lipidic cubic phase
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Author(s):  
Peter Berntsen ◽  
Connie Darmanin ◽  
Eugeniu Balaur ◽  
Leonie Flueckiger ◽  
Alex Kozlov ◽  
...  

Author(s):  
Shuto Kozaka ◽  
Rie Wakabayashi ◽  
Noriho Kamiya ◽  
Masahiro Goto

IUCrJ ◽  
2021 ◽  
Vol 8 (1) ◽  
pp. 22-32
Author(s):  
Thomas Cleveland IV ◽  
Emily Blick ◽  
Susan Krueger ◽  
Anna Leung ◽  
Tamim Darwish ◽  
...  

Lipidic cubic phase (LCP) crystallization methods have been essential in obtaining crystals of certain membrane proteins, particularly G-protein-coupled receptors. LCP crystallization is generally optimized across a large number of potential variables, one of which may be the choice of the solubilizing detergent. A better fundamental understanding of the behavior of detergents in the LCP may guide and simplify the detergent selection process. This work investigates the distribution of protein and detergent in LCP using the membrane protein bacteriorhodopsin (bR), with the LCP prepared from highly deuterated monoolein to allow contrast-matched small-angle neutron scattering. Contrast-matching allows the scattering from the LCP bilayer itself to be suppressed, so that the distribution and behavior of the protein and detergent can be directly studied. The results showed that, for several common detergents, the detergent micelle dissociates and incorporates into the LCP bilayer essentially as free detergent monomers. In addition, the detergent octyl glucoside dissociates from bR, and neither the protein nor detergent forms clusters in the LCP. The lack of detergent assemblies in the LCP implies that, upon incorporation, micelle sizes and protein/detergent interactions become less important than they would be in solution crystallization. Crystallization screening confirmed this idea, with crystals obtained from bR in the presence of most detergents tested. Thus, in LCP crystallization, detergents can be selected primarily on the basis of protein stabilization in solution, with crystallization suitability a lesser consideration.


IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 976-984
Author(s):  
Ming-Yue Lee ◽  
James Geiger ◽  
Andrii Ishchenko ◽  
Gye Won Han ◽  
Anton Barty ◽  
...  

Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase; however, like most techniques, it has limitations. Here we attempt to address some of these limitations related to the use of a vacuum chamber and the need for attenuation of the XFEL beam, in order to further improve the efficiency of this method. Using an optimized SFX experimental setup in a helium atmosphere, the room-temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution is determined and compared with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, the capability of utilizing high XFEL beam transmissions is demonstrated, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete dataset. The experimental setup presented herein can be applied to future SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.


Structure ◽  
2020 ◽  
Vol 28 (10) ◽  
pp. 1149-1159.e4 ◽  
Author(s):  
Lan Zhu ◽  
Guanhong Bu ◽  
Liang Jing ◽  
Dan Shi ◽  
Ming-Yue Lee ◽  
...  

2019 ◽  
Vol 75 (10) ◽  
pp. 937-946 ◽  
Author(s):  
Rebecka Andersson ◽  
Cecilia Safari ◽  
Petra Båth ◽  
Robert Bosman ◽  
Anastasya Shilova ◽  
...  

Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.


2019 ◽  
Author(s):  
Lan Zhu ◽  
Guanhong Bu ◽  
Liang Jing ◽  
Dan Shi ◽  
Tamir Gonen ◽  
...  

AbstractThe lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. Because crystals grown in LCP can be limited in size, improved techniques for structure determination from these small crystals are important. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-TEM to collect electron diffraction data and determine high-resolution structures from very thin micro and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP. Proteinase K in LCP was used as a model system, and several LCP sample preparation strategies were tested. Among these, treatment with 2-Methyl-2,4-pentanediol (MPD) and lipase were both able to reduce the viscosity of the LCP and produce quality cryo-EM grids with well-diffracting crystals. These results set the stage for the use of MicroED to analyze other microcrystalline samples grown in LCP.


2019 ◽  
Vol 52 (4) ◽  
pp. 864-868
Author(s):  
Satomi Niwa ◽  
Kazuki Takeda

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.


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