Time-Resolved Macromolecular Crystallography at Pulsed X-ray Sources

Ultrabright pulses produced in X-ray free-electron lasers (XFELs) offer new possibilities for industry and research, particularly for biochemistry and pharmaceuticals. The unprecedented brilliance of these next-generation sources enables structure determination from sub-micron crystals as well as radiation-sensitive proteins. The European X-Ray Free-Electron Laser (EuXFEL), with its first light in 2017, ushered in a new era for ultrabright X-ray sources by providing an unparalleled megahertz-pulse repetition rate, with orders of magnitude more pulses per second than previous XFEL sources. This rapid pulse frequency has significant implications for structure determination; not only will data collection be faster (resulting in more structures per unit time), but experiments requiring large quantities of data, such as time-resolved structures, become feasible in a reasonable amount of experimental time. Early experiments at the SPB/SFX instrument of the EuXFEL demonstrate how such closely-spaced pulses can be successfully implemented in otherwise challenging experiments, such as time-resolved studies.

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Pump-Probe Time-Resolved Serial Femtosecond Crystallography at X-Ray Free Electron Lasers

Crystals ◽

10.3390/cryst10070628 ◽

2020 ◽

Vol 10 (7) ◽

pp. 628

Author(s):

Suraj Pandey ◽

Ishwor Poudyal ◽

Tek Narsingh Malla

Keyword(s):

Free Electron ◽

Reaction Dynamics ◽

Reaction Coordinate ◽

Biological Macromolecules ◽

Free Electron Lasers ◽

Time Resolved ◽

Pump Probe ◽

X Ray ◽

Recent Developments ◽

Serial Femtosecond Crystallography

With time-resolved crystallography (TRX), it is possible to follow the reaction dynamics in biological macromolecules by investigating the structure of transient states along the reaction coordinate. X-ray free electron lasers (XFELs) have enabled TRX experiments on previously uncharted femtosecond timescales. Here, we review the recent developments, opportunities, and challenges of pump-probe TRX at XFELs.

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Direct protein crystallization on ultrathin membranes for diffraction measurements at X-ray free-electron lasers

Journal of Applied Crystallography ◽

10.1107/s1600576717005799 ◽

2017 ◽

Vol 50 (3) ◽

pp. 909-918 ◽

Cited By ~ 12

Author(s):

Nadia Opara ◽

Isabelle Martiel ◽

Stefan A. Arnold ◽

Thomas Braun ◽

Henning Stahlberg ◽

...

Keyword(s):

Free Electron ◽

Protein Crystallization ◽

Protein Crystallography ◽

Liquid Jets ◽

X Rays ◽

Free Electron Lasers ◽

Time Resolved ◽

X Ray ◽

Drop On Demand ◽

Novel Approach

A new era of protein crystallography started when X-ray free-electron lasers (XFELs) came into operation, as these provide an intense source of X-rays that facilitates data collection in the `diffract-before-destroy' regime. In typical experiments, crystals sequentially delivered to the beam are exposed to X-rays and destroyed. Therefore, the novel approach of serial crystallography requires thousands of nearly identical samples. Currently applied sample-delivery methods, in particular liquid jets or drop-on-demand systems, suffer from significant sample consumption of the precious crystalline material. Direct protein microcrystal growth by the vapour diffusion technique inside arrays of nanolitre-sized wells is a method specifically tailored to crystallography at XFELs. The wells, with X-ray transparent Si3N4windows as bottoms, are fabricated in silicon chips. Their reduced dimensions can significantly decrease protein specimen consumption. Arrays provide crystalline samples positioned in an ordered way without the need to handle fragile crystals. The nucleation process inside these microfabricated cavities was optimized to provide high membrane coverage and a quasi-random crystal distribution. Tight sealing of the chips and protection of the crystals from dehydration were achieved, as confirmed by diffraction experiments at a protein crystallography beamline. Finally, the test samples were shown to be suitable for time-resolved measurements at an XFEL at femtosecond resolution.

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The expanding toolkit for structural biology: synchrotrons, X-ray lasers and cryoEM

IUCrJ ◽

10.1107/s2052252519002422 ◽

2019 ◽

Vol 6 (2) ◽

pp. 167-177 ◽

Cited By ~ 12

Author(s):

Stephen P. Muench ◽

Svetlana V. Antonyuk ◽

S. Samar Hasnain

Keyword(s):

High Resolution ◽

Membrane Proteins ◽

Structural Biology ◽

Free Electron ◽

Structural Information ◽

Protein Complexes ◽

Short Review ◽

X Rays ◽

Free Electron Lasers ◽

X Ray

Structural biology continues to benefit from an expanding toolkit, which is helping to gain unprecedented insight into the assembly and organization of multi-protein machineries, enzyme mechanisms and ligand/inhibitor binding. The combination of results from X-ray free-electron lasers (XFELs), modern synchrotron crystallographic beamlines and cryo-electron microscopy (cryoEM) is proving to be particularly powerful. The highly brilliant undulator beamlines at modern synchrotron facilities have empowered the crystallographic revolution of high-throughput structure determination at high resolution. The brilliance of the X-rays at these crystallographic beamlines has enabled this to be achieved using microcrystals, but at the expense of an increased absorbed X-ray dose and a consequent vulnerability to radiation-induced changes. The advent of serial femtosecond crystallography (SFX) with X-ray free-electron lasers provides a new opportunity in which damage-free structures can be obtained from much smaller crystals (2 µm) and more complex macromolecules, including membrane proteins and multi-protein complexes. For redox enzymes, SFX provides a unique opportunity by providing damage-free structures at both cryogenic and ambient temperatures. The promise of being able to visualize macromolecular structures and complexes at high resolution without the need for crystals using X-rays has remained a dream, but recent technological advancements in cryoEM have made this come true and hardly a month goes by when the structure of a new/novel macromolecular assembly is not revealed. The uniqueness of cryoEM in providing structural information for multi-protein complexes, particularly membrane proteins, has been demonstrated by examples such as respirasomes. The synergistic use of cryoEM and crystallography in lead-compound optimization is highlighted by the example of the visualization of antimalarial compounds in cytochromebc1. In this short review, using some recent examples including our own work, we share the excitement of these powerful structural biology methods.

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Single-pulse phase-contrast imaging at free-electron lasers in the hard X-ray regime

Journal of Synchrotron Radiation ◽

10.1107/s160057752001557x ◽

2021 ◽

Vol 28 (1) ◽

pp. 52-63

Author(s):

Johannes Hagemann ◽

Malte Vassholz ◽

Hannes Hoeppe ◽

Markus Osterhoff ◽

Juan M. Rosselló ◽

...

Keyword(s):

Free Electron ◽

Near Field ◽

Principal Component ◽

X Rays ◽

Free Electron Lasers ◽

Contrast Imaging ◽

Optical Pump ◽

Time Resolved ◽

Full Field ◽

X Ray

X-ray free-electron lasers (XFELs) have opened up unprecedented opportunities for time-resolved nano-scale imaging with X-rays. Near-field propagation-based imaging, and in particular near-field holography (NFH) in its high-resolution implementation in cone-beam geometry, can offer full-field views of a specimen's dynamics captured by single XFEL pulses. To exploit this capability, for example in optical-pump/X-ray-probe imaging schemes, the stochastic nature of the self-amplified spontaneous emission pulses, i.e. the dynamics of the beam itself, presents a major challenge. In this work, a concept is presented to address the fluctuating illumination wavefronts by sampling the configuration space of SASE pulses before an actual recording, followed by a principal component analysis. This scheme is implemented at the MID (Materials Imaging and Dynamics) instrument of the European XFEL and time-resolved NFH is performed using aberration-corrected nano-focusing compound refractive lenses. Specifically, the dynamics of a micro-fluidic water-jet, which is commonly used as sample delivery system at XFELs, is imaged. The jet exhibits rich dynamics of droplet formation in the break-up regime. Moreover, pump–probe imaging is demonstrated using an infrared pulsed laser to induce cavitation and explosion of the jet.

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Electronic Excitations and Radiation Damage in Macromolecular Crystallography

Crystals ◽

10.3390/cryst8070273 ◽

2018 ◽

Vol 8 (7) ◽

pp. 273 ◽

Cited By ~ 1

Author(s):

José Brandão-Neto ◽

Leonardo Bernasconi

Keyword(s):

Chemical Information ◽

X Rays ◽

Electronic Excitations ◽

Macromolecular Crystallography ◽

X Ray Diffraction ◽

X Ray ◽

Atomic Structures ◽

Successful Approach ◽

Structural Research

Macromolecular crystallography at cryogenic temperatures has so far provided the majority of the experimental evidence that underpins the determination of the atomic structures of proteins and other biomolecular assemblies by means of single crystal X-ray diffraction experiments. One of the core limitations of the current methods is that crystal samples degrade as they are subject to X-rays, and two broad groups of effects are observed: global and specific damage. While the currently successful approach is to operate outside the range where global damage is observed, specific damage is not well understood and may lead to poor interpretation of the chemistry and biology of the system under study. In this work, we present a phenomenological model in which specific damage is understood as the result of a single process, the steady excitation of crystal electrons caused by X-ray absorption, which acts as a trigger for the bulk effects that manifest themselves in the form of global damage and obscure the interpretation of chemical information from XFEL and synchrotron structural research.

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Electronic Excitations and Radiation Damage in Macromolecular Crystallography

10.20944/preprints201806.0353.v1 ◽

2018 ◽

Author(s):

José Brandão-Neto ◽

Leonardo Bernasconi

Keyword(s):

Chemical Information ◽

X Rays ◽

Electronic Excitations ◽

Macromolecular Crystallography ◽

X Ray Diffraction ◽

X Ray ◽

Atomic Structures ◽

Successful Approach ◽

Structural Research

Macromolecular crystallography at cryogenic temperatures has so far provided the majority of the experimental evidence that underpins the determination of the atomic structures of proteins and other biomolecular assemblies by means of single crystal X-ray diffraction experiments. One of the core limitations of the current methods is that crystal samples degrade as they are subject to X-rays, and two broad groups of effects are observed: global and specific damage. While the currently successful approach is to operate outside the range where global damage is observed, specific damage is not well understood and may lead to poor interpretation of the chemistry and biology of the system under study. In this work, we present a phenomenological model in which specific damage is understood as the result of a single process, the steady excitation of crystal electrons caused by X-ray absorption, which acts as a trigger for the bulk effects that manifest themselves in the form of global damage and obscure the interpretation of chemical information from XFEL and synchrotron structural research.

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X-Ray Free-Electron Lasers for the Structure and Dynamics of Macromolecules

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-013118-110744 ◽

2019 ◽

Vol 88 (1) ◽

pp. 35-58 ◽

Cited By ~ 41

Author(s):

Henry N. Chapman

Keyword(s):

Free Electron ◽

Conformational Dynamics ◽

Cryogenic Cooling ◽

X Rays ◽

Free Electron Lasers ◽

X Ray ◽

Virus Particles ◽

Structure And Dynamics ◽

Small Crystals ◽

Serial Crystallography

X-ray free-electron lasers provide femtosecond-duration pulses of hard X-rays with a peak brightness approximately one billion times greater than is available at synchrotron radiation facilities. One motivation for the development of such X-ray sources was the proposal to obtain structures of macromolecules, macromolecular complexes, and virus particles, without the need for crystallization, through diffraction measurements of single noncrystalline objects. Initial explorations of this idea and of outrunning radiation damage with femtosecond pulses led to the development of serial crystallography and the ability to obtain high-resolution structures of small crystals without the need for cryogenic cooling. This technique allows the understanding of conformational dynamics and enzymatics and the resolution of intermediate states in reactions over timescales of 100 fs to minutes. The promise of more photons per atom recorded in a diffraction pattern than electrons per atom contributing to an electron micrograph may enable diffraction measurements of single molecules, although challenges remain.

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Accelerator-based X-ray sources: synchrotron radiation, X-ray free electron lasers and beyond

Philosophical Transactions of The Royal Society A Mathematical Physical and Engineering Sciences ◽

10.1098/rsta.2018.0231 ◽

2019 ◽

Vol 377 (2147) ◽

pp. 20180231 ◽

Cited By ~ 2

Author(s):

Tetsuya Ishikawa

Keyword(s):

Synchrotron Radiation ◽

Storage Ring ◽

Free Electron ◽

Continuous Wave ◽

Transitional Period ◽

Light Sources ◽

X Rays ◽

Free Electron Lasers ◽

Beam Emittance ◽

X Ray

The evolution of synchrotron radiation (SR) sources and related sciences is discussed to explain the ‘generation’ of the SR sources. Most of the contemporary SR sources belong to the third generation, where the storage rings are optimized for the use of undulator radiation. The undulator development allowed to reduction of the electron energy of the storage ring necessary for delivering 10 keV X-rays from the initial 6–8 GeV to the current 3 Gev. Now is the transitional period from the double-bend-achromat lattice-based storage ring to the multi-bend-achromat lattice to achieve much smaller electron beam emittance. Free electron lasers are the other important accelerator-based light sources which recently reached hard X-ray regime by using self-amplified spontaneous emission scheme. Future accelerator-based X-ray sources should be continuous wave X-ray free electron lasers and pulsed X-ray free electron lasers. Some pathways to reach the future case are discussed. This article is part of the theme issue ‘Fifty years of synchrotron science: achievements and opportunities’.

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