Resolution Extension by Image Summing in Serial Femtosecond Crystallography of Two-Dimensional Membrane-Protein Crystals

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Description

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

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.

Date Created
2018-01
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Femtosecond X-ray diffraction from two-dimensional protein crystals

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Description
X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due

X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.
Date Created
2014-02-28
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Fixed-Target Protein Serial Microcrystallography With an X-Ray Free Electron Laser

Description

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

Date Created
2014-08-12
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Instrument design in selected works for solo multiple percussion

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Description
Instrument design is intrinsic to multiple percussion solo performance preparation, from formulating a physical layout of instruments that best suit each work's technical requirements to fabricating unique instruments to fulfill each composer's sonic specifications. Several works in the multiple percussion

Instrument design is intrinsic to multiple percussion solo performance preparation, from formulating a physical layout of instruments that best suit each work's technical requirements to fabricating unique instruments to fulfill each composer's sonic specifications. Several works in the multiple percussion repertoire require setups comprised partly or entirely of performer-built instruments. Given that performers have varying degrees of expertise with instrument design and construction, the specialized instruments created do not necessarily meet the level of care with which many of the masterworks in the percussion field were created. Even with the many articles, books, and other publications regarding the instrument design issues of specific works, solo percussion literature is so varied that many set-ups are created using a set of nebulous guidelines. Developing solutions to the problems inherent in multiple percussion instrument design is clearly a continuing effort. Instrument and setup design within selected works for solo multiple percussion is the focus of this document and will be addressed through specific examples from literature commonly performed on concert stages and educational institutions. The scope of this document is limited to the widely applicable design issues of three pieces: Maki Ishii's Thirteen Drums: for Percussion Solo, Op. 66 (1985), David Lang's The Anvil The Chorus: for Percussion Solo (1991), and Steve Reich's Music for Pieces of Wood: for claves (1973). The set-up designs for these pieces suggested by the author are largely the focus for which other material in this document is preparatory.
Date Created
2012
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