Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers

Hattne, Johan; Echols, Nathaniel; Tran, Rosalie; Kern, Jan; Gildea, Richard J.; Brewster, Aaron S.; Alonso-Mori, Roberto; Glöckner, Carina; Hellmich, Julia; Laksmono, Hartawan; Sierra, Raymond G.; Lassalle‐Kaiser, Benedikt; Lampe, Alyssa; Han, Guangye; Gul, Sheraz; DiFiore, D.; Milathianaki, Despina; Fry, Alan; Miahnahri, Alan; White, William E.; Schafer, Donald W.; Seibert, M.; Koglin, Jason E.; Sokaras, Dimosthenis; Weng, Tsu Chien; Sellberg, Jonas A.; Latimer, Matthew J.; Glatzel, Pieter; Zwart, Petrus H.; Grosse‐Kunstleve, Ralf W.; Bogan, Michael J.; Messerschmidt, Marc; Williams, Garth J.; Boutet, Sébastien; Messinger, Johannes; Zouni, Athina; Yano, Junko; Bergmann, Uwe; Yachandra, Vittal K.; Adams, Paul D.; Sauter, Nicholas K.

doi:10.1038/nmeth.2887

Cited by 143 publications

(197 citation statements)

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“…The goniometerbased instrumentation described here provides an efficient and flexible framework with which to carry out these experiments, using automated strategies tailored to handle a variety of sample requirements, crystal sizes, and experimental goals. These developments, coupled with recent improvements in data-processing algorithms (30), make it possible to derive high-resolution structures, unadulterated by the effects of radiation exposure, using only 100-1,000 still diffraction images. The exposure of over 930 randomly oriented myoglobin crystals using only 32 grids demonstrates the utility of high-density sample containers, such as grids to optimize throughput and sample consumption.…”

Section: Discussionmentioning

confidence: 99%

“…Diffraction data for all crystals were collected at 100 K using the goniomemeter-based setup at LCLS XPP (SI Materials and Methods). Datasets were processed using both cctbx.xfel (30) and IPMOSFLM/ SCALA (34), and the structures were solved using molecular replacement (SI Materials and Methods).…”

Section: Methodsmentioning

confidence: 99%

“…This highly automated system enables the experiment to be performed from the XPP control room without depending on personnel access to the experimental area. Diffraction images were analyzed in quasi-real time to determine unit cell parameters, diffraction resolution, and dataset completeness using the cctbx.xfel package (30).…”

Section: Instrumentationmentioning

confidence: 99%

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Goniometer-based femtosecond crystallography with X-ray free electron lasers

Cohen

Soltis

González

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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130

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The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiationsensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β 2 -adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.femtosecond diffraction | crystallography | XFEL | structural biology U sing extremely bright, short-timescale X-ray pulses produced by X-ray free-electron lasers (XFELs), femtosecond crystallography (FX) is an emerging method that expands the structural information accessible from very small or very radiation-sensitive macromolecular crystals. Central to this method is the "diffraction before destruction" (1) process in which a still diffraction image is produced by a single X-ray pulse before significant radiation-induced electronic and atomic rearrangements occur within the crystal (1-3). At the Linac Coherent Light Source (LCLS) at SLAC, a single ∼50-fs-long X-ray pulse can expose a crystal to as many X-ray photons as a typical synchrotron beam line produces in about a second. Exposing small crystals to these intense ultrashort pulses circumvents the dose limitations of conventional X-ray diffraction experiments (4) and may produce useful data to resolutions beyond what is achievable at synchrotrons (5). This innovation provides a pathway to obtain atomic information from proteins that only form micrometer-to nanometer-sized crystals, such as many membrane proteins and large multiprotein complexes. Moreover, XFELs enable "diffraction before reduction" data collection to address another major challenge in structural enzymology by providing a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzyme active sites (6), such as high-valency reaction intermediates that may be significantly photoreduced during a single X-ray exposure at a synchrotron, even at very small doses (7-11). Furthermore, the use of short (tens of femtoseconds) X-ray pulses further complements the structural characterization of biochemical reaction processes by providing access to a time domain two to three orders of magnitude faster (12, 13) than currently accessible using synchrotro...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Goniometer-based femtosecond crystallography with X-ray free electron lasers

Cohen

Soltis

González

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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130

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“…The detector geometry is progressively refined by comparing the location of measured Bragg peaks to the location of peaks predicted once the crystal reciprocal lattice vectors have been determined using autoindexing [11,12]. This method can be applied using either a dedicated calibration sample or, in the case of an SFX experiment, from the serial crystallography data itself.…”

Section: Introductionmentioning

confidence: 99%

Accurate determination of segmented X-ray detector geometry

Yefanov¹,

Mariani²,

Gati³

et al. 2015

Opt. Express

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Recent advances in X-ray detector technology have resulted in the introduction of segmented detectors composed of many small detector modules tiled together to cover a large detection area. Due to mechanical tolerances and the desire to be able to change the module layout to suit the needs of different experiments, the pixels on each module might not align perfectly on a regular grid. Several detectors are designed to permit detector sub-regions (or modules) to be moved relative to each other for different experiments. Accurate determination of the location of detector elements relative to the beam-sample interaction point is critical for many types of experiment, including X-ray crystallography, coherent diffractive imaging (CDI), small angle X-ray scattering (SAXS) and spectroscopy. For detectors with moveable modules, the relative positions of pixels are no longer fixed, necessitating the development of a simple procedure to calibrate detector geometry after reconfiguration. We describe a simple and robust method for determining the geometry of segmented X-ray detectors using measurements obtained by serial crystallography. By comparing the location of observed Bragg peaks to the spot locations predicted from the crystal indexing procedure, the position, rotation and distance of each module relative to the interaction region can be refined. We show that the refined detector geometry greatly improves the results of experiments. #245734Received 17 Boutet, M. Messerschmidt, and I. Schlichting, "De novo protein crystal structure determination from X-ray freeelectron laser data," Nature 505(7482), 244-247 (2013). 23. M. Schmidt, K. Pande, S. Basu, and J. Tenboer, "Room temperature structures beyond 1.5 Å by serial femtosecond crystallography," Struct. Dyn. 2(4), 041708 (2015).

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“…However, by Bragg's law, only those parts of peaks that intersect the Ewald sphere diffract for a given orientation of crystal. Typically, the widths of the Bragg reflections are wider than the thickness of the Ewald sphere, so the diffracted intensity is only partially representative of the full integrated Bragg spot (9). In standard crystallography this is solved by oscillating the crystal through a small angle (e.g., a few tenths of a degree) over the course of the measurement so the Bragg peak passes fully through the Ewald sphere at a uniform rate.…”

mentioning

confidence: 99%

Expanding the femtosecond crystallography toolkit

Grüner

2014

Proc. Natl. Acad. Sci. U.S.A.

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The "21 st century of biology" is built on advances in protein structure determination developed over the last five decades. Exponential growth in the number of determined protein structures has largely been driven by new technologies in protein X-ray crystallography, including synchrotron X-ray sources, X-ray detectors, computational tools, and methods of handling protein crystals. In consequence, the time to acquire a routine, complete crystallographic dataset has gone from years to minutes. However, many important macromolecular structures are still undetermined and many challenges remain, especially in cases where crystals are either hard to obtain, particularly sensitive to X-ray damage, or exhibit transient states that are difficult to capture by structural analysis. The latest tool in the crystallographic arsenal, the X-ray free electron laser (XFEL) (1), promises to help surmount these challenges (2), provided efficient ways are found to handle crystals in the demanding XFEL environment. In PNAS, Cohen et al. (3) show that crystal handling technology developed primarily for use at synchrotron storage ring X-ray sources can also be effectively adapted for use at XFELs.Short-wavelength XFELs suitable for crystallography have only become available since the Linac Coherent Light Source turned on at the Department of Energy's California SLAC laboratory in 2009. The relevant difference between storage ring and XFEL sources is that storage ring sources deliver many low-intensity X-ray pulses per second, whereas XFELs do the same with a much smaller number of ultra-short pulses that are each millions of times as intense as storage ring pulses. The result is that the XFEL can deliver roughly as many X-rays in a single tens-of-femtoseconds duration pulse as a storage ring source delivers over the course of a second. At the LCLS this occurs 120 times per second.

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Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers

Cited by 143 publications

References 62 publications

Goniometer-based femtosecond crystallography with X-ray free electron lasers

Goniometer-based femtosecond crystallography with X-ray free electron lasers

Accurate determination of segmented X-ray detector geometry

Expanding the femtosecond crystallography toolkit

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