Publications by authors named "Marc Messerschmidt"

99 Publications

Segmented flow generator for serial crystallography at the European X-ray free electron laser.

Nat Commun 2020 09 9;11(1):4511. Epub 2020 Sep 9.

School of Molecular Sciences, Arizona State University, Tempe, AZ, 85287-1604, USA.

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.
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http://dx.doi.org/10.1038/s41467-020-18156-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481229PMC
September 2020

Ptychographic X-ray speckle tracking with multi-layer Laue lens systems.

J Appl Crystallogr 2020 Aug 8;53(Pt 4):927-936. Epub 2020 Jul 8.

DESY, Notkestrasse 85, 22607 Hamburg, Germany.

The ever-increasing brightness of synchrotron radiation sources demands improved X-ray optics to utilize their capability for imaging and probing biological cells, nano-devices and functional matter on the nanometre scale with chemical sensitivity. Hard X-rays are ideal for high-resolution imaging and spectroscopic applications owing to their short wavelength, high penetrating power and chemical sensitivity. The penetrating power that makes X-rays useful for imaging also makes focusing them technologically challenging. Recent developments in layer deposition techniques have enabled the fabrication of a series of highly focusing X-ray lenses, known as wedged multi-layer Laue lenses. Improvements to the lens design and fabrication technique demand an accurate, robust, and at-wavelength characterization method. To this end, a modified form of the speckle tracking wavefront metrology method has been developed. The ptychographic X-ray speckle tracking method is capable of operating with highly divergent wavefields. A useful by-product of this method is that it also provides high-resolution and aberration-free projection images of extended specimens. Three separate experiments using this method are reported, where the ray path angles have been resolved to within 4 nrad with an imaging resolution of 45 nm (full period). This method does not require a high degree of coherence, making it suitable for laboratory-based X-ray sources. Likewise, it is robust to errors in the registered sample positions, making it suitable for X-ray free-electron laser facilities, where beam-pointing fluctuations can be problematic for wavefront metrology.
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http://dx.doi.org/10.1107/S1600576720006925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401788PMC
August 2020

Author Correction: Membrane protein megahertz crystallography at the European XFEL.

Nat Commun 2020 Jan 30;11(1):703. Epub 2020 Jan 30.

Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ, 85287-5001, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-14436-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992783PMC
January 2020

Membrane protein megahertz crystallography at the European XFEL.

Nat Commun 2019 11 4;10(1):5021. Epub 2019 Nov 4.

Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ, 85287-5001, USA.

The world's first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.
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http://dx.doi.org/10.1038/s41467-019-12955-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828683PMC
November 2019

Coherent diffractive imaging of microtubules using an X-ray laser.

Nat Commun 2019 06 13;10(1):2589. Epub 2019 Jun 13.

Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, 40530, Gothenburg, Sweden.

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.
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http://dx.doi.org/10.1038/s41467-019-10448-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565740PMC
June 2019

The Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography instrument of the European XFEL: initial installation.

J Synchrotron Radiat 2019 May 12;26(Pt 3):660-676. Epub 2019 Apr 12.

European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany.

The European X-ray Free-Electron Laser (FEL) became the first operational high-repetition-rate hard X-ray FEL with first lasing in May 2017. Biological structure determination has already benefitted from the unique properties and capabilities of X-ray FELs, predominantly through the development and application of serial crystallography. The possibility of now performing such experiments at data rates more than an order of magnitude greater than previous X-ray FELs enables not only a higher rate of discovery but also new classes of experiments previously not feasible at lower data rates. One example is time-resolved experiments requiring a higher number of time steps for interpretation, or structure determination from samples with low hit rates in conventional X-ray FEL serial crystallography. Following first lasing at the European XFEL, initial commissioning and operation occurred at two scientific instruments, one of which is the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument. This instrument provides a photon energy range, focal spot sizes and diagnostic tools necessary for structure determination of biological specimens. The instrumentation explicitly addresses serial crystallography and the developing single particle imaging method as well as other forward-scattering and diffraction techniques. This paper describes the major science cases of SPB/SFX and its initial instrumentation - in particular its optical systems, available sample delivery methods, 2D detectors, supporting optical laser systems and key diagnostic components. The present capabilities of the instrument will be reviewed and a brief outlook of its future capabilities is also described.
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http://dx.doi.org/10.1107/S1600577519003308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510195PMC
May 2019

MHz data collection of a microcrystalline mixture of different jack bean proteins.

Sci Data 2019 04 3;6(1):18. Epub 2019 Apr 3.

Max Planck Institute for Medical Research, Jahnstrasse 29, 69120, Heidelberg, Germany.

We provide a detailed description of a serial femtosecond crystallography (SFX) dataset collected at the European X-ray free-electron laser facility (EuXFEL). The EuXFEL is the first high repetition rate XFEL delivering MHz X-ray pulse trains at 10 Hz. The short spacing (<1 µs) between pulses requires fast flowing microjets for sample injection and high frame rate detectors. A data set was recorded of a microcrystalline mixture of at least three different jack bean proteins (urease, concanavalin A, concanavalin B). A one megapixel Adaptive Gain Integrating Pixel Detector (AGIPD) was used which has not only a high frame rate but also a large dynamic range. This dataset is publicly available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development and for data analysis training for prospective XFEL users.
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http://dx.doi.org/10.1038/s41597-019-0010-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472352PMC
April 2019

X-ray focusing with efficient high-NA multilayer Laue lenses.

Light Sci Appl 2018 23;7:17162. Epub 2018 Mar 23.

Alfred-Wegener Institute, Helmholtz Center for Polar and Marine Research, Bussestr. 27, Bremerhaven 27570, Germany.

Multilayer Laue lenses are volume diffraction elements for the efficient focusing of X-rays. With a new manufacturing technique that we introduced, it is possible to fabricate lenses of sufficiently high numerical aperture (NA) to achieve focal spot sizes below 10 nm. The alternating layers of the materials that form the lens must span a broad range of thicknesses on the nanometer scale to achieve the necessary range of X-ray deflection angles required to achieve a high NA. This poses a challenge to both the accuracy of the deposition process and the control of the materials properties, which often vary with layer thickness. We introduced a new pair of materials-tungsten carbide and silicon carbide-to prepare layered structures with smooth and sharp interfaces and with no material phase transitions that hampered the manufacture of previous lenses. Using a pair of multilayer Laue lenses (MLLs) fabricated from this system, we achieved a two-dimensional focus of 8.4 × 6.8 nm at a photon energy of 16.3 keV with high diffraction efficiency and demonstrated scanning-based imaging of samples with a resolution well below 10 nm. The high NA also allowed projection holographic imaging with strong phase contrast over a large range of magnifications. An error analysis indicates the possibility of achieving 1 nm focusing.
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http://dx.doi.org/10.1038/lsa.2017.162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6060042PMC
March 2018

Structure-factor amplitude reconstruction from serial femtosecond crystallography of two-dimensional membrane-protein crystals.

IUCrJ 2019 Jan 1;6(Pt 1):34-45. Epub 2019 Jan 1.

Paul Scherrer Institute, 5232 Villigen PSI, Switzerland.

Serial femtosecond crystallography of two-dimensional membrane-protein crystals at X-ray free-electron lasers has the potential to address the dynamics of functionally relevant large-scale motions, which can be sterically hindered in three-dimensional crystals and suppressed in cryocooled samples. In previous work, diffraction data limited to a two-dimensional reciprocal-space slice were evaluated and it was demonstrated that the low intensity of the diffraction signal can be overcome by collecting highly redundant data, thus enhancing the achievable resolution. Here, the application of a newly developed method to analyze diffraction data covering three reciprocal-space dimensions, extracting the reciprocal-space map of the structure-factor amplitudes, is presented. Despite the low resolution and completeness of the data set, it is shown by molecular replacement that the reconstructed amplitudes carry meaningful structural information. Therefore, it appears that these intrinsic limitations in resolution and completeness from two-dimensional crystal diffraction may be overcome by collecting highly redundant data along the three reciprocal-space axes, thus allowing the measurement of large-scale dynamics in pump-probe experiments.
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http://dx.doi.org/10.1107/S2052252518014641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6327180PMC
January 2019

X-ray Emission Spectroscopy at X-ray Free Electron Lasers: Limits to Observation of the Classical Spectroscopic Response for Electronic Structure Analysis.

J Phys Chem Lett 2019 Feb 16;10(3):441-446. Epub 2019 Jan 16.

Department of Physics and Astronomy , Purdue University , West Lafayette , Indiana 47907 , United States.

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 Mn 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.
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http://dx.doi.org/10.1021/acs.jpclett.8b03595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047744PMC
February 2019

Megahertz serial crystallography.

Authors:
Max O Wiedorn Dominik Oberthür Richard Bean Robin Schubert Nadine Werner Brian Abbey Martin Aepfelbacher Luigi Adriano Aschkan Allahgholi Nasser Al-Qudami Jakob Andreasson Steve Aplin Salah Awel Kartik Ayyer Saša Bajt Imrich Barák Sadia Bari Johan Bielecki Sabine Botha Djelloul Boukhelef Wolfgang Brehm Sandor Brockhauser Igor Cheviakov Matthew A Coleman Francisco Cruz-Mazo Cyril Danilevski Connie Darmanin R Bruce Doak Martin Domaracky Katerina Dörner Yang Du Hans Fangohr Holger Fleckenstein Matthias Frank Petra Fromme Alfonso M Gañán-Calvo Yaroslav Gevorkov Klaus Giewekemeyer Helen Mary Ginn Heinz Graafsma Rita Graceffa Dominic Greiffenberg Lars Gumprecht Peter Göttlicher Janos Hajdu Steffen Hauf Michael Heymann Susannah Holmes Daniel A Horke Mark S Hunter Siegfried Imlau Alexander Kaukher Yoonhee Kim Alexander Klyuev Juraj Knoška Bostjan Kobe Manuela Kuhn Christopher Kupitz Jochen Küpper Janine Mia Lahey-Rudolph Torsten Laurus Karoline Le Cong Romain Letrun P Lourdu Xavier Luis Maia Filipe R N C Maia Valerio Mariani Marc Messerschmidt Markus Metz Davide Mezza Thomas Michelat Grant Mills Diana C F Monteiro Andrew Morgan Kerstin Mühlig Anna Munke Astrid Münnich Julia Nette Keith A Nugent Theresa Nuguid Allen M Orville Suraj Pandey Gisel Pena Pablo Villanueva-Perez Jennifer Poehlsen Gianpietro Previtali Lars Redecke Winnie Maria Riekehr Holger Rohde Adam Round Tatiana Safenreiter Iosifina Sarrou Tokushi Sato Marius Schmidt Bernd Schmitt Robert Schönherr Joachim Schulz Jonas A Sellberg M Marvin Seibert Carolin Seuring Megan L Shelby Robert L Shoeman Marcin Sikorski Alessandro Silenzi Claudiu A Stan Xintian Shi Stephan Stern Jola Sztuk-Dambietz Janusz Szuba Aleksandra Tolstikova Martin Trebbin Ulrich Trunk Patrik Vagovic Thomas Ve Britta Weinhausen Thomas A White Krzysztof Wrona Chen Xu Oleksandr Yefanov Nadia Zatsepin Jiaguo Zhang Markus Perbandt Adrian P Mancuso Christian Betzel Henry Chapman Anton Barty

Nat Commun 2018 10 2;9(1):4025. Epub 2018 Oct 2.

Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607, Hamburg, Germany.

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 β-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.
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http://dx.doi.org/10.1038/s41467-018-06156-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168542PMC
October 2018

Megahertz data collection from protein microcrystals at an X-ray free-electron laser.

Nat Commun 2018 08 28;9(1):3487. Epub 2018 Aug 28.

Max Planck Institute for Medical Research, Jahnstrasse 29, 69120, Heidelberg, Germany.

X-ray free-electron lasers (XFELs) enable novel experiments because of their high peak brilliance and femtosecond pulse duration. However, non-superconducting XFELs offer repetition rates of only 10-120 Hz, placing significant demands on beam time and sample consumption. We describe serial femtosecond crystallography experiments performed at the European XFEL, the first MHz repetition rate XFEL, delivering 1.128 MHz X-ray pulse trains at 10 Hz. Given the short spacing between pulses, damage caused by shock waves launched by one XFEL pulse on sample probed by subsequent pulses is a concern. To investigate this issue, we collected data from lysozyme microcrystals, exposed to a ~15 μm XFEL beam. Under these conditions, data quality is independent of whether the first or subsequent pulses of the train were used for data collection. We also analyzed a mixture of microcrystals of jack bean proteins, from which the structure of native, magnesium-containing concanavalin A was determined.
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http://dx.doi.org/10.1038/s41467-018-05953-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113309PMC
August 2018

Ultrafast nonthermal heating of water initiated by an X-ray Free-Electron Laser.

Proc Natl Acad Sci U S A 2018 05 14;115(22):5652-5657. Epub 2018 May 14.

Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron, DE-22607 Hamburg, Germany;

The bright ultrafast pulses of X-ray Free-Electron Lasers allow investigation into the structure of matter under extreme conditions. We have used single pulses to ionize and probe water as it undergoes a phase transition from liquid to plasma. We report changes in the structure of liquid water on a femtosecond time scale when irradiated by single 6.86 keV X-ray pulses of more than 10 J/cm These observations are supported by simulations based on molecular dynamics and plasma dynamics of a water system that is rapidly ionized and driven out of equilibrium. This exotic ionic and disordered state with the density of a liquid is suggested to be structurally different from a neutral thermally disordered state.
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http://dx.doi.org/10.1073/pnas.1711220115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984484PMC
May 2018

Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals.

IUCrJ 2018 Jan 1;5(Pt 1):103-117. Epub 2018 Jan 1.

Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550, USA.

Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump-probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.
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http://dx.doi.org/10.1107/S2052252517017043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755582PMC
January 2018

Nanocrystallography measurements of early stage synthetic malaria pigment.

J Appl Crystallogr 2017 Oct 28;50(Pt 5):1533-1540. Epub 2017 Sep 28.

ARC Centre of Excellence in Advanced Molecular Imaging, Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.

The recent availability of extremely intense, femtosecond X-ray free-electron laser (XFEL) sources has spurred the development of serial femtosecond nanocrystallography (SFX). Here, SFX is used to analyze nanoscale crystals of β-hematin, the synthetic form of hemozoin which is a waste by-product of the malaria parasite. This analysis reveals significant differences in β-hematin data collected during SFX and synchrotron crystallography experiments. To interpret these differences two possibilities are considered: structural differences between the nanocrystal and larger crystalline forms of β-hematin, and radiation damage. Simulation studies show that structural inhomogeneity appears at present to provide a better fit to the experimental data. If confirmed, these observations will have implications for designing compounds that inhibit hemozoin formation and suggest that, for some systems at least, additional information may be gained by comparing structures obtained from nanocrystals and macroscopic crystals of the same molecule.
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http://dx.doi.org/10.1107/S1600576717012663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627683PMC
October 2017

Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene.

J Vis Exp 2017 08 22(126). Epub 2017 Aug 22.

Australian Research Council (ARC) Centre of Excellence in Advanced Molecular Imaging, Department of Chemistry and Physics, La Trobe Institute for Molecular Sciences, La Trobe University;

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the results of femtosecond X-ray free electron laser (XFEL) experiments. An increasing number of experimental observations have shown that although nuclear motion can be negligible, given a short enough incident pulse duration, electronic motion cannot be ignored. The current and widely accepted models assume that although electrons undergo dynamics driven by interaction with the pulse, their motion could largely be considered 'random'. This would then allow the supposedly incoherent contribution from the electronic motion to be treated as a continuous background signal and thus ignored. The original aim of our experiment was to precisely measure the change in intensity of individual Bragg peaks, due to X-ray induced electronic damage in a model system, crystalline C60. Contrary to this expectation, we observed that at the highest X-ray intensities, the electron dynamics in C60 were in fact highly correlated, and over sufficiently long distances that the positions of the Bragg reflections are significantly altered. This paper describes in detail the methods and protocols used for these experiments, which were conducted both at the Linac Coherent Light Source (LCLS) and the Australian Synchrotron (AS) as well as the crystallographic approaches used to analyse the data.
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http://dx.doi.org/10.3791/56296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614354PMC
August 2017

Single-shot determination of focused FEL wave fields using iterative phase retrieval.

Opt Express 2017 Jul;25(15):17892-17903

Determining fluctuations in focus properties is essential for many experiments at Self-Amplified-Spontaneous-Emission (SASE) based Free-Electron-Lasers (FELs), in particular for imaging single non-crystalline biological particles. We report on a diffractive imaging technique to fully characterize highly focused, single-shot pulses using an iterative phase retrieval algorithm, and benchmark it against an existing Hartmann wavefront sensor. The results, both theoretical and experimental, demonstrate the effectiveness of this technique to provide a comprehensive and convenient shot-to-shot measurement of focused-pulse wave fields and source-point positional variations without the need for manipulative optics between the focus and the detector.
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http://dx.doi.org/10.1364/OE.25.017892DOI Listing
July 2017

Structural insights into the extracellular recognition of the human serotonin 2B receptor by an antibody.

Proc Natl Acad Sci U S A 2017 08 17;114(31):8223-8228. Epub 2017 Jul 17.

Department of Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089;

Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å resolution structure of a complex between the human 5-hydroxytryptamine 2B (5-HT) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femtosecond crystallography with an X-ray free-electron laser. The antibody binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies.
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http://dx.doi.org/10.1073/pnas.1700891114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5547598PMC
August 2017

How Cubic Can Ice Be?

J Phys Chem Lett 2017 Jul 30;8(14):3216-3222. Epub 2017 Jun 30.

William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University , Columbus, Ohio 43210, United States.

Using an X-ray laser, we investigated the crystal structure of ice formed by homogeneous ice nucleation in deeply supercooled water nanodrops (r ≈ 10 nm) at ∼225 K. The nanodrops were formed by condensation of vapor in a supersonic nozzle, and the ice was probed within 100 μs of freezing using femtosecond wide-angle X-ray scattering at the Linac Coherent Light Source free-electron X-ray laser. The X-ray diffraction spectra indicate that this ice has a metastable, predominantly cubic structure; the shape of the first ice diffraction peak suggests stacking-disordered ice with a cubicity value, χ, in the range of 0.78 ± 0.05. The cubicity value determined here is higher than those determined in experiments with micron-sized drops but comparable to those found in molecular dynamics simulations. The high cubicity is most likely caused by the extremely low freezing temperatures and by the rapid freezing, which occurs on a ∼1 μs time scale in single nanodroplets.
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http://dx.doi.org/10.1021/acs.jpclett.7b01142DOI Listing
July 2017

Flow-aligned, single-shot fiber diffraction using a femtosecond X-ray free-electron laser.

Cytoskeleton (Hoboken) 2017 Dec 19;74(12):472-481. Epub 2017 Jun 19.

Institute of Molecular and Cell Biology, Biopolis, A*STAR (Agency for Science, Technology and Research), 138673, Singapore.

A major goal for X-ray free-electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one-dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F-actin, and amyloid fibrils), which when intersected by femtosecond X-ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F-actin can be flow-aligned to a disorientation of approximately 5 degrees. Using this XFEL-based technique, we determine that gelsolin amyloids are comprised of stacked β-strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual α-synuclein amyloids.
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http://dx.doi.org/10.1002/cm.21378DOI Listing
December 2017

Diffraction data of core-shell nanoparticles from an X-ray free electron laser.

Sci Data 2017 04 11;4:170048. Epub 2017 Apr 11.

Complex Systems Division, Beijing Computational Science Research Center, ZPark II, Haidian, Beijing 100193, China.

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.
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http://dx.doi.org/10.1038/sdata.2017.48DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5387922PMC
April 2017

Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser.

Proc Natl Acad Sci U S A 2017 02 15;114(9):2247-2252. Epub 2017 Feb 15.

Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany;

To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.
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http://dx.doi.org/10.1073/pnas.1609243114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338516PMC
February 2017

De novo phasing with X-ray laser reveals mosquito larvicide BinAB structure.

Nature 2016 11 28;539(7627):43-47. Epub 2016 Sep 28.

UCLA-DOE Institute for Genomics and Proteomics, Department of Biological Chemistry, University of California, Los Angeles, California 90095-1570, USA.

BinAB is a naturally occurring paracrystalline larvicide distributed worldwide to combat the devastating diseases borne by mosquitoes. These crystals are composed of homologous molecules, BinA and BinB, which play distinct roles in the multi-step intoxication process, transforming from harmless, robust crystals, to soluble protoxin heterodimers, to internalized mature toxin, and finally to toxic oligomeric pores. The small size of the crystals-50 unit cells per edge, on average-has impeded structural characterization by conventional means. Here we report the structure of Lysinibacillus sphaericus BinAB solved de novo by serial-femtosecond crystallography at an X-ray free-electron laser. The structure reveals tyrosine- and carboxylate-mediated contacts acting as pH switches to release soluble protoxin in the alkaline larval midgut. An enormous heterodimeric interface appears to be responsible for anchoring BinA to receptor-bound BinB for co-internalization. Remarkably, this interface is largely composed of propeptides, suggesting that proteolytic maturation would trigger dissociation of the heterodimer and progression to pore formation.
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http://dx.doi.org/10.1038/nature19825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161637PMC
November 2016

X-ray laser-induced electron dynamics observed by femtosecond diffraction from nanocrystals of Buckminsterfullerene.

Sci Adv 2016 09 9;2(9):e1601186. Epub 2016 Sep 9.

ARC Centre of Excellence in Advanced Molecular Imaging, School of Physics, University of Melbourne, Parkville, Victoria 3010, Australia.

X-ray free-electron lasers (XFELs) deliver x-ray pulses with a coherent flux that is approximately eight orders of magnitude greater than that available from a modern third-generation synchrotron source. The power density of an XFEL pulse may be so high that it can modify the electronic properties of a sample on a femtosecond time scale. Exploration of the interaction of intense coherent x-ray pulses and matter is both of intrinsic scientific interest and of critical importance to the interpretation of experiments that probe the structures of materials using high-brightness femtosecond XFEL pulses. We report observations of the diffraction of extremely intense 32-fs nanofocused x-ray pulses by a powder sample of crystalline C60. We find that the diffraction pattern at the highest available incident power significantly differs from the one obtained using either third-generation synchrotron sources or XFEL sources operating at low output power and does not correspond to the diffraction pattern expected from any known phase of crystalline C60. We interpret these data as evidence of a long-range, coherent dynamic electronic distortion that is driven by the interaction of the periodic array of C60 molecular targets with intense x-ray pulses of femtosecond duration.
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http://dx.doi.org/10.1126/sciadv.1601186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5017826PMC
September 2016

Establishing nonlinearity thresholds with ultraintense X-ray pulses.

Sci Rep 2016 09 13;6:33292. Epub 2016 Sep 13.

Paul Scherrer Institut, Villigen, Switzerland.

X-ray techniques have evolved over decades to become highly refined tools for a broad range of investigations. Importantly, these approaches rely on X-ray measurements that depend linearly on the number of incident X-ray photons. The advent of X-ray free electron lasers (XFELs) is opening the ability to reach extremely high photon numbers within ultrashort X-ray pulse durations and is leading to a paradigm shift in our ability to explore nonlinear X-ray signals. However, the enormous increase in X-ray peak power is a double-edged sword with new and exciting methods being developed but at the same time well-established techniques proving unreliable. Consequently, accurate knowledge about the threshold for nonlinear X-ray signals is essential. Herein we report an X-ray spectroscopic study that reveals important details on the thresholds for nonlinear X-ray interactions. By varying both the incident X-ray intensity and photon energy, we establish the regimes at which the simplest nonlinear process, two-photon X-ray absorption (TPA), can be observed. From these measurements we can extract the probability of this process as a function of photon energy and confirm both the nature and sub-femtosecond lifetime of the virtual intermediate electronic state.
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http://dx.doi.org/10.1038/srep33292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5020491PMC
September 2016

Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser.

Sci Data 2016 Aug 1;3:160060. Epub 2016 Aug 1.

Department of Cell and Molecular Biology, Laboratory of Molecular Biophysics, Uppsala University, Husargatan 3 (Box 596), SE-751 24 Uppsala, Sweden.

Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms.
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http://dx.doi.org/10.1038/sdata.2016.60DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968188PMC
August 2016

Serial femtosecond crystallography datasets from G protein-coupled receptors.

Sci Data 2016 Aug 1;3:160057. Epub 2016 Aug 1.

Departments of Chemistry and Physics &Astronomy, The Bridge Institute, University of Southern California, Los Angeles, California 90089, USA.

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.
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http://dx.doi.org/10.1038/sdata.2016.57DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968193PMC
August 2016

Transient lattice contraction in the solid-to-plasma transition.

Sci Adv 2016 Jan 29;2(1):e1500837. Epub 2016 Jan 29.

Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA.; Pulse Institute, Stanford University and SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA.; Argonne National Laboratory, Lemont, IL 60439, USA.; Department of Physics and Astronomy, Northwestern University, Evanston, IL 60208, USA.

In condensed matter systems, strong optical excitations can induce phonon-driven processes that alter their mechanical properties. We report on a new phenomenon where a massive electronic excitation induces a collective change in the bond character that leads to transient lattice contraction. Single large van der Waals clusters were isochorically heated to a nanoplasma state with an intense 10-fs x-ray (pump) pulse. The structural evolution of the nanoplasma was probed with a second intense x-ray (probe) pulse, showing systematic contraction stemming from electron delocalization during the solid-to-plasma transition. These findings are relevant for any material in extreme conditions ranging from the time evolution of warm or hot dense matter to ultrafast imaging with intense x-ray pulses or, more generally, any situation that involves a condensed matter-to-plasma transition.
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http://dx.doi.org/10.1126/sciadv.1500837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846449PMC
January 2016