scholarly journals Usefulness of oils for cleaning the host matrix and for cryoprotection of lipidic cubic phase crystals

2019 ◽  
Vol 52 (4) ◽  
pp. 864-868
Author(s):  
Satomi Niwa ◽  
Kazuki Takeda
Keyword(s):  
Data Collection ◽  
Membrane Protein ◽  
Crystal Surface ◽  
Phase Method ◽  
Cubic Phase ◽  
Data Sets ◽  
Accurate Data ◽  
Host Matrix ◽  
Crystal Stability ◽  
Lipidic Cubic Phase

The lipidic cubic phase method is an effective approach for membrane protein crystallography. The in meso grown crystals are usually cryocooled directly without removing the host matrix from the harvested crystal surface. However, the host matrix often causes the appearance of scattering rings and an increase in background scattering during the data collection. Moreover, the frozen host matrix sometimes becomes opaque and it can hinder conventional crystal centering. In this study, several oils were examined for their ability to clean the host matrix and to provide cryoprotection for crystals grown in the lipidic cubic phase. Several of the tested oils appeared to be useful in terms of their effect on crystal stability and background scattering. This method should be of value for the collection of highly accurate data sets.

scholarly journals Serial femtosecond crystallography of soluble proteins in lipidic cubic phase

IUCrJ ◽  
2015 ◽  
Vol 2 (5) ◽  
pp. 545-551 ◽  
Author(s):  
Raimund Fromme ◽  
Andrii Ishchenko ◽  
Markus Metz ◽  
Shatabdi Roy Chowdhury ◽  
Shibom Basu ◽  
...  
Keyword(s):  
Data Collection ◽  
Protein Structure Determination ◽  
Soluble Proteins ◽  
Cubic Phase ◽  
Data Sets ◽  
Carrier Medium ◽  
X Ray ◽  
Membrane Mimetic ◽  
Lipidic Cubic Phase ◽  
Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is demonstrated that LCP can also be used as a suitable carrier medium for microcrystals of soluble proteins, enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.

scholarly journals A fast, simple and robust protocol for growing crystals in the lipidic cubic phase

2012 ◽  
Vol 45 (6) ◽  
pp. 1330-1333 ◽  
Author(s):  
Margaret Aherne ◽  
Joseph A. Lyons ◽  
Martin Caffrey
Keyword(s):  
Quality Control ◽  
Data Collection ◽  
Protein Crystallization ◽  
Cubic Phase ◽  
Sandwich Plates ◽  
Protein Targets ◽  
Test Protein ◽  
Membrane Protein Crystallization ◽  
Lipidic Cubic Phase ◽  
Robust Protocol

A simple and inexpensive protocol for producing crystals in the sticky and viscous mesophase used for membrane protein crystallization by thein mesomethod is described. It provides crystals that appear within 15–30 min of setup at 293 K. The protocol gives the experimenter a convenient way of gaining familiarity and a level of comfort with the lipidic cubic mesophase, which can be daunting as a material when first encountered. Having used the protocol to produce crystals of the test protein, lysozyme, the experimenter can proceed with confidence to apply the method to more valuable membrane (and soluble) protein targets. The glass sandwich plates prepared using this robust protocol can further be used to practice harvesting and snap-cooling ofin meso-grown crystals, to explore diffraction data collection with mesophase-embedded crystals, and for an assortment of quality control and calibration applications when used in combination with a crystallization robot.

From test tube to plate: a simple procedure for the rapid preparation of microcrystallization experiments using the cubic phase method

2002 ◽  
Vol 35 (5) ◽  
pp. 637-640 ◽  
Author(s):  
Peter Nollert
Keyword(s):  
Liquid Crystalline ◽  
Simple Procedure ◽  
Protein Crystallization ◽  
Test Tube ◽  
Phase Method ◽  
Cubic Phase ◽  
Rapid Preparation ◽  
Lipidic Cubic Phase ◽  
Crystallization Conditions ◽  
Set Up

Crystals of transmembrane proteins for X-ray diffraction experiments may be grown either by employing mixed protein–detergent complexes, or in a matrix of liquid-crystalline membraneous material forming a lipidic cubic phase (in cubo). Widespread use of thein cubomethod has been severely hampered by its tediousness and the large amounts of protein required. Here a simple procedure is presented that by virtue of its simplicity and small setup size substantially reduces the preparation time as well as the amount of protein. Crystallization trials are set up in conventional multi-well plates using a semi-automatic dispenser-driven microsyringe. The microprocedure is amenable to full automation and further miniaturization. Its feasibility is demonstrated by screening for new crystallization conditions for bacteriorhodopsin using volumes ofca200 nl of lipidic cubic phase. New crystallization conditions were identified that avoid the necessity of weighing solid precipitation agents.

scholarly journals De novo protein structure determination by heavy-atom soaking in lipidic cubic phase and SIRAS phasing using serial synchrotron crystallography

IUCrJ ◽  
2018 ◽  
Vol 5 (5) ◽  
pp. 524-530 ◽  
Author(s):  
S. Botha ◽  
D. Baitan ◽  
K. E. J. Jungnickel ◽  
D. Oberthür ◽  
C. Schmidt ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Data Collection ◽  
Diffraction Data ◽  
Heavy Atom ◽  
Proteinase K ◽  
Cubic Phase ◽  
Anomalous Scattering ◽  
Experimental Phasing ◽  
Lipidic Cubic Phase ◽  
Serial Crystallography

During the past few years, serial crystallography methods have undergone continuous development and serial data collection has become well established at high-intensity synchrotron-radiation beamlines and XFEL radiation sources. However, the application of experimental phasing to serial crystallography data has remained a challenging task owing to the inherent inaccuracy of the diffraction data. Here, a particularly gentle method for incorporating heavy atoms into micrometre-sized crystals utilizing lipidic cubic phase (LCP) as a carrier medium is reported. Soaking in LCP prior to data collection offers a new, efficient and gentle approach for preparing heavy-atom-derivative crystals directly before diffraction data collection using serial crystallography methods. This approach supports effective phasing by utilizing a reasonably low number of diffraction patterns. Using synchrotron radiation and exploiting the anomalous scattering signal of mercury for single isomorphous replacement with anomalous scattering (SIRAS) phasing resulted in high-quality electron-density maps that were sufficient for building a complete structural model of proteinase K at 1.9 Å resolution using automatic model-building tools.

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