scholarly journals Mix and Inject: Reaction Initiation by Diffusion for Time-Resolved Macromolecular Crystallography

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Marius Schmidt
Keyword(s):  
Chemical Kinetics ◽  
Structure Determination ◽  
Macromolecular Crystallography ◽  
Time Resolved ◽  
X Ray ◽  
Transient States ◽  
Kinetic Mechanisms ◽  
Active Substrate ◽  
Reaction Initiation

Time-resolved macromolecular crystallography unifies structure determination with chemical kinetics, since the structures of transient states and chemical and kinetic mechanisms can be determined simultaneously from the same data. To start a reaction in an enzyme, typically, an initially inactive substrate present in the crystal is activated. This has particular disadvantages that are circumvented when active substrate is directly provided by diffusion. However, then it is prohibitive to use macroscopic crystals because diffusion times become too long. With small micro- and nanocrystals diffusion times are adequately short for most enzymes and the reaction can be swiftly initiated. We demonstrate here that a time-resolved crystallographic experiment becomes feasible by mixing substrate with enzyme nanocrystals which are subsequently injected into the X-ray beam of a pulsed X-ray source.

scholarly journals Double-focusing mixing jet for XFEL study of chemical kinetics

2014 ◽  
Vol 21 (6) ◽  
pp. 1364-1366 ◽  
Author(s):  
Dingjie Wang ◽  
Uwe Weierstall ◽  
Lois Pollack ◽  
John Spence
Keyword(s):  
Chemical Kinetics ◽  
Chemical Kinetic ◽  
Time Resolved ◽  
X Ray ◽  
Liquid Mixing ◽  
Combine Structure ◽  
Kinetic Mechanisms ◽  
Injection Methods ◽  
Double Focusing

Several liquid sample injection methods have been developed to satisfy the requirements for serial femtosecond X-ray nanocrystallography, which enables radiation-damage-free determination of molecular structure at room temperature. Time-resolved nanocrystallography would combine structure analysis with chemical kinetics by determining the structures of the transient states and chemical kinetic mechanisms simultaneously. A windowless liquid mixing jet device has been designed for this purpose. It achieves fast uniform mixing of substrates and enzymes in the jet within 250 µs, with an adjustable delay between mixing and probing by the X-ray free-electron laser beam of up to 1 s for each frame of a `movie'. The principle of the liquid mixing jet device is illustrated using numerical simulation, and experimental results are presented using a fluorescent dye.

scholarly journals NSLS-II MX beamlines FMX for micro-crystallography & AMX for highly automated MX

2014 ◽  
Vol 70 (a1) ◽  
pp. C1733-C1733
Author(s):  
Martin Fuchs ◽  
Robert Sweet ◽  
Lonny Berman ◽  
Dileep Bhogadi ◽  
Wayne Hendrickson ◽  
...  
Keyword(s):  
Energy Range ◽  
Structure Determination ◽  
Optical Systems ◽  
Photon Flux ◽  
X Rays ◽  
Macromolecular Crystallography ◽  
Binding Studies ◽  
X Ray ◽  
Array Detectors ◽  
Broad Energy Range

We present the final design of the x-ray optical systems and experimental stations of the two macromolecular crystallography (MX) beamlines, FMX and AMX, at the National Synchrotron Light Source-II (NSLS-II). Along with its companion x-ray scattering beamline, LIX, this suite of Advanced Beamlines for Biological Investigations with X-rays (ABBIX, [1]) will begin user operation in 2016. The pair of MX beamlines with complementary and overlapping capabilities is located at canted undulators (IVU21) in sector 17-ID. The Frontier Microfocusing Macromolecular Crystallography beamline (FMX) will deliver a photon flux of ~5x10^12 ph/s at a wavelength of 1 Å into a spot of 1 - 50 µm size. It will cover a broad energy range from 5 - 30 keV, corresponding to wavelengths from 0.4 - 2.5 Å. The highly Automated Macromolecular Crystallography beamline (AMX) will be optimized for high throughput applications, with beam sizes from 4 - 100 µm, an energy range of 5 - 18 keV (0.7 - 2.5 Å), and a flux at 1 Å of ~10^13 ph/s. Central components of the in-house-developed experimental stations are a 100 nm sphere of confusion goniometer with a horizontal axis, piezo-slits to provide dynamic beam size changes during diffraction experiments, a dedicated secondary goniometer for crystallization plates, and sample- and plate-changing robots. FMX and AMX will support a broad range of biomedical structure determination methods from serial crystallography on micron-sized crystals, to structure determination of complexes in large unit cells, to rapid sample screening and data collection of crystals in trays, for instance to characterize membrane protein crystals and to conduct ligand-binding studies. Together with the solution scattering program at LIX, the new beamlines will offer unique opportunities for advanced diffraction experiments with micro- and mini-beams, with next generation hybrid pixel array detectors and emerging crystal delivery methods such as acoustic droplet ejection. This work is supported by the US National Institutes of Health.

scholarly journals The Role of Validation in Macromolecular Crystallography

1998 ◽  
Vol 54 (6) ◽  
pp. 1109-1118 ◽  
Author(s):  
Eleanor Dodson
Keyword(s):  
Structure Determination ◽  
Atomic Resolution ◽  
File Format ◽  
Macromolecular Crystallography ◽  
X Ray

The importance of validation techniques in X-ray structure determination and their relation to refinement procedures are discussed, with particular reference to atomic resolution structures. The requirements of deposition and publication, and the role of validation tools in this are analysed. The need for a rigorously defined file format is emphasized.

scholarly journals Time-Resolved Macromolecular Crystallography at Pulsed X-ray Sources

2019 ◽  
Vol 20 (6) ◽  
pp. 1401 ◽  
Author(s):  
Marius Schmidt
Keyword(s):  
Structural Biology ◽  
Free Electron ◽  
Reaction Dynamics ◽  
X Rays ◽  
Free Electron Lasers ◽  
Macromolecular Crystallography ◽  
Time Resolved ◽  
X Ray ◽  
Methodological Aspects

The focus of structural biology is shifting from the determination of static structures to the investigation of dynamical aspects of macromolecular function. With time-resolved macromolecular crystallography (TRX), intermediates that form and decay during the macromolecular reaction can be investigated, as well as their reaction dynamics. Time-resolved crystallographic methods were initially developed at synchrotrons. However, about a decade ago, extremely brilliant, femtosecond-pulsed X-ray sources, the free electron lasers for hard X-rays, became available to a wider community. TRX is now possible with femtosecond temporal resolution. This review provides an overview of methodological aspects of TRX, and at the same time, aims to outline the frontiers of this method at modern pulsed X-ray sources.

RADDOSE-3D: time- and space-resolved modelling of dose in macromolecular crystallography

2013 ◽  
Vol 46 (4) ◽  
pp. 1225-1230 ◽  
Author(s):  
Oliver B. Zeldin ◽  
Markus Gerstel ◽  
Elspeth F. Garman
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Diffraction Experiment ◽  
Macromolecular Crystallography ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
Online Methods ◽  
Crystal Volume ◽  
Number Of Iterations

RADDOSE-3D allows the macroscopic modelling of an X-ray diffraction experiment for the purpose of better predicting radiation-damage progression. The distribution of dose within the crystal volume is calculated for a number of iterations in small angular steps across one or more data collection wedges, providing a time-resolved picture of the dose state of the crystal. The code is highly modular so that future contributions from the community can be easily integrated into it, in particular to incorporate online methods for determining the shape of macromolecular crystals and better protocols for imaging real experimental X-ray beam profiles.

scholarly journals Towards the time resolved X-ray structure determination of proteins

2012 ◽  
Vol 1817 ◽  
pp. S29-S30
Author(s):  
Raimund Fromme ◽  
Ingo Grotjohann ◽  
Christopher Kupitz ◽  
Shibom Basu ◽  
Petra Fromme
Keyword(s):  
Structure Determination ◽  
Time Resolved ◽  
X Ray

Time-resolved macromolecular crystallography: introductory remarks and a little history

1992 ◽  
Vol 340 (1657) ◽  
pp. 169-173 ◽  
Keyword(s):  
Crystallographic Data ◽  
Macromolecular Crystallography ◽  
Time Resolved ◽  
X Ray ◽  
Early Use ◽  
Laue Method

X -ray crystallographic data can be collected rapidly at synchrotrons by the Laue method. The early use of the Laue method by Ewald, Nishikawa, Wyckoff and Pauling is reviewed.

scholarly journals First Experiments in Structural Biology at the European X-ray Free-Electron Laser

2020 ◽  
Vol 10 (10) ◽  
pp. 3642 ◽  
Author(s):  
Grant Mills ◽  
Richard Bean ◽  
Adrian P. Mancuso
Keyword(s):  
Structural Biology ◽  
Structure Determination ◽  
Free Electron ◽  
Free Electron Laser ◽  
Pulse Frequency ◽  
Free Electron Lasers ◽  
Time Resolved ◽  
New Era ◽  
X Ray ◽  
Rapid Pulse

Ultrabright pulses produced in X-ray free-electron lasers (XFELs) offer new possibilities for industry and research, particularly for biochemistry and pharmaceuticals. The unprecedented brilliance of these next-generation sources enables structure determination from sub-micron crystals as well as radiation-sensitive proteins. The European X-Ray Free-Electron Laser (EuXFEL), with its first light in 2017, ushered in a new era for ultrabright X-ray sources by providing an unparalleled megahertz-pulse repetition rate, with orders of magnitude more pulses per second than previous XFEL sources. This rapid pulse frequency has significant implications for structure determination; not only will data collection be faster (resulting in more structures per unit time), but experiments requiring large quantities of data, such as time-resolved structures, become feasible in a reasonable amount of experimental time. Early experiments at the SPB/SFX instrument of the EuXFEL demonstrate how such closely-spaced pulses can be successfully implemented in otherwise challenging experiments, such as time-resolved studies.

scholarly journals Advances in long-wavelength native phasing at X-ray free-electron lasers

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 965-975 ◽  
Author(s):  
Karol Nass ◽  
Robert Cheng ◽  
Laura Vera ◽  
Aldo Mozzanica ◽  
Sophie Redford ◽  
...  
Keyword(s):  
Data Quality ◽  
Structure Determination ◽  
Free Electron ◽  
De Novo ◽  
Geometry Optimization ◽  
Protein Structure Determination ◽  
Time Resolved ◽  
X Ray ◽  
Long Wavelength ◽  
Order Of Magnitude

Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for de novo protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized de novo structure determination of challenging proteins at XFELs. Improvements presented in this work can be used in other types of SFX experiments that require accurate measurements of weak signals, for example time-resolved studies.

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