scholarly journals Visualizing soaking process of protein crystal

2014 ◽  
Vol 70 (a1) ◽  
pp. C1661-C1661
Author(s):  
Ryuta Mizutani ◽  
Yusuke Shimizu ◽  
Rino Saiga ◽  
Go Ueno ◽  
Yuki Nakamura ◽  
...  
Keyword(s):  
Process Model ◽  
Superficial Layer ◽  
Protein Crystal ◽  
Time Resolved ◽  
Crystal Core ◽  
Lysozyme Crystals ◽  
Density Development ◽  
Synchrotron Radiation Microtomography ◽  
Lysozyme Crystal ◽  
Platinum Derivative

Time-resolved visualization of the soaking process of tetragonal lysozyme crystal was performed by synchrotron radiation microtomography. Mother liquor containing hexachloroplatinate was introduced into a capillary bearing lysozyme crystals to visualize crystals undergoing soaking. The platinum distribution was first observed in the superficial layer of crystal and then gradually penetrated into the crystal core. The crystal structure of the platinum derivative in each soaking period was determined by time-resolved crystallography. A total of five platinum sites were identified in Bijvoet difference maps. These sites were classified into two groups on the basis of the time dependence of electron density development. A soaking process model consisting of binding-rate-driven and equilibrium-driven layers is proposed to describe the results. This study suggests that the structures of soaked crystals vary depending on the crystal position from which diffractions were taken.

Crystallization of high-quality protein crystals using an external electric field

2015 ◽  
Vol 48 (5) ◽  
pp. 1507-1513 ◽  
Author(s):  
H. Koizumi ◽  
S. Uda ◽  
K. Fujiwara ◽  
M. Tachibana ◽  
K. Kojima ◽  
...  
Keyword(s):  
Electric Field ◽  
External Electric Field ◽  
Applied Field ◽  
Local Strain ◽  
Protein Crystal ◽  
X Ray Diffraction ◽  
High Quality Protein ◽  
Hen Egg White ◽  
Lysozyme Crystals

The effect of a 20 kHz external electric field on the quality of tetragonal hen egg white (HEW) lysozyme crystals was investigated using X-ray diffraction rocking-curve measurements. The full width at half-maximum was found to be larger for high-order reflections but smaller for low-order reflections. In particular, it was revealed that a large amount of local strain is accumulated in tetragonal HEW lysozyme crystals grown under an applied field at 20 kHz. Comparison with previous results obtained for crystals grown with an applied field at 1 MHz [Koizumi, Uda, Fujiwara, Tachibana, Kojima & Nozawa (2013).J. Appl. Cryst.46, 25–29] indicated that improvement of the protein crystal quality could be achieved by selection of an appropriate frequency for the applied electric field, which has a significant effect on the growth of the solid.

Protein crystal dynamics studied by time-resolved analysis of X-ray diffuse scattering

Nature ◽  
1989 ◽  
Vol 338 (6217) ◽  
pp. 665-666 ◽  
Author(s):  
G. U. Nienhaus ◽  
J. Heinzl ◽  
E. Huenges ◽  
F. Parak
Keyword(s):  
Diffuse Scattering ◽  
Protein Crystal ◽  
Time Resolved ◽  
X Ray ◽  
Crystal Dynamics

Synchrotron X-ray crystallography techniques: time-resolved aspects of data collection

1992 ◽  
Vol 340 (1657) ◽  
pp. 221-232 ◽  
Keyword(s):  
Data Collection ◽  
Three Dimensional ◽  
Protein Crystal ◽  
Measure Data ◽  
Laue Pattern ◽  
Data Sets ◽  
Time Resolved ◽  
Spatial Overlap ◽  
X Ray Crystallography ◽  
Unit Cells

Synchrotron X-radiation (SR) is intense, polychromatic and collimated. These properties are exploited routinely now to measure data at high resolution from proteins or from large unit cells (e.g. viruses), particularly using a monochromatized short wavelength beam. The time needed to measure protein crystal data-sets (in rotation geometry) can be quick (hours or minutes) compared with laboratory sources (weeks or days). Even so, more rapid data collection is of interest for timeresolved macromolecular crystallography. White beam : stationary crystal (Laue) geometry at SR sources offers shorter exposure times (seconds and less with film). Laue data-sets can be sensitive to subtle structural differences. Technical challenges still presented by SR Laue patterns include the energy overlap of low-resolution data and the spatial overlap of spots, both of which affect the completeness of data-sets. Some energy deconvolution is already possible by the use of multiple films. The spatial overlap problem can be alleviated by the use of three-dimensional detectors, such as a ‘toast-rack’ of plates. Monitoring of a crystalline process, via the Laue pattern, requires time slicing detector systems (e.g. based on CCDS) to be developed.

scholarly journals Protein Crystal Motions from Time-Resolved Diffracted X-ray Blinking

2020 ◽  
Vol 118 (3) ◽  
pp. 487a
Author(s):  
Yuji C. Sasaki ◽  
Masahiro Kuramochi ◽  
Kazuhiro Mio ◽  
Hiroshi Sekiguchi ◽  
Ayana Sato-Tomita ◽  
...  
Keyword(s):  
Protein Crystal ◽  
Time Resolved ◽  
X Ray

scholarly journals Reconstructing three-dimensional protein crystal intensities from sparse unoriented two-axis X-ray diffraction patterns

2017 ◽  
Vol 50 (4) ◽  
pp. 985-993 ◽  
Author(s):  
Ti-Yen Lan ◽  
Jennifer L. Wierman ◽  
Mark W. Tate ◽  
Hugh T. Philipp ◽  
Veit Elser ◽  
...  
Keyword(s):  
Radiation Damage ◽  
Parallel Computation ◽  
Three Dimensional ◽  
Protein Crystal ◽  
X Ray Diffraction ◽  
X Ray ◽  
Small Crystals ◽  
Diffraction Patterns ◽  
Memory Scaling ◽  
Lysozyme Crystal

Recently, there has been a growing interest in adapting serial microcrystallography (SMX) experiments to existing storage ring (SR) sources. For very small crystals, however, radiation damage occurs before sufficient numbers of photons are diffracted to determine the orientation of the crystal. The challenge is to merge data from a large number of such `sparse' frames in order to measure the full reciprocal space intensity. To simulate sparse frames, a dataset was collected from a large lysozyme crystal illuminated by a dim X-ray source. The crystal was continuously rotated about two orthogonal axes to sample a subset of the rotation space. With the EMC algorithm [expand–maximize–compress; Loh & Elser (2009).Phys. Rev. E,80, 026705], it is shown that the diffracted intensity of the crystal can still be reconstructed even without knowledge of the orientation of the crystal in any sparse frame. Moreover, parallel computation implementations were designed to considerably improve the time and memory scaling of the algorithm. The results show that EMC-based SMX experiments should be feasible at SR sources.

scholarly journals Combining X-rays, neutrons and electrons, and NMR, for precision and accuracy in structure–function studies

2021 ◽  
Vol 77 (3) ◽  
pp. 173-185
Author(s):  
John R. Helliwell
Keyword(s):  
Structure Function ◽  
Crystal Structures ◽  
Structure Prediction ◽  
Protein Crystal ◽  
X Rays ◽  
Time Resolved ◽  
X Ray ◽  
Precision And Accuracy ◽  
Electron Microscopes

The distinctive features of the physics-based probes used in understanding the structure of matter focusing on biological sciences, but not exclusively, are described in the modern context. This is set in a wider scope of holistic biology and the scepticism about `reductionism', what is called the `molecular level', and how to respond constructively. These topics will be set alongside the principles of accuracy and precision, and their boundaries. The combination of probes and their application together is the usual way of realizing accuracy. The distinction between precision and accuracy can be blurred by the predictive force of a precise structure, thereby lending confidence in its potential accuracy. These descriptions will be applied to the comparison of cryo and room-temperature protein crystal structures as well as the solid state of a crystal and the same molecules studied by small-angle X-ray scattering in solution and by electron microscopy on a sample grid. Examples will include: time-resolved X-ray Laue crystallography of an enzyme Michaelis complex formed directly in a crystal equivalent to in vivo; a new iodoplatin for radiation therapy predicted from studies of platin crystal structures; and the field of colouration of carotenoids, as an effective assay of function, i.e. their colouration, when unbound and bound to a protein. The complementarity of probes, as well as their combinatory use, is then at the foundation of real (biologically relevant), probe-artefacts-free, structure–function studies. The foundations of our methodologies are being transformed by colossal improvements in technologies of X-ray and neutron sources and their beamline instruments, as well as improved electron microscopes and NMR spectrometers. The success of protein structure prediction from gene sequence recently reported by CASP14 also opens new doors to change and extend the foundations of the structural sciences.

scholarly journals Serial Femtosecond Crystallography user's consortium apparatus at European XFEL

2014 ◽  
Vol 70 (a1) ◽  
pp. C1748-C1748
Author(s):  
Marc Messerschmidt ◽  
Leonard Chavas ◽  
Sunil Ananthaneni ◽  
Hamidreza Dadgostar ◽  
Heinz Graafsma ◽  
...  
Keyword(s):  
Protein Crystal ◽  
Pixel Detector ◽  
Single Particles ◽  
Time Resolved ◽  
X Ray ◽  
Overall Design ◽  
Laser Facility ◽  
Common Problems ◽  
Automated Procedures ◽  
Serial Femtosecond Crystallography

The Serial Femtosecond Crystallography (SFX) user's consortium apparatus is to be installed within the Single Particles, Clusters and Biomolecules (SPB) instrument of the European X-ray Free-Electron Laser facility (XFEL.EU) [1, 2]. The XFEL.EU will provide ultra-short, highly intense, coherent X-ray pulses at an unprecedented repetition rate. The experimental setup and methodological approaches of many scientific areas will be transformed, including structural biology that could potentially overcome common problems and bottlenecks encountered in crystallography, such as creating large crystals, dealing with radiation damage, or understanding sub-picosecond time-resolved phenomena. The key concept of the SFX method is based on the kinetic insertion of protein crystal samples in solution via a gas dynamic virtual nozzle jet and recording diffraction signals of individual, randomly oriented crystals passing through the XFEL beam, as first demonstrated by Chapman et al. [3]. The SFX-apparatus will refocus the beam spent by the SPB instrument into a second interaction region, in some cases enabling two parallel experiments. The planned photon energy range at the SPB instrument is from 3 to 16 keV. The Adaptive Gain Integrating Pixel Detector (AGIPD) is to be implemented in the SPB instrument, including a 4 Megapixel version for the SFX-apparatus. The AGIPD is designed to store over 350 data frames from successive pulses, and aims to collect more than 3,000 images per second. Together with the implementation of automated procedures for sample exchange and injection, high-throughput nanocrystallography experiments can be integrated at the SFX-apparatus. In this work, we review the overall design of the SFX-apparatus and discuss the main parameters and challenges

scholarly journals The Study of the Mechanism of Protein Crystallization in Space by Using Microchannel to Simulate Microgravity Environment

Crystals ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 400 ◽  
Author(s):  
Yong Yu ◽  
Kai Li ◽  
Hai Lin ◽  
Ji-Cheng Li
Keyword(s):  
Growth Rates ◽  
Simulated Microgravity ◽  
Crystal Surface ◽  
Protein Crystallization ◽  
Experimental Results ◽  
Protein Crystals ◽  
Microgravity Environment ◽  
Microgravity Conditions ◽  
Lysozyme Crystals ◽  
Lysozyme Crystal

Space is expected to be a convection-free, quiescent environment for the production of large-size and high-quality protein crystals. However, the mechanisms by which the diffusion environment in space improves the quality of the protein crystals are not fully understood. The interior of a microfluidic device can be used to simulate a microgravity environment to investigate the protein crystallization mechanism that occurs in space. In the present study, lysozyme crystals were grown in a prototype microchannel device with a height of 50 μm in a glass-polydimethylsiloxane (PDMS)-glass sandwich structure. Comparative experiments were also conducted in a sample pool with a height of 2 mm under the same growth conditions. We compared the crystal morphologies and growth rates of the grown crystals in the two sample pools. The experimental results showed that at very low initial supersaturation, the morphology and growth rates of lysozyme crystals under the simulated microgravity conditions is similar to that on Earth. With increasing initial supersaturation, a convection-free, quiescent environment is better for lysozyme crystal growth. When the initial supersaturation exceeded a threshold, the growth of the lysozyme crystal surface under the simulated microgravity conditions never completely transform from isotropic to anisotropic. The experimental results showed that the convection may have a dual effect on the crystal morphology. Convection can increase the roughness of the crystal surface and promote the transformation of the crystal form from circular to tetragonal during the crystallization process.

scholarly journals X-ray diffraction in temporally and spatially resolved biomolecular science

2015 ◽  
Vol 177 ◽  
pp. 429-441 ◽  
Author(s):  
John R. Helliwell ◽  
Alice Brink ◽  
Surasak Kaenket ◽  
Victoria Laurina Starkey ◽  
Simon W. M. Tanley
Keyword(s):  
Single Molecule ◽  
Structural Changes ◽  
Protein Crystallography ◽  
European Synchrotron Radiation Facility ◽  
Protein Crystal ◽  
Structural Studies ◽  
X Ray Diffraction ◽  
Microscopy Imaging ◽  
Time Resolved ◽  
X Ray

Time-resolved Laue protein crystallography at the European Synchrotron Radiation Facility (ESRF) opened up the field of sub-nanosecond protein crystal structure analyses. There are a limited number of such time-resolved studies in the literature. Why is this? The X-ray laser now gives us femtosecond (fs) duration pulses, typically 10 fs up to ∼50 fs. Their use is attractive for the fastest time-resolved protein crystallography studies. It has been proposed that single molecules could even be studied with the advantage of being able to measure X-ray diffraction from a ‘crystal lattice free’ single molecule, with or without temporal resolved structural changes. This is altogether very challenging R&D. So as to assist this effort we have undertaken studies of metal clusters that bind to proteins, both ‘fresh’ and after repeated X-ray irradiation to assess their X-ray-photo-dynamics, namely Ta6Br12, K2PtI6 and K2PtBr6 bound to a test protein, hen egg white lysozyme. These metal complexes have the major advantage of being very recognisable shapes (pseudo spherical or octahedral) and thereby offer a start to (probably very difficult) single molecule electron density map interpretations, both static and dynamic. A further approach is to investigate the X-ray laser beam diffraction strength of a well scattering nano-cluster; an example from nature being the iron containing ferritin. Electron crystallography and single particle electron microscopy imaging offers alternatives to X-ray structural studies; our structural studies of crustacyanin, a 320 kDa protein carotenoid complex, can be extended either by electron based techniques or with the X-ray laser representing a fascinating range of options. General outlook remarks concerning X-ray, electron and neutron macromolecular crystallography as well as ‘NMR crystallography’ conclude the article.

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