Controlling the Diffusive Field to Grow a Higher Quality Protein Crystal in Microgravity

2012 ◽  
Vol 323-325 ◽  
pp. 549-554 ◽  
Author(s):  
Hiroaki Tanaka ◽  
Koji Inaka ◽  
Naoki Furubayashi ◽  
Mari Yamanaka ◽  
Sachiko Takahashi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Three Dimensional ◽  
Protein Crystal ◽  
Microgravity Environment ◽  
High Quality ◽  
X Ray ◽  
Crystallization Experiments ◽  
Protein Molecules ◽  
Filtering Effect ◽  
Internal Order

Growing high quality crystals is a bottleneck in the multi-stepped process of three-dimensional structural analyses of protein. It is known that a microgravity environment may maintain ideal depletion zones of protein and impurity around a growing crystal and the filtering effect of these depletion zones may contribute to obtaining high-resolution X-ray diffracting crystals with superior internal order. The effects of these depletion zones around growing crystals are thought to be the main mechanisms for the improvement of crystal quality in microgravity. A competition between the diffusion of protein molecules in the solution (indexed by the diffusion coefficient, D) and the adsorption of those into the growing crystal (indexed by the kinetic coeffcient, β) decides the extent of depletion zones. Lower D values and higher β values indicate that these effects are more obvious in numerical analyses. Therefore we use the D/β value as an index for these effects. The most effective method of lowering the D/β value is using viscous precipitant reagents, such as a high molecular weight polyethylene glycol (PEG) to decrease the D value and using highly homogenous protein samples to increase the β value. In this report, we briefly introduce simple yet practical methods of estimating D and β values followed by a numerical analysis to understand the filtration effects, and the results of crystallization experiments in microgravity when controlling the diffusive field around the growing crystals using the D/β value as an index.

Phase Diagram Visualization via Continuously-Fed Crystallization: Experiments and Model

2009 ◽  
Author(s):  
Masano T. Sugiyama ◽  
Victor H. Barocas
Keyword(s):  
Phase Diagram ◽  
Three Dimensional ◽  
Protein Crystal ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Single Experiment ◽  
X Ray ◽  
High Quality Protein ◽  
Crystallization Experiments ◽  
And Diffusion

A protein’s atomic level three-dimensional structure is typically determined by X-ray diffraction of a high-quality protein crystal (Figure 1a). The X-ray bream is diffracted inside the crystal producing spots at various locations (Figure 1b) which can be used to back out the location of the electron cloud of the protein which is used to solve the structure (Figure 1c). The current bottleneck in these structural determination projects is in growing a diffraction quality protein crystal. Current growth methods involve testing hundreds of conditions in this multi-parametric process without knowledge of the protein phase diagram (PD). In this work, a microfluidic crystallization chamber is manufactured to allow phase diagram visualization to predict the PD of a protein in a single experiment. This phase diagram visualizer (PDV) screens a large range of protein and salt concentrations by controlling the transport mechanism (convection and diffusion) of both salt and protein. The PDV has been fabricated and its flow and concentration profiles have been determined using a computational model. The PDV has predicted the metastable line of the phase diagram for tetragonal lysozyme (known PD) and the solubility line for triclinic lysozyme (unknown PD).

scholarly journals Semi-automated protein crystal mounting device for the sulfur single-wavelength anomalous diffraction method

2010 ◽  
Vol 43 (2) ◽  
pp. 341-346 ◽  
Author(s):  
Yu Kitago ◽  
Nobuhisa Watanabe ◽  
Isao Tanaka
Keyword(s):  
Diffraction Method ◽  
Systematic Errors ◽  
Protein Crystal ◽  
X Rays ◽  
Anomalous Diffraction ◽  
X Ray ◽  
Frozen Solution ◽  
Custom Made ◽  
Protein Molecules ◽  
X Ray Absorption

Use of longer-wavelength X-rays has advantages for the detection of small anomalous signals from light atoms, such as sulfur, in protein molecules. However, the accuracy of the measured diffraction data decreases at longer wavelengths because of the greater X-ray absorption. The capillary-top mounting method (formerly the loopless mounting method) makes it possible to eliminate frozen solution around the protein crystal and reduces systematic errors in the evaluation of small anomalous differences. However, use of this method requires custom-made tools and a large amount of skill. Here, the development of a device that can freeze the protein crystal semi-automatically using the capillary-top mounting method is described. This device can pick up the protein crystal from the crystallization drop using a micro-manipulator, and further procedures, such as withdrawal of the solution around the crystal by suction and subsequent flash freezing of the protein crystal, are carried out automatically. This device makes it easy for structural biologists to use the capillary-top mounting method for sulfur single-wavelength anomalous diffraction phasing using longer-wavelength X-rays.

Introduction

1986 ◽  
Vol 317 (1540) ◽  
pp. 293-294 ◽  
Keyword(s):  
Direct Method ◽  
Hydrolytic Enzyme ◽  
Hydrolytic Enzymes ◽  
Three Dimensional ◽  
X Ray ◽  
Enzyme Active Site ◽  
X Ray Crystallography ◽  
Protein Molecules ◽  
Biological Catalysis ◽  
The 1960S

Our understanding of the function of protein molecules was revolutionized in the 1960s by the use of X-ray crystallography to give a three-dimensional picture of their structures at atomic resolution. The structure of myoglobin was rapidly followed by the structure of several hydrolytic enzymes such as lysozyme, carboxypeptidase, ribonuclease, chymotrypsin, and subtilisin; and, not long after, by the much more complicated structure of haemoglobin, composed of four myoglobin-like molecules interacting with each other. The first hydrolytic enzyme structures showed us how enzymes perform biological catalysis by immobilizing their substrates at the enzyme active site, and gave us definite ideas about the specific functions of different parts of the protein molecules. These ideas had to be treated as hypotheses, because there was no direct method to check them. A few particular points could be proved by cunning but tedious experiments.

Numerical Analysis of the Diffusive Field around a Growing Protein Crystal in Microgravity

2012 ◽  
Vol 323-325 ◽  
pp. 565-569 ◽  
Author(s):  
Koji Inaka ◽  
Hiroaki Tanaka ◽  
Sachiko Takahashi ◽  
Satoshi Sano ◽  
Masaru Sato ◽  
...  
Keyword(s):  
Numerical Analysis ◽  
Kinetic Constant ◽  
Protein Crystal ◽  
Microgravity Environment ◽  
X Ray Diffraction ◽  
X Ray ◽  
Microgravity Experiments ◽  
Space Agency ◽  
Filtration Effect

t is believed that a microgravity environment may maintain ideal depletion zones of protein (PDZ) and impurity (IDZ) around growing crystals and may contribute to growing high-quality crystals. This can lead to an X-ray diffraction data collection of higher resolution with lower mosaicity, because of the better internal order and fewer defects in the crystals when compared to ground-grown crystals. The extent of these depletion zones are dependent on a competition between the diffusion of the molecules in the solution (indexed by the diffusion coefficient, D) and the adsorption of those into the growing crystal (indexed by the kinetic constant, β). If we use the D/β value as an index of the extent of PDZ and IDZ, a lower D/β value is ideal for maintaining PDZ and IDZ. Using experimental results, we could easily obtain the D/β value. When we combined the D/β value with the quality of protein crystals obtained in microgravity experiments provided by Japanese Space Agency (JAXA), we found that the effects of microgravity contributed to obtaining superior crystals especially if the D/β value was less than 3 mm. The numerical analysis of the PDZ and IDZ shows that the radius of the crystal (R) is also related to the PDZ and the IDZ. If the Rβ/D value is large, both the PDZ and the IDZ provide a filtration effect, but if the Rβ/D value is small, only the IDZ does.

STUDY OF COMPLEX Cu-O LATTICE IN La8-xSrxCu8O20 BY HIGH RESOLUTION X-RAY ABSORPTION SPECTROSCOPY

2000 ◽  
Vol 14 (29n31) ◽  
pp. 3656-3661 ◽  
Author(s):  
N. L. SAINI ◽  
A. LANZARA ◽  
K. B. GARG ◽  
A. BIANCONI ◽  
T. ITO ◽  
...  
Keyword(s):  
High Resolution ◽  
Single Crystals ◽  
Absorption Spectroscopy ◽  
Local Structure ◽  
Three Dimensional ◽  
Xanes Spectroscopy ◽  
High Quality ◽  
X Ray ◽  
X Ray Absorption

Cu K-edge XANES spectroscopy has been used to study local structure around the Cu-site in three-dimensional oxygen deficient perovkite La 8- x Sr x Cu 8 O 20 system. The combination of E//ab and E//c polarization and high resolution XANES spectra on high quality single crystals has allowed us to distinguish the various features associated with the complex Cu-O networks in the system which have then been compared with those of the La 2- x Sr x CuO 4 system.

scholarly journals Comparison of the Quality of Protein Crystals Grown by CLPC Seeds Method

Crystals ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 501 ◽  
Author(s):  
Li ◽  
Yan ◽  
Liu ◽  
Wu ◽  
Liu ◽  
...  
Keyword(s):  
Diffraction Data ◽  
Protein Crystal ◽  
Protein Crystals ◽  
X Ray Diffraction ◽  
High Quality ◽  
X Ray ◽  
High Quality Protein

We present a systematic quality comparison of protein crystals obtained with and without cross-linked protein crystal (CLPC) seeds. Four proteins were used to conduct the experiments, and the results showed that crystals obtained in the presence of CLPC seeds exhibited a better morphology. In addition, the X-ray diffraction data showed that the CLPC seeds method is a powerful tool to obtain high-quality protein crystals. Therefore, we recommend the use of CLPC seeds in preparing high-quality diffracting protein crystals.

scholarly journals A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals

2013 ◽  
Vol 69 (7) ◽  
pp. 1223-1230 ◽  
Author(s):  
Igor Nederlof ◽  
Eric van Genderen ◽  
Yao-Wang Li ◽  
Jan Pieter Abrahams
Keyword(s):  
Electron Diffraction ◽  
Data Collection ◽  
Diffraction Data ◽  
Three Dimensional ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Electron Diffraction Data ◽  
Single Shot ◽  
X Ray ◽  
Area Detector

When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e− Å−2), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ∼100 nm thick protein crystal. A highly parallel 200 keV electron beam (λ = 0.025 Å) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4°. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLMandSCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins.

scholarly journals Femtosecond X-ray diffraction from two-dimensional protein crystals

IUCrJ ◽  
2014 ◽  
Vol 1 (2) ◽  
pp. 95-100 ◽  
Author(s):  
Matthias Frank ◽  
David B. Carlson ◽  
Mark S. Hunter ◽  
Garth J. Williams ◽  
Marc Messerschmidt ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Bragg Diffraction ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Two Dimensional ◽  
X Ray Diffraction ◽  
X Ray ◽  
Linac Coherent Light Source ◽  
Diffraction Patterns ◽  
Better Than

X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

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.

Present and Future Accelerator-Based X-ray Sources: A Perspective

2019 ◽  
Vol 10 (01) ◽  
pp. 33-48
Author(s):  
J. B. Hastings ◽  
L. Rivkin ◽  
G. Aeppli
Keyword(s):  
Data Science ◽  
High Temperature Superconductors ◽  
Three Dimensional ◽  
Structural Studies ◽  
X Ray ◽  
Protein Molecules ◽  
Complex Protein ◽  
Image Production ◽  
Photon Science ◽  
Computation Data

Accelerator-based X-ray sources have contributed uniquely to the physical, engineering and life sciences. There has been a constant development of the sources themselves as well as of the necessary X-ray optics and detectors. These advances have combined to push X-ray science to the forefront in structural studies, achieving atomic resolution for complex protein molecules, to meV scale dynamics addressing problems ranging from geoscience to high-temperature superconductors, and to spatial resolutions approaching 10[Formula: see text]nm for elemental mapping as well as three-dimensional structures. Here we discuss accelerator-based photon science in the frame of imaging and highlight the importance of optics, detectors and computation/data science as well as the source technology. We look to a bright future for X-ray systems, integrating all components from accelerator sources to digital image production algorithms, and highlight aspects that make them unique scientific tools.

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