scholarly journals Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

10.3791/54463 ◽  
2016 ◽  
Author(s):  
Andrii Ishchenko ◽  
Vadim Cherezov ◽  
Wei Liu
Keyword(s):  
Cubic Phase ◽  
Lipidic Cubic Phase ◽  
Serial Femtosecond Crystallography

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 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

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 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 Preparation of microcrystals in lipidic cubic phase for serial femtosecond crystallography

2014 ◽  
Vol 9 (9) ◽  
pp. 2123-2134 ◽  
Author(s):  
Wei Liu ◽  
Andrii Ishchenko ◽  
Vadim Cherezov
Keyword(s):  
Cubic Phase ◽  
Lipidic Cubic Phase ◽  
Serial Femtosecond Crystallography

scholarly journals Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Uwe Weierstall ◽  
Daniel James ◽  
Chong Wang ◽  
Thomas A. White ◽  
Dingjie Wang ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cubic Phase ◽  
Lipidic Cubic Phase ◽  
Serial Femtosecond Crystallography

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.

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