The frontiers of time-resolved macromolecular crystallography: movies and chirped X-ray pulses

2002 ◽  
Vol 122 ◽  
pp. 65-77 ◽  
Author(s):  
Keith Moffat
Keyword(s):  
Macromolecular Crystallography ◽  
Time Resolved ◽  
X Ray

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.

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.

The emergence of the synchrotron Laue method for rapid data collection from protein crystals

1993 ◽  
Vol 442 (1914) ◽  
pp. 177-192 ◽  
Keyword(s):  
Multiplicity Distribution ◽  
Spectral Curve ◽  
Three Dimensional ◽  
Protein Crystal ◽  
Photographic Film ◽  
Macromolecular Crystallography ◽  
Time Resolved ◽  
X Ray ◽  
Laue Geometry ◽  
Laue Method

Synchrotron X-radiation (SR) is intense, polychromatic and collimated. It is widely exploited, in macromolecular crystallography, particularly using a monochromatized short wavelength beam. The spectral curve of SR, however, ideally lends itself to use of Laue geometry, i. e. the original diffraction experimental arrangement based on a stationary crystal and a polychromatic X-ray beam. Rapid exposure times and time-resolved crystallography studies, e. g. of enzymes, are now possible. Historical objections to the use of Laue diffraction data, particularly the multiplicity distribution, have been found not to be as limiting as once thought. The credentials of the Laue method have been established through a variety of Laue crystal structure analyses, involving photographic film as detector. Recently a three-dimensional arrangement of films, known as a toast-rack, has been used to alleviate problems with spatially overlapping spots. This paper provides a review of these results and then reports several developments. In particular, one of the first Laue analyses using an image plate as detector, namely of a cobalt substituted concanavalin A crystal, is discussed. Recent experimental developments, also at the Daresbury synchrotron, are then described. First, a large toast-rack has been used to record Laue data from a protein crystal. Secondly, a transmission X-ray mirror has been constructed from thin mylar (1.5 μm) and used to provide a λ max filter instead of using aluminium foils. Thirdly, since the Laue method suffers from poor sampling of the low resolution data, a new method (known as LOT) has been introduced.

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 Kilohertz Macromolecular Crystallography Using an EIGER Detector at Low X-ray Fluxes

Crystals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1146
Author(s):  
Krishna P. Khakurel ◽  
Shirly Espinoza ◽  
Martin Savko ◽  
Vitaly Polovinkin ◽  
Jan Dohnalek ◽  
...  
Keyword(s):  
Frame Rate ◽  
Macromolecular Crystallography ◽  
Experimental Proof ◽  
Time Resolved ◽  
X Ray ◽  
High Repetition ◽  
Pixel Detectors ◽  
Low X ◽  
Significant Effort ◽  
Made In

Time-resolved in-house macromolecular crystallography is primarily limited by the capabilities of the in-house X-ray sources. These sources can only provide a time-averaged structure of the macromolecules. A significant effort has been made in the development of in-house laser-driven ultrafast X-ray sources, with one of the goals as realizing the visualization of the structural dynamics of macromolecules at a very short timescale within the laboratory-scale infrastructure. Most of such in-house ultrafast X-ray sources are operated at high repetition rates and usually deliver very low flux. Therefore, the necessity of a detector that can operate at the repetition rate of the laser and perform extremely well under low flux conditions is essential. Here, we present experimental results demonstrating the usability of the hybrid-pixel detectors, such as Eiger X 1M, and provide experimental proof that they can be successfully operated to collect macromolecular crystallographic data up to a detector frame rate of 3 kHz from synchrotron sources. Our results also show that the data reduction and structural analysis are successful at such high frame rates and fluxes as low as 108 photons/s, which is comparable to the values expected from a typical laser-driven X-ray source.

Microtubule nucleation sequence studied by time-resolved x-ray scattering and electron microscopy

1983 ◽  
Vol 41 ◽  
pp. 766-767
Author(s):  
Eva-Maria Mandelkow ◽  
Eckhard Mandelkow ◽  
Joan Bordas
Keyword(s):  
Electron Microscopy ◽  
Dynamic Equilibrium ◽  
Time Resolved ◽  
X Ray ◽  
Additional Information ◽  
X Ray Scattering ◽  
Low Concentrations ◽  
Microtubule Growth ◽  
Ray Patterns ◽  
Ray Scattering

When a solution of microtubule protein is changed from non-polymerising to polymerising conditions (e.g. by temperature jump or mixing with GTP) there is a series of structural transitions preceding microtubule growth. These have been detected by time-resolved X-ray scattering using synchrotron radiation, and they may be classified into pre-nucleation and nucleation events. X-ray patterns are good indicators for the average behavior of the particles in solution, but they are difficult to interpret unless additional information on their structure is available. We therefore studied the assembly process by electron microscopy under conditions approaching those of the X-ray experiment. There are two difficulties in the EM approach: One is that the particles important for assembly are usually small and not very regular and therefore tend to be overlooked. Secondly EM specimens require low concentrations which favor disassembly of the particles one wants to observe since there is a dynamic equilibrium between polymers and subunits.

Time-Resolved Cryo-Electron Microscopy of Oscillating Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 502-503
Author(s):  
Eva-Maria Mandelkow ◽  
Ron Milligan
Keyword(s):  
Electron Microscopy ◽  
Dynamic Instability ◽  
Entire Solution ◽  
Reaction Scheme ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
A Chain ◽  
Ray Scattering

Microtubules form part of the cytoskeleton of eukaryotic cells. They are hollow libers of about 25 nm diameter made up of 13 protofilaments, each of which consists of a chain of heterodimers of α-and β-tubulin. Microtubules can be assembled in vitro at 37°C in the presence of GTP which is hydrolyzed during the reaction, and they are disassembled at 4°C. In contrast to most other polymers microtubules show the behavior of “dynamic instability”, i.e. they can switch between phases of growth and phases of shrinkage, even at an overall steady state [1]. In certain conditions an entire solution can be synchronized, leading to autonomous oscillations in the degree of assembly which can be observed by X-ray scattering (Fig. 1), light scattering, or electron microscopy [2-5]. In addition such solutions are capable of generating spontaneous spatial patterns [6].In an earlier study we have analyzed the structure of microtubules and their cold-induced disassembly by cryo-EM [7]. One result was that disassembly takes place by loss of protofilament fragments (tubulin oligomers) which fray apart at the microtubule ends. We also looked at microtubule oscillations by time-resolved X-ray scattering and proposed a reaction scheme [4] which involves a cyclic interconversion of tubulin, microtubules, and oligomers (Fig. 2). The present study was undertaken to answer two questions: (a) What is the nature of the oscillations as seen by time-resolved cryo-EM? (b) Do microtubules disassemble by fraying protofilament fragments during oscillations at 37°C?

Time-resolved X-ray reciprocal space mapping of the crystal under external electric field

2018 ◽  
Vol 189 (02) ◽  
pp. 187-194 ◽  
Author(s):  
Nikita V. Marchenkov ◽  
Anton G. Kulikov ◽  
Ivan I. Atknin ◽  
Arsen A. Petrenko ◽  
Alexander E. Blagov ◽  
...  
Keyword(s):  
Electric Field ◽  
External Electric Field ◽  
Space Mapping ◽  
Reciprocal Space ◽  
Reciprocal Space Mapping ◽  
Time Resolved ◽  
X Ray
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