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

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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Femtosecond crystallography of membrane proteins in the lipidic cubic phase

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2013.0314 ◽

2014 ◽

Vol 369 (1647) ◽

pp. 20130314 ◽

Cited By ~ 36

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.

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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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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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Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319012695 ◽

2019 ◽

Vol 75 (10) ◽

pp. 937-946 ◽

Cited By ~ 3

Author(s):

Rebecka Andersson ◽

Cecilia Safari ◽

Petra Båth ◽

Robert Bosman ◽

Anastasya Shilova ◽

...

Keyword(s):

Membrane Proteins ◽

Protein Structures ◽

Cubic Phase ◽

Crystal Formation ◽

X Ray Diffraction ◽

Time Resolved ◽

X Ray ◽

X Ray Crystallography ◽

Lipidic Cubic Phase ◽

Serial 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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Cubic InGaN Grown by MOCVD

MRS Internet Journal of Nitride Semiconductor Research ◽

10.1557/s1092578300002520 ◽

1999 ◽

Vol 4 (S1) ◽

pp. 239-243

Author(s):

J.B. Li ◽

Hui Yang ◽

L.X. Zheng ◽

D.P. Xu ◽

Y.T. Wang

Keyword(s):

Room Temperature ◽

Emission Peak ◽

Buffer Layers ◽

Cubic Phase ◽

Yellow Luminescence ◽

X Ray Diffraction ◽

High Quality ◽

X Ray ◽

Cubic Gan ◽

Pl Emission

We report on the growth of high-quality cubic phase InGaN on GaAs by MOCVD. The cubic InGaN layers are grown on cubic GaN buffer layers on GaAs (001) substrates. The surface morphology of the films are mirror-like. The cubic nature of the InGaN films is obtained by X-ray diffraction (XRD) measurements. The InGaN layers show strong photoluminescence (PL) at room temperature. Neither emission peak from wurtzite GaN nor yellow luminescence is observed in our films. The highest In content as determined by XRD is about 17% with an PL emission wavelength of 450 nm. The FWHM of the cubic InGaN PL peak are 153 meV and 216 meV for 427 nm and 450 nm emissions, respectively. It is found that the In compositions determined from XRD are not in agreement with those estimated from PL measurements. The reasons for this disagreement are discussed.

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The X-Ray Powder Diffraction Patterns and Crystal Structure for Al2M3Y(M=Cu, Ni)

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.950.48 ◽

2014 ◽

Vol 950 ◽

pp. 48-52

Author(s):

De Gui Li ◽

Ming Qin ◽

Liu Qing Liang ◽

Zhao Lu ◽

Shu Hui Liu ◽

...

Keyword(s):

Crystal Structure ◽

Powder Diffraction ◽

Diffraction Data ◽

Hexagonal Structure ◽

Argon Atmosphere ◽

X Ray Diffraction ◽

High Quality ◽

X Ray ◽

Arc Melting ◽

Diffraction Patterns

The Al2M3Y(M=Cu, Ni) compound was synthesized by arc melting under argon atmosphere. The high-quality powder X-ray diffraction data of Al2M3Y have been presented. The refinement of the X-ray diffraction patterns for the Al2M3Y compound show that the Al2M3Y has hexagonal structure, space groupP6/mmm(No.191), with a = b = 5.1618(2) Å, c = 4.1434(1) Å,V= 95.6 Å3,Z= 1,ڑx= 5.7922 g/cm3,F30= 155.5(0.0057, 34), RIR = 2.31 for Al2Cu3Y, and with a = b = 5.0399(1) Å, c = 4.0726(1) Å,V= 89.59 Å3,Z= 1,ڑx= 5.9118 g/cm3,F30= 135.7(0.0072, 30), RIR = 2.54 for Al2Ni3Y.

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X-ray powder diffraction study of K2MSiO4, M=Mg, Zn, Co, Cd

Powder Diffraction ◽

10.1017/s0885715600008927 ◽

1996 ◽

Vol 11 (1) ◽

pp. 51-55 ◽

Cited By ~ 5

Author(s):

W. A. Dollase

Keyword(s):

Powder Diffraction ◽

Diffraction Data ◽

Rietveld Method ◽

Site Occupancy ◽

Cubic Phase ◽

Variable Degree ◽

Cubic Symmetry ◽

X Ray ◽

Constrained Model ◽

Diffraction Patterns

The title materials are stuffed cristobalites possessing moderate to extreme pseudosymmetry. On the bases of their X-ray powder diffraction patterns, the Mg, Zn, and Cd compounds had been previously reported as cubic and, more recently, the Zn phase as orthorhombic. Newly measured X-ray powder diffraction data demonstrate that all (including the hitherto unknown Co analog) have the Pca21 structure of Na2BeSiO4 at room temperature, but with a widely variable degree of cubic pseudosymmetry. Observed X-ray diffraction data are in good agreement with those calculated by the Rietveld method using a constrained model with Pca21 M2+/Si site occupancy and pseudocentrosymmetric Pcab atom locations. For the most nearly cubic phase, the Cd compound, there is too little deviation in the pattern from cubic symmetry to support atom coordinate refinement even with the constrained model. In these derivatives of the stuffed cristobalite structure family, M2+ and Si atoms form an ordered tetrahedral array which avoids M2+–O–M2+ connections. Potassium atoms fill all of the intervening large cavity sites.

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Microscope detection options for colorless protein crystals grown in lipidic cubic phases

Journal of Applied Crystallography ◽

10.1107/s0021889803013724 ◽

2003 ◽

Vol 36 (5) ◽

pp. 1295-1296 ◽

Cited By ~ 5

Author(s):

Peter Nollert

Keyword(s):

Membrane Proteins ◽

Crystal Nucleation ◽

Protein Crystal ◽

Protein Crystals ◽

Cubic Phase ◽

Polarization Microscopy ◽

Cubic Phases ◽

Lipidic Cubic Phase ◽

Surface Distortions ◽

Microscopic Techniques

The use of lipidic cubic phases as crystal nucleation and growth matrices is becoming popular and has yielded crystals of soluble and membrane proteins. So far, all of the membrane proteins crystallized by this method have been colored. This feature has facilitated the detection of the often encountered microcrystals in initial screening rounds. Indeed, small colorless protein crystals have poor optical contrast as a result of the small differences in refractive index of the protein crystal and the surrounding lipidic cubic phase. While a perfect preparation of a lipidic cubic phase is transparent and optically isotropic, in a crystallization setup it frequently disguises crystals due to cracks, inclusions, surface distortions and phase boundaries. Here, several specialized microscopic techniques and illumination conditions are compared and it is found that sufficient contrast is generated by cross polarization microscopy and by Hoffman modulation contrast microscopy for the detection of colorless protein crystals.

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