scholarly journals Processing of Protein Crystals using by Deep-UV Pulsed Laser

2014 ◽  
Vol 70 (a1) ◽  
pp. C334-C334
Author(s):  
Yoshiaki Kawano ◽  
Takaaki Hikima ◽  
Kunio Hirata ◽  
Seiki Baba ◽  
Hironori Murakami ◽  
...  
Keyword(s):  
Diffraction Data ◽  
Processing System ◽  
Spherical Shape ◽  
Systematic Errors ◽  
Mirror System ◽  
Protein Crystal ◽  
Protein Crystals ◽  
X Rays ◽  
Uv Laser ◽  
Pass Through

The absorption of X-rays which pass through the protein crystal is possibly the largest source of systematic errors in macromolecular crystallography. Therefore we are developing protein crystal processing system using Pulsed UV Laser Soft Ablation (PULSA) technique [1] to reduce the systematic error as well as background scattering from cryoprotectant agents. For high-quality diffraction data collection from organic material, crystals are usually processed to spherical shape in order to keep X-ray path length in crystal constant. This dramatically reduces systematic errors caused by `absorption of X-rays'. Although shaping crystal was thought to be effective for protein crystallography, there was no usual technique to achieve this because protein crystals are exceedingly fragile against mechanical stress. We are developing protein crystal processing system using PULSA technique. In this system, short pulsed UV-laser (maximum power: 1.0 μJ/pulse, wavelength: 193.4 nm, duration: less than 1.3 nsec) is raised by NSL-193L (Nikon Corporation) and focused on 4 μmφ (FWHM). The focused laser is controlled by galvanomic mirror system and irradiates a sample. Combining this mirror system with four-axis goniometer enables to process crystal to arbitrary shape that is easily defined on GUI. Several protein crystals have been successfully processed into spherical, column and square pole shape, etc. In the case of crystal processed into column shape (diameter is 50 μm), in addition to reducing absorption effects, signal-noise ratio of diffraction data can be increased by removing cryoprotectant agent surrounding the crystal. This work was supported by "Platform for Drug Discovery, Informatics, and Structural Life Science" from MEXT, Japan.

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.

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 Radiation damage in protein crystals examined under various conditions by different methods

2009 ◽  
Vol 16 (2) ◽  
pp. 129-132 ◽  
Author(s):  
Elspeth F. Garman ◽  
Colin Nave
Keyword(s):  
Radiation Damage ◽  
Electronic State ◽  
Room Temperature ◽  
Protein Crystal ◽  
Protein Crystals ◽  
X Rays ◽  
X Ray Diffraction ◽  
Radiation Physics ◽  
X Ray ◽  
The Past

Investigation of radiation damage in protein crystals has progressed in several directions over the past couple of years. There have been improvements in the basic procedures such as calibration of the incident X-ray intensity and calculation of the dose likely to be deposited in a crystal of known size and composition with this intensity. There has been increased emphasis on using additional techniques such as optical, Raman or X-ray spectroscopy to complement X-ray diffraction. Apparent discrepancies between the results of different techniques can be explained by the fact that they are sensitive to different length scales or to changes in the electronic state rather than to movement of atoms. Investigations have been carried out at room temperature as well as cryo-temperatures and, in both cases, with the introduction of potential scavenger molecules. These and other studies are leading to an overall description of the changes which can occur when a protein crystal is irradiated with X-rays at both cryo- and room temperatures. Results from crystallographic and spectroscopic radiation-damage experiments can be reconciled with other studies in the field of radiation physics and chemistry.

Recent X-ray diffraction studies of muscle

1968 ◽  
Vol 1 (2) ◽  
pp. 177-216 ◽  
Author(s):  
Jean Hanson
Keyword(s):  
Electron Microscopy ◽  
Diffraction Method ◽  
Artificial Muscle ◽  
Regular Structure ◽  
Normal Activity ◽  
Protein Crystal ◽  
Natural State ◽  
X Rays ◽  
X Ray Diffraction ◽  
Muscle Biology

An intact living muscle has such a regular structure that it diffracts light or X-rays, thereby providing patterns that contain uniquely valuable information. Interpretation of these patterns is not straightforward, but is helped by light microscopy and electron microscopy, which can often provide similar though less reliable information. At all levels of complexity, from that of the fibrils to that of the molecules, structure in a muscle is orderly. No other natural cell assembly is so suited to study by the diffraction method, and the results obtained in recent years are an outstanding example of how this method can elucidate a biological problem. In contrast to protein crystallography, where the system studied is artificial, muscle can be examined in its natural state, during normal activity. The levels of structure explored as yet in muscle are above that of the atoms in the molecules. Such structure is more commonly investigated by electron microscopy, and the application of the diffraction method to living muscle has provided a valuable check on the preparative artifacts that worry the microscopist. The great complexity of a muscle, as compared with a protein crystal, and the fact that the system is only semi-crystalline, giving a much less detailed diffraction pattern, make the problems of interpretation especially difficult. But a great deal of useful information is available about other properties of muscle and its constituents, and the flourishing state of muscle biology at present is a major factor contributing to the successful application of the diffraction method.

A Novel Embedded System Architecture for Fire Detection Based on Image Processing

2014 ◽  
Vol 608-609 ◽  
pp. 454-458
Author(s):  
Wei Bai ◽  
Chen Yuan Hu
Keyword(s):  
Image Processing ◽  
Embedded System ◽  
System Architecture ◽  
Cooperative Control ◽  
Processing System ◽  
Fire Detection ◽  
System Level ◽  
High Definition ◽  
The Embedded System ◽  
Pass Through

This paper presents novel logic/software co-work architecture for embedded high definition image processing platform, which is built by the considerations of system level, board hardware level, and the tasks partition between CPU processing and programmable logic based on the latest launched System on Chip Field Programmable Gate Array (Soc FPGA) – Xilinx ZC7020. For this case, we comprehensive analyze of the critical data paths: the uniform Advanced Extensible Interface (AXI) processing between processing system (PS) and processing logic (PL), including high definition video pass through PL to PS and PS software processing send to PL for speed up. We have included the transplant of opensource Linux, multiprocessing cooperative control and boot loader in PS side. Since the general platform is proposed, a fire detection approach based on high definition image processing is implemented. Experiment results indicated the feasibility and universality of the embedded system architecture.

scholarly journals Softer but stronger? Sulfur SAD with low energy X-rays of 2.7 Å

2014 ◽  
Vol 70 (a1) ◽  
pp. C604-C604
Author(s):  
Dorothee Liebschner ◽  
Naohiro Matsugaki ◽  
Miki Senda ◽  
Yusuke Yamada ◽  
Toshiya Senda
Keyword(s):  
Absorption Edge ◽  
Protein Crystal ◽  
X Rays ◽  
Long Wavelength ◽  
Heavy Atoms ◽  
Statistical Validity ◽  
Experimental Phasing ◽  
Recent Developments ◽  
Long Time ◽  
Anomalous Signal

Single wavelength anomalous diffraction (SAD) is a powerful experimental phasing technique used in macromolecular crystallography (MX). SAD is based on the absorption of X-rays by heavy atoms, which can be either incorporated into the protein (crystal) or naturally present in the structure, such as sulfur or metal ions. In particular, sulfur seems to be an attractive candidate for phasing, because most proteins contain a considerable number of S atoms. However, the K-absorption edge of sulfur is around 5.1 Å wavelength (2.4 keV), which is far from the optimal wavelength of most MX-beamlines at synchrotrons. Therefore, phasing experiments have to be performed further away from the absorption edge, which results in weaker anomalous signal. This explains why S-SAD was not commonly used for a long time, although its feasibility was illustrated by the ground-breaking study by Hendrickson and Teeter [1]. Recent developments in instrumentation, software and methodology made it possible to measure intensities more accurately, and, as a consequence, S-SAD has lately obtained more and more attention [2]. The beamline BL-1A at Photon factory (KEK, Japan) is designed to take full advantage of a long wavelength X-ray beam at around 3 Å to further enhance anomalous signals. We performed S-SAD experiments at BL-1A using two different wavelengths (1.9 Å and 2.7 Å) and compared their phasing capabilities. This methodological study was performed with ferredoxin reductase crystals of various sizes. In order to guarantee statistical validity and to exclude the influence of a particular sample, we repeated the comparison with several crystals. The novelty in the approach consists in using very long wavelengths (2.7 Å), not fully exploited in the literature so far. According to our study, the 2.7 Å wavelength shows - despite strong absorption effects of the diffracted X-rays - more successful phasing results than at 1.9 Å.

scholarly journals Promises and pitfalls of In-situ diffraction

2014 ◽  
Vol 70 (a1) ◽  
pp. C1140-C1140
Author(s):  
James Holton
Keyword(s):  
Protein Crystal ◽  
Main Beam ◽  
X Rays ◽  
Thin Walls ◽  
Future Success ◽  
Crystallization Experiment ◽  
Cryo Preservation ◽  
Dry Out ◽  
In Situ Diffraction

The ultimate test of any crystallization experiment is how well the crystals (if any) diffract x-rays. In particular, we would like this diffraction data to be as free from caveats as possible, so that we know one condition really does produce better crystals than another, and not that we had a great crystal somewhere, but it was messed up in the harvesting and cryo-preservation process. So why doesn't everyone do in-situ diffraction? The reason is because of background. The principle impediment to observing weak high-resolution spots is that they get lost in the background scattering, so every effort must be made to minimize it. Unfortunately, plastic, water, oil, amorphous protein and protein crystal all generate a similar number of background x-rays per unit thickness. This is because they are all made of similarly light elements (oxygen, carbon, nitrogen) and have similar densities (0.9 to 1.2 g/cm^3). There is no such thing as a "low background" material, and everything the main beam touches on its way in, through and out of whatever is holding the crystal generates background. This is why loop mounts are so popular: the total path of the x-rays through non-crystalline stuff in a typical loop mount is generally not much thicker than the crystal, and reducing this thickness further has diminishing returns because the background is now dominated by that from disorder in the crystal lattice itself. So, why not make ultra-thin in-situ trays? Not only are thin walls difficult to manufacture cheaply, but they also dry out a lot faster, which is problematic for growing the crystals in the first place. The future success of in-situ diffraction requires trays that are not only thin-walled and low-permeability, but cheap.

Single-wavelength phasing strategy for quasi-racemic protein crystal diffraction data

2011 ◽  
Vol 68 (1) ◽  
pp. 62-68 ◽  
Author(s):  
Michael R. Sawaya ◽  
Brad L. Pentelute ◽  
Stephen B. H. Kent ◽  
Todd O. Yeates
Keyword(s):  
Diffraction Data ◽  
Protein Crystal
Sign in / Sign up

Export Citation Format

Share Document