Abstract:Serial crystallography has enabled the study of complex biological questions through the determination of biomolecular structures at room temperature using low X-ray doses. Furthermore, it has enabled the study of protein dynamics by the capture of atomically resolved and time-resolved molecular movies. However, the study of many biologically relevant targets is still severely hindered by high sample consumption and lengthy data-collection times. By combining serial synchrotron crystallography (SSX) with 3D pr… Show more
“…This setup enables the transport of crystals mounted in loops, grids or capillaries in a controlled humidity environment to the synchrotron beamline for robotic sample mounting. As three-dimensional (3D) printers become more accessible, custom 3D printed trays provide a viable alternative (Monteiro et al ., 2020). Fig.…”
Section: Before: How To Prepare For Rtx Data Collectionmentioning
confidence: 99%
“…While most of these were originally developed for XFEL beamlines, many are amenable to being adapted by microfocus synchrotron beamlines. These include high-viscosity injection (Botha et al ., 2015; Nogly et al ., 2015; Kovácsová et al ., 2017; Martin-Garcia et al ., 2017), flowing crystals through quartz capillaries (Stellato et al ., 2014), low sample consumption microfluidic flow-focusing (Pawate et al ., 2015; de Wijn et al ., 2019; Monteiro et al ., 2019) using 3D printed devices (Monteiro et al ., 2020) and stable ultrasonic acoustic levitation of microliter droplets (Tsujino and Tomizaki, 2016). Ongoing work addresses problems with clogging and flow-speed of microfluidics, bubble generation, viscous stream focusing, deposition of varied-sized crystals, and sample consumption to enable the routine, uninterrupted collection of serial crystallography data.…”
Section: During: How To Collect Rtx Datamentioning
X-ray crystallography enables detailed structural studies of proteins to understand and modulate their function. Conducting crystallographic experiments at cryogenic temperatures has practical benefits but potentially limits the identification of functionally important alternative protein conformations that can be revealed only at room temperature (RT). This review discusses practical aspects of preparing, acquiring, and analyzing X-ray crystallography data at RT to demystify preconceived impracticalities that freeze progress of routine RT data collection at synchrotron sources. Examples are presented as conceptual and experimental templates to enable the design of RT-inspired studies; they illustrate the diversity and utility of gaining novel insights into protein conformational landscapes. An integrative view of protein conformational dynamics enables opportunities to advance basic and biomedical research.
“…This setup enables the transport of crystals mounted in loops, grids or capillaries in a controlled humidity environment to the synchrotron beamline for robotic sample mounting. As three-dimensional (3D) printers become more accessible, custom 3D printed trays provide a viable alternative (Monteiro et al ., 2020). Fig.…”
Section: Before: How To Prepare For Rtx Data Collectionmentioning
confidence: 99%
“…While most of these were originally developed for XFEL beamlines, many are amenable to being adapted by microfocus synchrotron beamlines. These include high-viscosity injection (Botha et al ., 2015; Nogly et al ., 2015; Kovácsová et al ., 2017; Martin-Garcia et al ., 2017), flowing crystals through quartz capillaries (Stellato et al ., 2014), low sample consumption microfluidic flow-focusing (Pawate et al ., 2015; de Wijn et al ., 2019; Monteiro et al ., 2019) using 3D printed devices (Monteiro et al ., 2020) and stable ultrasonic acoustic levitation of microliter droplets (Tsujino and Tomizaki, 2016). Ongoing work addresses problems with clogging and flow-speed of microfluidics, bubble generation, viscous stream focusing, deposition of varied-sized crystals, and sample consumption to enable the routine, uninterrupted collection of serial crystallography data.…”
Section: During: How To Collect Rtx Datamentioning
X-ray crystallography enables detailed structural studies of proteins to understand and modulate their function. Conducting crystallographic experiments at cryogenic temperatures has practical benefits but potentially limits the identification of functionally important alternative protein conformations that can be revealed only at room temperature (RT). This review discusses practical aspects of preparing, acquiring, and analyzing X-ray crystallography data at RT to demystify preconceived impracticalities that freeze progress of routine RT data collection at synchrotron sources. Examples are presented as conceptual and experimental templates to enable the design of RT-inspired studies; they illustrate the diversity and utility of gaining novel insights into protein conformational landscapes. An integrative view of protein conformational dynamics enables opportunities to advance basic and biomedical research.
“…This new approach allows for the fast, affordable, and high-resolution fabrication of microchannels. For example, Monteiro et al 31 succesfully implemented a 3D printed ow focusing microuidic device for serial synchrotron crystallography data collection. The device allowed for stable data acquisition over long periods of time and was successfully used for a proof of concept study involving rapid-mixing, timeresolved protein diffraction experiments.…”
“…concentric or overhanging features) using such fabrication methods. A 3Dprinted device recently demonstrated compatibility with serial synchrotron crystallography and reported mixing timescales between 0.18 and 2 s (Monteiro et al, 2020). A limitation of the 2D hydrodynamic focusing with additional bottom and top layers scheme is the significant premixing (and reaction completion) which occurs prior to and during sample stream focusing (Hertzog et al, 2006;Park et al, 2006).…”
Determination of electronic structures during chemical reactions remains challenging in studies which involve reactions in the millisecond timescale, toxic chemicals, and/or anaerobic conditions. In this study, a three-dimensionally (3D) microfabricated microfluidic mixer platform that is compatible with time-resolved X-ray absorption and emission spectroscopy (XAS and XES, respectively) is presented. This platform, to initiate reactions and study their progression, mixes a high flow rate (0.50–1.5 ml min−1) sheath stream with a low-flow-rate (5–90 µl min−1) sample stream within a monolithic fused silica chip. The chip geometry enables hydrodynamic focusing of the sample stream in 3D and sample widths as small as 5 µm. The chip is also connected to a polyimide capillary downstream to enable sample stream deceleration, expansion, and X-ray detection. In this capillary, sample widths of 50 µm are demonstrated. Further, convection–diffusion-reaction models of the mixer are presented. The models are experimentally validated using confocal epifluorescence microscopy and XAS/XES measurements of a ferricyanide and ascorbic acid reaction. The models additionally enable prediction of the residence time and residence time uncertainty of reactive species as well as mixing times. Residence times (from initiation of mixing to the point of X-ray detection) during sample stream expansion as small as 2.1 ± 0.3 ms are also demonstrated. Importantly, an exploration of the mixer operational space reveals a theoretical minimum mixing time of 0.91 ms. The proposed platform is applicable to the determination of the electronic structure of conventionally inaccessible reaction intermediates.
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