scholarly journals Millisecond mix-and-quench crystallography (MMQX) enables time-resolved studies of PEPCK with remote data collection

IUCrJ ◽  
2021 ◽  
Vol 8 (5) ◽  
Author(s):  
Jonathan A. Clinger ◽  
David W. Moreau ◽  
Matthew J. McLeod ◽  
Todd Holyoak ◽  
Robert E. Thorne
Keyword(s):  
Data Collection ◽  
Protein Dynamics ◽  
Time Resolution ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Structural Studies ◽  
Widespread Application ◽  
Entry Barrier ◽  
Time Resolved ◽  
Oxaloacetic Acid ◽  
Serial Crystallography

Time-resolved crystallography of biomolecules in action has advanced rapidly as methods for serial crystallography have improved, but the large number of crystals and the complex experimental infrastructure that are required remain serious obstacles to its widespread application. Here, millisecond mix-and-quench crystallography (MMQX) has been developed, which yields millisecond time-resolved data using far fewer crystals and routine remote synchrotron data collection. To demonstrate the capabilities of MMQX, the conversion of oxaloacetic acid to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase (PEPCK) is observed with a time resolution of 40 ms. By lowering the entry barrier to time-resolved crystallography, MMQX should enable a broad expansion in structural studies of protein dynamics.

scholarly journals Millisecond Mix-and-Quench Crystallography (MMQX) Enables Time-Resolved Studies of PEPCK With Remote Data Collection

2021 ◽  
Author(s):  
Jonathan A Clinger ◽  
David W. Moreau ◽  
Matthew J McLeod ◽  
Todd Holyoak ◽  
Robert E Thorne
Keyword(s):  
Data Collection ◽  
Protein Dynamics ◽  
Time Resolution ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Structural Studies ◽  
Widespread Application ◽  
Entry Barrier ◽  
Time Resolved ◽  
Oxaloacetic Acid ◽  
Serial Crystallography

Time-resolved crystallography of biomolecules in action has advanced rapidly as methods for serial crystallography have improved, but the large number of crystals and complex experimental infrastructure required remain serious obstacles to widespread application. We have developed Millisecond Mix-and-Quench crystallography (MMQX), which yields millisecond time-resolved data using far fewer crystals and routine remote synchrotron data collection. To demonstrate the capabilities of MMQX, the conversion of oxaloacetic acid to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase is observed with a time resolution of 40 ms. MMQX, by lowering the entry barrier to time-resolved crystallography, should enable broad expansion in structural studies of protein dynamics.

scholarly journals Time-resolved structural studies with serial crystallography: A new light on retinal proteins

2015 ◽  
Vol 2 (4) ◽  
pp. 041718 ◽  
Author(s):  
Valérie Panneels ◽  
Wenting Wu ◽  
Ching-Ju Tsai ◽  
Przemek Nogly ◽  
Jan Rheinberger ◽  
...  
Keyword(s):  
Structural Studies ◽  
Time Resolved ◽  
Serial Crystallography ◽  
Retinal Proteins

scholarly journals 3D-MiXD: 3D-printed X-ray-compatible microfluidic devices for rapid, low-consumption serial synchrotron crystallography data collection in flow

IUCrJ ◽  
2020 ◽  
Vol 7 (2) ◽  
pp. 207-219 ◽  
Author(s):  
Diana C. F. Monteiro ◽  
David von Stetten ◽  
Claudia Stohrer ◽  
Marta Sans ◽  
Arwen R. Pearson ◽  
...  
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Time Resolved ◽  
X Ray ◽  
Biologically Relevant ◽  
3D Printed ◽  
Serial Crystallography ◽  
Low X ◽  
Very High

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 printing, a new experimental platform has been created that tackles these challenges. An affordable 3D-printed, X-ray-compatible microfluidic device (3D-MiXD) is reported that allows data to be collected from protein microcrystals in a 3D flow with very high hit and indexing rates, while keeping the sample consumption low. The miniaturized 3D-MiXD can be rapidly installed into virtually any synchrotron beamline with only minimal adjustments. This efficient collection scheme in combination with its mixing geometry paves the way for recording molecular movies at synchrotrons by mixing-triggered millisecond time-resolved SSX.

scholarly journals Towards time-resolved serial crystallography in a microfluidic device

2015 ◽  
Vol 71 (7) ◽  
pp. 823-830 ◽  
Author(s):  
Ashtamurthy S. Pawate ◽  
Vukica Šrajer ◽  
Jeremy Schieferstein ◽  
Sudipto Guha ◽  
Robert Henning ◽  
...  
Keyword(s):  
Photoactive Yellow Protein ◽  
Structural Studies ◽  
Time Resolved ◽  
Protein Targets ◽  
Laser Illumination ◽  
Serial Data ◽  
Multivariable Analyses ◽  
Serial Crystallography ◽  
Collection Strategies ◽  
Reaction Initiation

Serial methods for crystallography have the potential to enable dynamic structural studies of protein targets that have been resistant to single-crystal strategies. The use of serial data-collection strategies can circumvent challenges associated with radiation damage and repeated reaction initiation. This work utilizes a microfluidic crystallization platform for the serial time-resolved Laue diffraction analysis of macroscopic crystals of photoactive yellow protein (PYP). Reaction initiation was achievedviapulsed laser illumination, and the resultant electron-density difference maps clearly depict the expected pR1/pRE46Qand pR2/pRCWstates at 10 µs and the pB1intermediate at 1 ms. The strategies presented here have tremendous potential for extension to chemical triggering methods for reaction initiation and for extension to dynamic, multivariable analyses.

scholarly journals Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

Crystals ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 215 ◽  
Author(s):  
Robert Cheng
Keyword(s):  
Data Collection ◽  
Collection Efficiency ◽  
Structural Data ◽  
Fast Time ◽  
Full Potential ◽  
Delivery Methods ◽  
Time Resolved ◽  
X Ray ◽  
Drop On Demand ◽  
Serial Crystallography

The advent of the X-ray free electron laser (XFEL) in the last decade created the discipline of serial crystallography but also the challenge of how crystal samples are delivered to X-ray. Early sample delivery methods demonstrated the proof-of-concept for serial crystallography and XFEL but were beset with challenges of high sample consumption, jet clogging and low data collection efficiency. The potential of XFEL and serial crystallography as the next frontier of structural solution by X-ray for small and weakly diffracting crystals and provision of ultra-fast time-resolved structural data spawned a huge amount of scientific interest and innovation. To utilize the full potential of XFEL and broaden its applicability to a larger variety of biological samples, researchers are challenged to develop better sample delivery methods. Thus, sample delivery is one of the key areas of research and development in the serial crystallography scientific community. Sample delivery currently falls into three main systems: jet-based methods, fixed-target chips, and drop-on-demand. Huge strides have since been made in reducing sample consumption and improving data collection efficiency, thus enabling the use of XFEL for many biological systems to provide high-resolution, radiation damage-free structural data as well as time-resolved dynamics studies. This review summarizes the current main strategies in sample delivery and their respective pros and cons, as well as some future direction.

Pump-Probe Measurements Using Silicon Nanocrystal Waveguides

2003 ◽  
Vol 770 ◽  
Author(s):  
Nathanael Smith ◽  
Max J. Lederer ◽  
Marek Samoc ◽  
Barry Luther-Davies ◽  
Robert G. Elliman
Keyword(s):  
Probe Beam ◽  
Time Resolution ◽  
Pump Beam ◽  
Silicon Nanocrystals ◽  
Continuous Wave ◽  
Optical Gain ◽  
Optical Pump ◽  
Time Resolved ◽  
Pump Probe ◽  
Probe Measurements

AbstractOptical pump-probe measurements were performed on planar slab waveguides containing silicon nanocrystals in an attempt to measure optical gain from photo-excited silicon nanocrystals. Two experiments were performed, one with a continuous-wave probe beam and a pulsed pump beam, giving a time resolution of approximately 25 ns, and the other with a pulsed pump and probe beam, giving a time resolution of approximately 10 ps. In both cases the intensity of the probe beam was found to be attenuated by the pump beam, with the attenuation increasing monotonically with increasing pump power. Time-resolved measurements using the first experimental arrangement showed that the probe signal recovered its initial intensity on a time scale of 45-70 μs, a value comparable to the exciton lifetime in Si nanocrystals. These data are shown to be consistent with an induced absorption process such as confined carrier absorption. No evidence for optical gain was observed.

scholarly journals Synchrotron radiation macromolecular crystallography: our science and spin offs

2014 ◽  
Vol 70 (a1) ◽  
pp. C10-C10
Author(s):  
John Helliwell
Keyword(s):  
Synchrotron Radiation ◽  
Protein Complexes ◽  
Bound Water ◽  
Water Structure ◽  
Public Understanding ◽  
Anomalous Scattering ◽  
Structural Studies ◽  
Macromolecular Crystallography ◽  
Time Resolved ◽  
Chemical Crystallography

I will give an overview of synchrotron radiation (SR) in macromolecular crystallography (MX) instrumentation, methods and applications from the early days to the present, including the evolution of SR sources and on to the `ultimate storage ring'. The build of dedicated beamlines for resonant anomalous scattering, large unit cells, ever smaller crystals and studies up to ultra-high resolution are core benefits. Results include a high output of PDB depositions, the successful use of microcrystals, pushing the frontiers of using high and low photon energies and time-resolved structural studies at even sub-nanosecond resolutions. These intensively physics based developments will be complemented by biological and chemical crystallography research results, encompassing catalysis and marine coloration, as well as the public understanding of our science and its impacts. Spin off benefits include services to the pharmaceutical industry and helping develop chemical crystallography uses of SR. The development of the Laue method with SR has led to pioneering spin off developments in neutron MX, including transfer of the well validated Daresbury Laue software to various neutron facilities worldwide. Neutron MX is gathering pace as new instrumentation and dedicated sample preparation facilities are in place at reactor and spallation neutron sources; smaller samples and much larger molecular weight protein complexes are now feasible for investigation so as to establish their protonation states and bound water structure. With the X-ray lasers, closely linked to the SR developments, we anticipate the use of ever smaller samples such as nanocrystals, nanoclusters and single molecules, as well as opening up femtosecond time-resolved diffraction structural studies. At the SR sources, a very high throughput assessment for the best crystal samples and tackling sub-micron crystals will become widespread.

scholarly journals Polyimide mesh-based sample holder with irregular crystal mounting holes for fixed-target serial crystallography

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ki Hyun Nam ◽  
Jihan Kim ◽  
Yunje Cho
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Picosecond Laser ◽  
Temperature Structure ◽  
Sample Holder ◽  
Fixed Target ◽  
Random Orientation ◽  
Physical Damage ◽  
Crystal Sample ◽  
Serial Crystallography

AbstractThe serial crystallography (SX) technique enables the determination of the room-temperature structure of a macromolecule while causing minimal radiation damage, as well as the visualization of the molecular dynamics by time-resolved studies. The fixed-target (FT) scanning approach is one method for SX sample delivery that minimizes sample consumption and minimizes physical damage to crystals during data collection. Settling of the crystals on the sample holder in random orientation is important for complete three dimensional data collection. To increase the random orientation of crystals on the sample holder, we developed a polyimide mesh-based sample holder with irregular crystal mounting holes for FT-SX. The polyimide mesh was fabricated using a picosecond laser. Each hole in the polyimide mesh has irregularly shaped holes because of laser thermal damage, which may cause more crystals to settle at random orientations compared to regular shaped sample holders. A crystal sample was spread onto a polyimide-mesh, and a polyimide film was added to both sides to prevent dehydration. Using this sample holder, FT-SX was performed at synchrotron and determined the room-temperature lysozyme structure at 1.65 Å. The polyimide mesh with irregularly shaped holes will allow for expanded applications in sample delivery for FT-SX experiments.

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