Acoustically Mounted Microcrystals Yield High-Resolution X-ray Structures

Soares, Alexei S.; Engel, M.; Stearns, Richard G.; Datwani, Sammy S.; Olechno, Joe; Ellson, Richard; Skinner, John M.; Allaire, Marc; Orville, Allen M.

doi:10.1021/bi200549x

Cited by 48 publications

(44 citation statements)

References 24 publications

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“…However, droplet speeds produced tend to be unstable on the necessary timescales, making synchronization difficult. Additional challenges faced by this method include large droplet size and thus high background 96,97 , crystal settling without the addition of a viscous solution 48 , and compatibility with high vacuum 48 . To date, neither the pulsed liquid jet nor acoustic droplet ejection has been employed at an XFEL successfully.…”

Section: Sample Delivery Methodsmentioning

confidence: 99%

Serial femtosecond crystallography: A revolution in structural biology

Martín-García

Conrad

Coe

et al. 2016

Archives of Biochemistry and Biophysics

113

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Macromolecular crystallography at synchrotron sources has proven to be the most influential method within structural biology, producing thousands of structures since its inception. While its utility has been instrumental in progressing our knowledge of structures of molecules, it suffers from limitations such as the need for large, well-diffracting crystals, and radiation damage that can hamper native structural determination. The recent advent of X-ray free electron lasers (XFELs) and their implementation in the emerging field of serial femtosecond crystallography (SFX) has given rise to a remarkable expansion upon existing crystallographic constraints, allowing structural biologists access to previously restricted scientific territory. SFX relies on exceptionally brilliant, micro-focused X-ray pulses, which are femtoseconds in duration, to probe nano/micrometer sized crystals in a serial fashion. This results in data sets comprised of individual snapshots, each capturing Bragg diffraction of single crystals in random orientations prior to their subsequent destruction. Thus structural elucidation while avoiding radiation damage, even at room temperature, can now be achieved. This emerging field has cultivated new methods for nanocrystallogenesis, sample delivery, and data processing. Opportunities and challenges within SFX are reviewed herein.

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Section: Sample Delivery Methodsmentioning

confidence: 99%

Serial femtosecond crystallography: A revolution in structural biology

Martín-García

Conrad

Coe

et al. 2016

Archives of Biochemistry and Biophysics

113

View full text Add to dashboard Cite

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“…While capillary and microfluidic devices can clog and damage crystals by subjecting them to shear force, in ADE the droplet emerges from the meniscus of the fluid without passing through an orifice. Some current applications of ADE in macromolecular crystallography (MX) include crystal growth (Villaseñor et al, 2012), microseeding (Villaseñor et al, 2010), ligand screening (Cole et al, 2014; Teplitsky et al, 2015; Yin et al, 2014), and discrete crystal deposition onto data collection media for synchrotron-based cryo-crystallography (Cuttitta et al, 2015; Heroux et al, 2014; Roessler et al, 2013; Soares et al, 2011). …”

Section: Resultsmentioning

confidence: 99%

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

et al. 2016

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SUMMARY X-Ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of micro-plates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.

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“…We previously demonstrated that protein crystals can be harvested by using acoustic droplet ejection (ADE) to propel small droplets containing crystals out of the mother liquor, through a short air column, and onto a micromesh 1 or onto a crystal transporting conveyor belt that is linked to the x-ray diffractometer. using sonar is modest compared with other techniques.…”

Section: Introductionmentioning

confidence: 99%

Acoustic Methods to Monitor Protein Crystallization and to Detect Protein Crystals in Suspensions of Agarose and Lipidic Cubic Phase

et al. 2016

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Improvements needed for automated crystallography include crystal detection and crystal harvesting. A technique that uses acoustic droplet ejection to harvest crystals was previously reported. Here a method is described for using the same acoustic instrument to detect protein crystals and to monitor crystal growth. Acoustic pulses were used to monitor the progress of crystallization trials and to detect the presence and location of protein crystals. Crystals were detected, and crystallization was monitored in aqueous solutions and in lipidic cubic phase. Using a commercially available acoustic instrument, crystals measuring ~150 µm or larger were readily detected. Simple laboratory techniques were used to increase the sensitivity to 50 µm by suspending the crystals away from the plastic surface of the crystallization plate. This increased the sensitivity by separating the strong signal generated by the plate bottom that can mask the signal from small protein crystals. It is possible to further boost the acoustic reflection from small crystals by reducing the wavelength of the incident sound pulse, but our current instrumentation does not allow this option. In the future, commercially available sound-emitting transducers with a characteristic frequency near 300 MHz should detect and monitor the growth of individual 3 µm crystals.

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Acoustically Mounted Microcrystals Yield High-Resolution X-ray Structures

Cited by 48 publications

References 24 publications

Serial femtosecond crystallography: A revolution in structural biology

Serial femtosecond crystallography: A revolution in structural biology

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

Acoustic Methods to Monitor Protein Crystallization and to Detect Protein Crystals in Suspensions of Agarose and Lipidic Cubic Phase

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