scholarly journals Isoprenoid-chained lipid EROCOC17+4: a new matrix for membrane protein crystallization and a crystal delivery medium in serial femtosecond crystallography

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kentaro Ihara ◽  
Masakatsu Hato ◽  
Takanori Nakane ◽  
Keitaro Yamashita ◽  
Tomomi Kimura-Someya ◽  
...  
Keyword(s):  
Protein Crystallization ◽  
Water System ◽  
Cubic Phase ◽  
Marked Contrast ◽  
X Ray ◽  
X Ray Scattering ◽  
Membrane Protein Crystallization ◽  
Almost All ◽  
Serial Femtosecond Crystallography ◽  
Delivery Medium

Abstract In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase (LCP). However, almost all previous crystallization trials have used monoacylglycerols, with 1-(cis-9-octadecanoyl)-rac-glycerol (MO) being the most widely used lipid. We now report that EROCOC17+4 mixed with 10% (w/w) cholesterol (Fig. 1) serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A2A receptor (A2AR). The structures of EROCOC17+4-matrix grown A2AR crystals were determined at 2.0 Å resolution by serial synchrotron rotation crystallography at a cryogenic temperature, and at 1.8 Å by LCP-serial femtosecond crystallography, using an X-ray free-electron laser at 4 and 20 °C sample temperatures, and are comparable to the structure of the MO-matrix grown A2AR crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC17+4/water system did not form the crystalline LC phase at least down to − 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline LC phase below about 17 °C. As the LC phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC17+4 will expand the utility of the in meso method.

scholarly journals Use of dynamic light scattering and small-angle X-ray scattering to characterize new surfactants in solution conditions for membrane-protein crystallization

2015 ◽  
Vol 71 (7) ◽  
pp. 838-846 ◽  
Author(s):  
Mohamed Dahani ◽  
Laurie-Anne Barret ◽  
Simon Raynal ◽  
Colette Jungas ◽  
Pétra Pernot ◽  
...  
Keyword(s):  
Light Scattering ◽  
Dynamic Light Scattering ◽  
Membrane Protein ◽  
Small Angle ◽  
Protein Crystallization ◽  
X Ray ◽  
X Ray Scattering ◽  
Membrane Protein Crystallization ◽  
Surfactant Phase ◽  
Ray Scattering

The structural and interactive properties of two novel hemifluorinated surfactants, F2H9-β-M and F4H5-β-M, the syntheses of which were based on the structure and hydrophobicity of the well known dodecyl-β-maltoside (DD-β-M), are described. The shape of their micellar assemblies was characterized by small-angle X-ray scattering and their intermicellar interactions in crystallizing conditions were measured by dynamic light scattering. Such information is essential for surfactant phase-diagram determination and membrane-protein crystallization.

Small-Angle X-ray Scattering Study of Photosystem I−Detergent Complexes:  Implications for Membrane Protein Crystallization

2007 ◽  
Vol 111 (16) ◽  
pp. 4211-4219 ◽  
Author(s):  
Hugh O'Neill ◽  
William T. Heller ◽  
Katherine E. Helton ◽  
Volker S. Urban ◽  
Elias Greenbaum
Keyword(s):  
Membrane Protein ◽  
Photosystem I ◽  
Small Angle ◽  
Protein Crystallization ◽  
X Ray ◽  
X Ray Scattering ◽  
Membrane Protein Crystallization ◽  
Scattering Study ◽  
Ray Scattering

scholarly journals Characterization of lipid matrices for membrane protein crystallization by high-throughput small angle X-ray scattering

Methods ◽  
2011 ◽  
Vol 55 (4) ◽  
pp. 342-349 ◽  
Author(s):  
Jeremiah S. Joseph ◽  
Wei Liu ◽  
Joshua Kunken ◽  
Thomas M. Weiss ◽  
Hiro Tsuruta ◽  
...  
Keyword(s):  
Membrane Protein ◽  
High Throughput ◽  
Small Angle ◽  
Protein Crystallization ◽  
X Ray ◽  
X Ray Scattering ◽  
Membrane Protein Crystallization ◽  
Ray Scattering

scholarly journals Lipidic cubic phase serial millisecond crystallography using synchrotron radiation

IUCrJ ◽  
2015 ◽  
Vol 2 (2) ◽  
pp. 168-176 ◽  
Author(s):  
Przemyslaw Nogly ◽  
Daniel James ◽  
Dingjie Wang ◽  
Thomas A. White ◽  
Nadia Zatsepin ◽  
...  
Keyword(s):  
Room Temperature ◽  
Cubic Phase ◽  
Temperature Structure ◽  
Free Electron Lasers ◽  
X Ray ◽  
Cubic Phases ◽  
Lipidic Cubic Phase ◽  
Effective Delivery ◽  
Serial Femtosecond Crystallography ◽  
Delivery Medium

Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.

scholarly journals A fully automated crystallization apparatus for small protein quantities

2021 ◽  
Vol 77 (1) ◽  
Author(s):  
Ryuichi Kato ◽  
Masahiko Hiraki ◽  
Yusuke Yamada ◽  
Mikio Tanabe ◽  
Toshiya Senda
Keyword(s):  
Protein Crystallization ◽  
Diffusion Method ◽  
Cubic Phase ◽  
Web Browser ◽  
X Ray ◽  
X Ray Crystallography ◽  
Membrane Protein Crystallization ◽  
Crystallization Conditions ◽  
Set Up ◽  
Very High

In 2003, a fully automated protein crystallization and monitoring system (PXS) was developed to support the structural genomics projects that were initiated in the early 2000s. In PXS, crystallization plates were automatically set up using the vapor-diffusion method, transferred to incubators and automatically observed according to a pre-set schedule. The captured images of each crystallization drop could be monitored through the internet using a web browser. While the screening throughput of PXS was very high, the demands of users have gradually changed over the ensuing years. To study difficult proteins, it has become important to screen crystallization conditions using small amounts of proteins. Moreover, membrane proteins have become one of the main targets for X-ray crystallography. Therefore, to meet the evolving demands of users, PXS was upgraded to PXS2. In PXS2, the minimum volume of the dispenser is reduced to 0.1 µl to minimize the amount of sample, and the resolution of the captured images is increased to five million pixels in order to observe small crystallization drops in detail. In addition to the 20°C incubators, a 4°C incubator was installed in PXS2 because crystallization results may vary with temperature. To support membrane-protein crystallization, PXS2 includes a procedure for the bicelle method. In addition, the system supports a lipidic cubic phase (LCP) method that uses a film sandwich plate and that was specifically designed for PXS2. These improvements expand the applicability of PXS2, reducing the bottleneck of X-ray protein crystallography.

scholarly journals Modeling of the Resonant X-ray Response of a Chiral Cubic Phase

Crystals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 214
Author(s):  
Timon Grabovac ◽  
Ewa Gorecka ◽  
Damian Pociecha ◽  
Nataša Vaupotič
Keyword(s):  
Absorption Edge ◽  
Unit Cell ◽  
Resonant Peak ◽  
Cubic Phase ◽  
Tensor Form ◽  
Resonant Enhancement ◽  
X Ray ◽  
X Ray Scattering ◽  
Thermotropic Liquid Crystals ◽  
Carbon Absorption

The structure of a continuous-grid chiral cubic phase made of achiral constituent molecules is a hot topic in the field of thermotropic liquid crystals. Several structural models have been proposed so far. Resonant X-ray scattering (RXS), which gives information on the molecular orientation in the unit cell, could be applied to select the most appropriate model. We modeled the RXS response for the recently proposed chiral cubic phase structure with an all-hexagon chiral continuous grid. A tensor form factor of a unit cell is constructed, which enables calculation of intensities of peaks for all Miller indices. We find that all the symmetry allowed peaks are resonantly enhanced, and their intensity is much stronger than the intensity of the symmetry forbidden (resonant) peaks. In particular, we predict that a strong resonant enhancement of the symmetry allowed peaks (011) and (002), not observed in a nonresonant scattering, could be observed by RXS at the carbon absorption edge. By RXS at the sulfur absorption edge, one might observe a resonant peak (113) and resonantly enhanced peak (233), and resonant enhancement of all the peaks that are observed in a nonresonant scattering, which probably hide the rest of the predicted resonant peaks.

scholarly journals Exploring the in meso crystallization mechanism by characterizing the lipid mesophase microenvironment during the growth of single transmembrane α-helical peptide crystals

2016 ◽  
Vol 374 (2072) ◽  
pp. 20150125 ◽  
Author(s):  
Leonie van 't Hag ◽  
Konstantin Knoblich ◽  
Shane A. Seabrook ◽  
Nigel M. Kirby ◽  
Stephen T. Mudie ◽  
...  
Keyword(s):  
Crystal Growth ◽  
Self Assembly ◽  
Lamellar Phase ◽  
Cubic Phase ◽  
Supporting Evidence ◽  
X Ray ◽  
X Ray Scattering ◽  
Standard Procedures ◽  
Helical Peptide ◽  
Saxs Analysis

The proposed mechanism for in meso crystallization of transmembrane proteins suggests that a protein or peptide is initially uniformly dispersed in the lipid self-assembly cubic phase but that crystals grow from a local lamellar phase, which acts as a conduit between the crystal and the bulk cubic phase. However, there is very limited experimental evidence for this theory. We have developed protocols to investigate the lipid mesophase microenvironment during crystal growth using standard procedures readily available in crystallography laboratories. This technique was used to characterize the microenvironment during crystal growth of the DAP12-TM peptide using synchrotron small angle X-ray scattering (SAXS) with a micro-sized X-ray beam. Crystal growth was found to occur from the gyroid cubic mesophase. For one in four crystals, a highly oriented local lamellar phase was observed, providing supporting evidence for the proposed mechanism for in meso crystallization. A new observation of this study was that we can differentiate diffraction peaks from crystals grown in meso , from peaks originating from the surrounding lipid matrix, potentially opening up the possibility of high-throughput SAXS analysis of in meso grown crystals. This article is part of the themed issue ‘Soft interfacial materials: from fundamentals to formulation’.

scholarly journals Phase Transition in Di-oleoylphosphatidylglycerol/Monoolein Membranes due to Interactions of Positively Charged Peptides at their Lipid Membrane-Interface

1970 ◽  
Vol 45 (3) ◽  
pp. 219-224
Author(s):  
Shah Md Masum ◽  
Masahito Yamazaki
Keyword(s):  
Phase Transition ◽  
Phase Transitions ◽  
Small Angle ◽  
Lipid Membrane ◽  
Cubic Phase ◽  
Membrane Interface ◽  
X Ray ◽  
X Ray Scattering ◽  
Charged Peptides ◽  
Positively Charged

To elucidate the factors that induce phase transitions in biomembranes due to interactions of proteins/peptides at the lipid membrane-interface, the effects of positively charged peptides on the cubic phase (Q229) of Dioleoylphosphatidylglycerol (DOPG)/Monoolein (MO) membranes were investigated. Small angle X-ray Scattering (SAXS) results revealed that 12 mol% DOPG/88 mol % MO membranes in excess water at 25°C is body centered cubic phase of crystallographic space group Im3m (Q229). In presence of peptide LLKKK, the lattices constant of Q229 phase was gradually decreased with an increase of peptide concentration and a phase transition from cubic (Q229) to cubic (Q224) phase occurred at R=0.080; (R= molar ratio of peptide to lipid). On the other hand the designed peptide WLFLLKKK and antimicrobial peptide Magainin-2 induced lamellar phase (Lα) in the same mixture membranes. These results indicate that the interactions of the these peptides with this mixture membrane are different: LLKKK induces electrostatic attractive interactions and that of WLFLLKKKK and Magainin-2 bound with the lipid membranes induce electrostatic repulsive interaction at the membrane-interface, might be the major factor inducing different phase transitions in 12 mol% DOPG/88mol% MO mixture membranes. Key words: Antimicrobial peptide Magain-2; Dioleoylphosphatidylglycerol; Monoolein; Cubic phases; Small angle X-ray Scattering DOI: 10.3329/bjsir.v45i3.6530Bangladesh J. Sci. Ind. Res. 45(3), 219-224, 2010

Small-Angle Neutron and X-ray Scattering from Amphiphilic Stimuli-Responsive Diamond-Type Bicontinuous Cubic Phase

2007 ◽  
Vol 129 (44) ◽  
pp. 13474-13479 ◽  
Author(s):  
Borislav Angelov ◽  
Angelina Angelova ◽  
Vasil M. Garamus ◽  
Geneviève Lebas ◽  
Sylviane Lesieur ◽  
...  
Keyword(s):  
Small Angle ◽  
Cubic Phase ◽  
Stimuli Responsive ◽  
X Ray ◽  
X Ray Scattering ◽  
Bicontinuous Cubic ◽  
Diamond Type ◽  
Ray Scattering
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