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 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 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 Exploring the structural relationship between encapsulated antimicrobial peptides and the bilayer membrane mimetic lipidic cubic phase: studies with gramicidin A′

RSC Advances ◽  
2016 ◽  
Vol 6 (73) ◽  
pp. 68685-68694 ◽  
Author(s):  
Thomas G. Meikle ◽  
Charlotte E. Conn ◽  
Frances Separovic ◽  
Calum J. Drummond
Keyword(s):  
Antimicrobial Peptides ◽  
Low Cost ◽  
Bilayer Membrane ◽  
Structural Relationship ◽  
Gramicidin A ◽  
Cubic Phase ◽  
Membrane Peptides ◽  
Membrane Mimetic ◽  
Lipidic Cubic Phase ◽  
Bicontinuous Cubic

Lipid based bicontinuous cubic mesophases provide a low-cost, robust membrane mimetic nanomaterial which allows for the incorporation of membrane peptides and proteins.

scholarly journals The Lipidic Cubic Phase as a Membrane Mimetic

2011 ◽  
Vol 100 (8) ◽  
pp. 2075 ◽  
Author(s):  
Nicole Höfer ◽  
David Aragão ◽  
Martin Caffrey
Keyword(s):  
Cubic Phase ◽  
Membrane Mimetic ◽  
Lipidic Cubic Phase

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 Femtosecond crystallography of membrane proteins in the lipidic cubic phase

2014 ◽  
Vol 369 (1647) ◽  
pp. 20130314 ◽  
Author(s):  
Wei Liu ◽  
Daniel Wacker ◽  
Chong Wang ◽  
Enrique Abola ◽  
Vadim Cherezov
Keyword(s):  
Membrane Proteins ◽  
Structure Determination ◽  
Cubic Phase ◽  
Technological Advances ◽  
Lipidic Cubic Phase ◽  
High Resolution Structure ◽  
Effects Of Radiation ◽  
G Protein Coupled ◽  
Serial Femtosecond Crystallography

Despite recent technological advances in heterologous expression, stabilization and crystallization of membrane proteins (MPs), their structural studies remain difficult and require new transformative approaches. During the past two years, crystallization in lipidic cubic phase (LCP) has started gaining a widespread acceptance, owing to the spectacular success in high-resolution structure determination of G protein-coupled receptors (GPCRs) and to the introduction of commercial instrumentation, tools and protocols. The recent appearance of X-ray free-electron lasers (XFELs) has enabled structure determination from substantially smaller crystals than previously possible with minimal effects of radiation damage, offering new exciting opportunities in structural biology. The unique properties of LCP material have been exploited to develop special protocols and devices that have established a new method of serial femtosecond crystallography of MPs in LCP (LCP-SFX). In this method, microcrystals are generated in LCP and streamed continuously inside the same media across the intersection with a pulsed XFEL beam at a flow rate that can be adjusted to minimize sample consumption. Pioneering studies that yielded the first room temperature GPCR structures, using a few hundred micrograms of purified protein, validate the LCP-SFX approach and make it attractive for structure determination of difficult-to-crystallize MPs and their complexes with interacting partners. Together with the potential of femtosecond data acquisition to interrogate unstable intermediate functional states of MPs, LCP-SFX holds promise to advance our understanding of this biomedically important class of proteins.

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.

scholarly journals Serial femtosecond crystallography of membrane proteins with a lipidic cubic phase injector

2013 ◽  
Vol 69 (a1) ◽  
pp. s140-s141
Author(s):  
Uwe Weierstall ◽  
Daniel James ◽  
Dingjie Wang ◽  
John C. H. Spence ◽  
R. B. Doak ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Cubic Phase ◽  
Lipidic Cubic Phase ◽  
Serial Femtosecond Crystallography
Sign in / Sign up

Export Citation Format

Share Document