Lipidic cubic phase injection for serial X-ray crystallography

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.

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A study of membrane lipids from dehydration-acclimated Brassica napus root cells: formation of a cubic phase under physiological conditions

Biochemistry and Cell Biology ◽

10.1139/o90-013 ◽

1990 ◽

Vol 68 (1) ◽

pp. 102-105 ◽

Cited By ~ 8

Author(s):

P. Norberg ◽

K. Larsson ◽

C. Liljenberg

Keyword(s):

Water Deficit ◽

Membrane Lipids ◽

Phase Behaviour ◽

Water Deficit Stress ◽

Cubic Phase ◽

Polarization Microscopy ◽

Root Cells ◽

X Ray ◽

X Ray Crystallography ◽

Microsomal Membranes

Rape seedlings were acclimated to evaporative dehydration by exposure to repeated moderate water-deficit stress. The stress program started after 19 days of growth and consisted of three, 24-h stress periods interspersed with 24-h rewatering periods. After the third stress period the roots were harvested and microsomal membranes were isolated. Control plants were grown under equivalent conditions without stress (nonacclimated cells). Total lipids were extracted from the membranes and investigated with X-ray crystallography and polarization microscopy at different degrees of hydration and temperatures. In excess water, the membrane lipids from both acclimated and nonacclimated cells exhibited a cubic phase. The lipids from the nonacclimated cells formed a hexagonal (HII) phase on dehydration. The lipids from the acclimated cells behaved in a different way during dehydration, where the cubic phase was transformed to an L2 phase via an intermediate HII phase. At increasing temperatures, the hydrated cubic phase started to form an L2 phase at 30 °C and was fully converted to the liquid-type state at 42 °C. The mesomorphic phase behaviour is discussed in relation to membrane activity.Key words: water-deficit stress, microsomal membranes, X-ray crystallography, polarization microscopy.

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Nanosecond pump–probe device for time-resolved serial femtosecond crystallography developed at SACLA

Journal of Synchrotron Radiation ◽

10.1107/s160057751701030x ◽

2017 ◽

Vol 24 (5) ◽

pp. 1086-1091 ◽

Cited By ~ 16

Author(s):

Minoru Kubo ◽

Eriko Nango ◽

Kensuke Tono ◽

Tetsunari Kimura ◽

Shigeki Owada ◽

...

Keyword(s):

Conformational Changes ◽

Free Electron ◽

Good Choice ◽

Cubic Phase ◽

Liquid Droplets ◽

Optical Pump ◽

Time Resolved ◽

X Ray ◽

X Ray Crystallography ◽

Serial Femtosecond Crystallography

X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.

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Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 µm size X-ray synchrotron beam

Journal of The Royal Society Interface ◽

10.1098/rsif.2009.0142.focus ◽

2009 ◽

Vol 6 (suppl_5) ◽

Cited By ~ 112

Author(s):

Vadim Cherezov ◽

Michael A. Hanson ◽

Mark T. Griffith ◽

Mark C. Hilgart ◽

Ruslan Sanishvili ◽

...

Keyword(s):

Membrane Proteins ◽

G Protein Coupled Receptors ◽

Cubic Phase ◽

High Quality ◽

Crystal Sample ◽

X Ray ◽

Synchrotron Beam ◽

Lipidic Cubic Phase ◽

Diffraction Patterns ◽

G Protein Coupled

Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals. Advent of the sub-10 µm minibeam at the APS GM/CA CAT has enabled the collection of high quality diffraction data from such microcrystals. Herein we describe the challenges and solutions related to growing, manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material. Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner. The resulting diffraction patterns have a significant reduction in background, with strong intensity and improvement in diffraction resolution compared with larger beam sizes. Three high-resolution structures of human G protein-coupled receptors serve as evidence of the utility of these techniques that will likely be useful for future structural determination efforts. We anticipate that further innovations of the technologies applied to microcrystallography will enable the solving of structures of ever more challenging targets.

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Harnessing the power of an X-ray laser for serial crystallography of membrane proteins crystallized in lipidic cubic phase

IUCrJ ◽

10.1107/s2052252520012701 ◽

2020 ◽

Vol 7 (6) ◽

pp. 976-984

Author(s):

Ming-Yue Lee ◽

James Geiger ◽

Andrii Ishchenko ◽

Gye Won Han ◽

Anton Barty ◽

...

Keyword(s):

Membrane Proteins ◽

Dynamic Range ◽

High Dynamic Range ◽

Cubic Phase ◽

Experimental Setup ◽

Temperature Structure ◽

Helium Atmosphere ◽

X Ray ◽

Lipidic Cubic Phase ◽

Time Required

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.

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Lipid Cubic Phase for Membrane Protein X-ray Crystallography

Membrane Biophysics ◽

10.1007/978-981-10-6823-2_7 ◽

2017 ◽

pp. 175-220 ◽

Cited By ~ 1

Author(s):

Jialu Zha ◽

Dianfan Li

Keyword(s):

Membrane Protein ◽

Cubic Phase ◽

X Ray ◽

X Ray Crystallography ◽

Protein X

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Lipidic cubic phase serial millisecond crystallography using synchrotron radiation

IUCrJ ◽

10.1107/s2052252514026487 ◽

2015 ◽

Vol 2 (2) ◽

pp. 168-176 ◽

Cited By ~ 145

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.

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Structure of membrane diacylglycerol kinase in lipid bilayers

Communications Biology ◽

10.1038/s42003-021-01802-1 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Jianping Li ◽

Yang Shen ◽

Yanke Chen ◽

Zhengfeng Zhang ◽

Shaojie Ma ◽

...

Keyword(s):

Lipid Bilayers ◽

Diacylglycerol Kinase ◽

Integral Membrane Protein ◽

Magic Angle Spinning ◽

Phospholipid Bilayers ◽

Solution Nmr ◽

Cubic Phase ◽

Magic Angle ◽

X Ray ◽

X Ray Crystallography

AbstractDiacylglycerol kinase (DgkA) is a small integral membrane protein, responsible for the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid. Its structures reported in previous studies, determined in detergent micelles by solution NMR and in monoolein cubic phase by X-ray crystallography, differ significantly. These differences point to the need to validate these detergent-based structures in phospholipid bilayers. Here, we present a well-defined homo-trimeric structure of DgkA in phospholipid bilayers determined by magic angle spinning solid-state NMR (ssNMR) spectroscopy, using an approach combining intra-, inter-molecular paramagnetic relaxation enhancement (PRE)-derived distance restraints and CS-Rosetta calculations. The DgkA structure determined in lipid bilayers is different from the solution NMR structure. In addition, although ssNMR structure of DgkA shows a global folding similar to that determined by X-ray, these two structures differ in monomeric symmetry and dynamics. A comparative analysis of DgkA structures determined in three different detergent/lipid environments provides a meaningful demonstration of the influence of membrane mimetic environments on the structure and dynamics of membrane proteins.

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MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP

10.1101/2020.09.27.316109 ◽

2020 ◽

Author(s):

Michael W. Martynowycz ◽

Anna Shiriaeva ◽

Xuanrui Ge ◽

Johan Hattne ◽

Brent L. Nannenga ◽

...

Keyword(s):

Adenosine Receptor ◽

G Protein Coupled Receptors ◽

Cubic Phase ◽

Transmembrane Receptors ◽

X Ray ◽

X Ray Crystallography ◽

Physiological Processes ◽

Sponge Phase ◽

Traditional Approaches ◽

G Protein Coupled

AbstractG Protein-Coupled Receptors (GPCRs), or 7-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without signaling partners bound requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here we show that the structure of a human A2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by cryoFIB milling. We determined the structure of the A2A receptor to 2.8 Å resolution and resolved an antagonist in its orthosteric ligand-binding site as well as 4 cholesterol molecules bound to the receptor. This study lays the groundwork for future GPCR structural studies using single microcrystals that would otherwise be impossible by other crystallographic methods.One sentence summaryFIB milled LCP-GPCR structure determined by MicroED

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Serial femtosecond crystallography of soluble proteins in lipidic cubic phase

IUCrJ ◽

10.1107/s2052252515013160 ◽

2015 ◽

Vol 2 (5) ◽

pp. 545-551 ◽

Cited By ~ 41

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.

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