstrated by the many structures available at this or better resolution. A central objective of structural biology is not only to solve static structures but to also observe their associated dynamics to infer and explore their functions. To examine reactions that occur in biological macromolecules, time-resolved methods are required. In time-resolved crystallography, a reaction is triggered inside a crystal and the progress of this reaction is then probed by short but highly intense X-ray pulses, shorter than both the dynamics studied and the reaction trigger. Time-resolved crystallographic experiments have been successfully carried out at synchrotron X-ray sources (Moffat, Annu Rev Biophys Biophys Chem 18:309–332, 1989; Moffat, Chem Rev 101:1569–1581, 2001; Schmidt, Synchrotron Radiat News 28:25–30, 2015). Mainly cyclic reversible, and light-activated reactions were examined. Irreversible (single path) reactions, for example those catalyzed by enzymes, remain difficult to investigate. The initiation of a reaction by adding a substrate or ligand to protein crystals remains a challenge, which prevents routine applications. The arrival of X-ray free electron lasers and micro-focus synchrotron beamlines, with their intense X-ray pulses, permit the use of significantly smaller crystals. With small crystals faster diffusion times are achieved which allow for straightforward investigations of these reactions. Several successful experiments have already been reported which show how the structures of transiently occupied intermediates and their dynamics can be investigated at room temperature in real time. In this chapter we will discuss the experimental setup, feasibility, and potential impact of the new facilities on the field of enzymology.
Christopher Kupitz, Marius Schmidt

Chapter 13. X-Ray Spectroscopy with XFELs

The use of XFELs in the last 10 years has produced multiple opportunities to undertake scientific questions that could not be addressed using other types of X-ray sources, in particular for femtosecond and picosecond processes and for radiation sensitive and scarce biological samples. X-ray spectroscopy, the experimental approach used in many of these studies, is well established and has been broadly used at synchrotron radiation sources worldwide for the last few decades. However to take advantage of spectroscopic tools at XFELs, synchrotron-based methods have to be adapted to the unique characteristics of the XFEL beam, like its pulsed nature and time structure, as well as the effects induced in the sample derived from these properties. In the few short years of XFEL operations, various studies relied on spectroscopic methods, both in the hard and in the soft X-ray regime. In this chapter, we will provide an inclusive review of recent XFEL spectroscopic studies on biological samples and focus on the description of the experimental aspects of such measurements. We will include a discussion on spectroscopy technique developments that are unique to XFELs with the potential to make an impact on the field.
Roberto Alonso-Mori, Junko Yano

Chapter 14. Single Molecule Imaging Using X-ray Free Electron Lasers

The potential to image single molecules in action with a resolution sufficiently high to reveal atomic information at room temperature without the need for crystallization is one of the most exciting applications of X-ray free electron lasers. Significant progress has been made towards this goal over the past years. Here we discuss the current status and describe the steps still required to realize atomic resolution X-ray single particle imaging.
Andrew Aquila, Anton Barty

Chapter 15. The Use of Angular Correlations to Extract Three-Dimensional Structural Information from X-Ray Solution Scattering

In this chapter we focus on scattering from non-crystalline solutions of molecules or nanoparticles in which the scattering objects are rotationally disordered.
Sebastian Doniach

Chapter 16. Future Directions of High Repetition Rate X-Ray Free Electron Lasers

A new scientific frontier opened in 2009 when the world’s first X-ray free electron laser (FEL), the Linac Coherent Light Source (LCLS) facility, began operations at SLAC National Accelerator Laboratory. The scientific start of LCLS has arguably been one of the most vigorous and successful of any new research facility, with a dramatic effect on a broad cross section of scientific fields, ranging from atomic and molecular science, ultrafast chemistry and catalysis, fluid dynamics, clean energy systems, structural biology, high energy-density science, photon science, and advanced materials [1].
Mike Dunne, Robert W. Schoenlein


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