Preparation, Crystallization, and X-ray Data Collection of Archaeal Oligopeptide Permease A

2021 ◽  
Vol 66 (7) ◽  
pp. 1300-1305
Author(s):  
H. Yokoyama ◽  
N. Kamei ◽  
K. Konishi ◽  
K. Hara ◽  
Y. Ishikawa ◽  
...  
Keyword(s):  
Data Collection ◽  
X Ray ◽  
Oligopeptide Permease

Design of a novel Peltier-based cooling device and its use in neutron diffraction data collection of perdeuterated yeast pyrophosphatase

2010 ◽  
Vol 43 (5) ◽  
pp. 1113-1120 ◽  
Author(s):  
Esko Oksanen ◽  
François Dauvergne ◽  
Adrian Goldman ◽  
Monika Budayova-Spano
Keyword(s):  
Data Collection ◽  
Neutron Diffraction ◽  
Diffraction Data ◽  
Catalytic Mechanism ◽  
Inorganic Pyrophosphatase ◽  
Neutron Diffraction Data ◽  
Cooling Device ◽  
Data Set ◽  
X Ray ◽  
X Ray Crystallography

H atoms play a central role in enzymatic mechanisms, but H-atom positions cannot generally be determined by X-ray crystallography. Neutron crystallography, on the other hand, can be used to determine H-atom positions but it is experimentally very challenging. Yeast inorganic pyrophosphatase (PPase) is an essential enzyme that has been studied extensively by X-ray crystallography, yet the details of the catalytic mechanism remain incompletely understood. The temperature instability of PPase crystals has in the past prevented the collection of a neutron diffraction data set. This paper reports how the crystal growth has been optimized in temperature-controlled conditions. To stabilize the crystals during neutron data collection a Peltier cooling device that minimizes the temperature gradient along the capillary has been developed. This device allowed the collection of a full neutron diffraction data set.

scholarly journals Protein microcrystallography using synchrotron radiation

IUCrJ ◽  
2017 ◽  
Vol 4 (5) ◽  
pp. 529-539 ◽  
Author(s):  
Masaki Yamamoto ◽  
Kunio Hirata ◽  
Keitaro Yamashita ◽  
Kazuya Hasegawa ◽  
Go Ueno ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Data Collection ◽  
Diffraction Data ◽  
Structure Determination ◽  
Structural Features ◽  
X Ray ◽  
Data Collection Strategy ◽  
Technological Developments ◽  
Novel Method

The progress in X-ray microbeam applications using synchrotron radiation is beneficial to structure determination from macromolecular microcrystals such as smallin mesocrystals. However, the high intensity of microbeams causes severe radiation damage, which worsens both the statistical quality of diffraction data and their resolution, and in the worst cases results in the failure of structure determination. Even in the event of successful structure determination, site-specific damage can lead to the misinterpretation of structural features. In order to overcome this issue, technological developments in sample handling and delivery, data-collection strategy and data processing have been made. For a few crystals with dimensions of the order of 10 µm, an elegant two-step scanning strategy works well. For smaller samples, the development of a novel method to analyze multiple isomorphous microcrystals was motivated by the success of serial femtosecond crystallography with X-ray free-electron lasers. This method overcame the radiation-dose limit in diffraction data collection by using a sufficient number of crystals. Here, important technologies and the future prospects for microcrystallography are discussed.

Intensity measurements

2002 ◽  
Author(s):  
David Blow
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Resolution Limit ◽  
Strategic Decisions ◽  
Experimental Conditions ◽  
X Ray ◽  
Intensity Measurements ◽  
Data Collection Technique ◽  
Molecular Structure Investigation ◽  
Disordered Crystal

Once a suitable crystal has been obtained, a molecular structure investigation requires measurement of the intensities of as many Bragg reflections as possible. In this chapter, some of the options that must be decided by the experimenter will be considered, and some of the criteria used to assess the accuracy and completeness of the data will be presented. The experimenter has to make a number of strategic decisions in collecting the crystal intensity data. These include: • What X-ray source should be used? • What X-ray detector should be used? • Under what conditions should the crystal be maintained? • How long should each crystal be exposed? • What data collection technique will be used? • What resolution limit should be applied? The choice of source and detector will depend largely on what is available, but the major decision is whether to use facilities in the home laboratory or whether to use a synchrotron at a central facility. The energy released by absorption of X-rays in a crystal inevitably damages it. The process of radiation damage increases crystal disorder and reduces the intensity of scattering. The experimenter will ultimately have to abandon data collection from the damaged and disordered crystal. Under ideal experimental conditions, all the useful diffraction data can be obtained from a crystal long before radiation damage takes its toll, and radiation damage does not create a practical problem. At the other end of the scale, it may be necessary to combine the measurements from many crystals in order to obtain a complete set of diffracted intensities. There is no definite criterion to decide when a crystal is so badly damaged that it must be discarded. But if the measurements are going to be of highest quality, any observable change is bad news. The most serious effects occur in the part of the diffraction pattern at the highest observed resolution, where the observed intensities of the Bragg reflections will be altered most rapidly. The first observable effect of radiation damage is usually a reduction of high angle intensities due to increased disorder.

scholarly journals Automated harvesting and processing of protein crystals through laser photoablation

2016 ◽  
Vol 72 (4) ◽  
pp. 454-466 ◽  
Author(s):  
Ulrich Zander ◽  
Guillaume Hoffmann ◽  
Irina Cornaciu ◽  
Jean-Pierre Marquette ◽  
Gergely Papp ◽  
...  
Keyword(s):  
Data Collection ◽  
Macromolecular Crystallography ◽  
X Ray Diffraction ◽  
X Ray ◽  
New Methods ◽  
Repeated Sample ◽  
Sample Recovery ◽  
Through Diffusion ◽  
X Ray Background ◽  
Low X

Currently, macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection. However, crystal harvesting and processing largely depend on manual operations. Here, a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments. In this approach, the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process, in many cases eliminating the use of cryoprotectant agents, while crystal-soaking experiments are performed through diffusion, precluding the need for repeated sample-recovery and transfer operations. Moreover, the high-precision laser enables new mounting strategies that are not accessible through other methods. This approach bridges an important gap in automation and can contribute to expanding the capabilities of modern macromolecular crystallography facilities.

scholarly journals Development of a shutterless continuous rotation method using an X-ray CMOS detector for protein crystallography

2009 ◽  
Vol 42 (6) ◽  
pp. 1165-1175 ◽  
Author(s):  
Kazuya Hasegawa ◽  
Kunio Hirata ◽  
Tetsuya Shimizu ◽  
Nobutaka Shimizu ◽  
Takaaki Hikima ◽  
...  
Keyword(s):  
Data Collection ◽  
Dynamic Range ◽  
Protein Structures ◽  
Protein Crystallography ◽  
New Method ◽  
Oxide Semiconductor ◽  
X Ray ◽  
Rotation Method ◽  
Continuous Rotation ◽  
Wide Range

A new shutterless continuous rotation method using an X-ray complementary metal-oxide semiconductor (CMOS) detector has been developed for high-speed, precise data collection in protein crystallography. The principle of operation and the basic performance of the X-ray CMOS detector (Hamamatsu Photonics KK C10158DK) have been shown to be appropriate to the shutterless continuous rotation method. The data quality of the continuous rotation method is comparable to that of the conventional oscillation method using a CCD detector and, furthermore, the combination with fine φ slicing improves the data accuracy without increasing the data-collection time. The new method is more sensitive to diffraction intensity because of the narrow dynamic range of the CMOS detector. However, the strong diffraction spots were found to be precisely measured by recording them on successive multiple images by selecting an adequate rotation step. The new method has been used to successfully determine three protein structures by multi- and single-wavelength anomalous diffraction phasing and has thereby been proved applicable in protein crystallography. The apparatus and method may become a powerful tool at synchrotron protein crystallography beamlines with important potential across a wide range of X-ray wavelengths.

Calibration tests and use of a Nicolet/Xentronics imaging proportional chamber mounted on a conventional source for protein crystallography

1989 ◽  
Vol 22 (2) ◽  
pp. 123-137 ◽  
Author(s):  
Z. Derewenda ◽  
J. R. Helliwell
Keyword(s):  
Data Collection ◽  
Single Crystal ◽  
Proportional Counter ◽  
Protein Crystallography ◽  
System Optimization ◽  
High Quality ◽  
X Ray ◽  
Proportional Chamber ◽  
A Performance

The results are presented of calibration tests and several single-crystal X-ray data collection experiments undertaken with a Nicolet/Xentronics Imaging Proportional Counter mounted on a conventional X-ray source. Considerable attention has been given to system optimization in collaboration with Nicolet, and this has led to a performance of high quality.

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