Lipidic Cubic Phase Research Articles

Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket

The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson's disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR-BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR-BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1-bound A2AR-BRIL prevented formation of the lattice observed with the ZM241385-bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson's disease.

Principles and Applications of Functional Lipidic Biomaterials in Molecular Recognition, Membrane Protein Crystallization and Drug Delivery

We have designed and synthesized a library of lipids with novel functionalities in order to correlate lipid molecular structure with the ensuing nanomaterial dynamics, stability and phase behavior. The designed lipids were assembled as within lipidic mesophases and their dispersions, forming functional biomaterials that were employed in areas ranging from molecular recognition, sequestration and enrichment of nucleic acids to biosensing. An exciting new class of biomaterials that are based on cyclopropanated lipids was developed. These form stable lipidic cubic phases (LCPs) at low temperature, opening the way to conduct biophysical and biochemical investigations on temperature sensitive bio-macromolecules, specifically membrane proteins. Furthermore, pH-sensitive lipidic matrices for hydrophilic as well as hydrophobic drug incorporation and release were designed, as well as stimuli-responsive lipids that can be incorporated into biomaterials. Efficient pH- and light-induced binding, release and sequestration of hydrophilic dyes were demonstrated. Significantly, these processes could be activated sequentially, thereby achieving high degree of temporal and dosage control. The scope of lipidic materials for drug delivery was expanded to azobenzene-containing hexagonal phases, in which “on demand” single-step as well as sequential light-triggered release and retention of embedded dye molecules were demonstrated. Finally, cubosomes were stabilized and functionalized with a novel, designed biotin-based block copolymer, resulting in dispersed biomaterials that were applied against the human adenocarcinoma cell line HeLa. These cubosomes are able to simultaneously transport paclitaxel, a potent anti-cancer drug, and a hydrophobic fluorescent dye in active targeting of cancer cells. Such biotinylated cubosomes are potentially applicable in diagnosis, drug delivery and monitoring of therapeutic response for active targeting versus cancer cells.

Crystallogenesis of Membrane Proteins Mediated by Polymer-Bounded Lipid Nanodiscs

Summary For some membrane proteins, detergent-mediated solubilization compromises protein stability and functionality, often impairing biophysical and structural analyses. Hence, membrane-protein structure determination is a continuing bottleneck in the field of protein crystallography. Here, as an alternative to approaches mediated by conventional detergents, we report the crystallogenesis of a recombinantly produced membrane protein that never left a lipid bilayer environment. We used styrene–maleic acid (SMA) copolymers to solubilize lipid-embedded proteins into SMA nanodiscs, purified these discs by affinity and size-exclusion chromatography, and transferred proteins into the lipidic cubic phase (LCP) for in meso crystallization. The 2.0-A structure of an α-helical seven-transmembrane microbial rhodopsin thus obtained is of high quality and virtually identical to the 2.2-A structure obtained from traditional detergent-based purification and subsequent LCP crystallization.

Crystallogenesis of Membrane Proteins Mediated by Polymer-Bounded Lipid Nanodiscs

For some membrane proteins, detergent-mediated solubilization compromises protein stability and functionality, often impairing biophysical and structural analyses. Hence, membrane-protein structure determination is a continuing bottleneck in the field of protein crystallography. Here, as an alternative to approaches mediated by conventional detergents, we report the crystallogenesis of a recombinantly produced membrane protein that never left a lipid bilayer environment. We used styrene-maleic acid (SMA) copolymers to solubilize lipid-embedded proteins into SMA nanodiscs, purified these discs by affinity and size-exclusion chromatography, and transferred proteins into the lipidic cubic phase (LCP) for in meso crystallization. The 2.0-Å structure of an α-helical seven-transmembrane microbial rhodopsin thus obtained is of high quality and virtually identical to the 2.2-Å structure obtained from traditional detergent-based purification and subsequent LCP crystallization.

Monoolein Cubic Phase Gels and Cubosomes Doped with Magnetic Nanoparticles–Hybrid Materials for Controlled Drug Release

Hybrid materials consisting of a monoolein lipidic cubic phase (LCP) incorporating two types of magnetic nanoparticles (NP) were designed as addressable drug delivery systems. The materials, prepared in the form of a gel, were subsequently used as a macroscopic layer modifying an electrode and, after dispersion to nanoscale, as magnetocubosomes. These two LCPs were characterized by small-angle X-ray scattering (SAXS), cross-polarized microscopy, magnetic measurements, and phase diagrams. The magnetic dopants were hydrophobic NPoleic and hydrophilic NPcitric, characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and their influence on the properties of the cubic phases was investigated. The removal of the anticancer drug, Doxorubicin (Dox) from the hybrid cubic phase gels was studied by electrochemical methods. The advantages of incorporating magnetic nanoparticles into the self-assembled lipid liquid crystalline phases include the ability to address the cubic phase nanoparticle containing large amounts of drug and to control the kinetics of the drug release.

High-resolution crystal structure of the human CB1 cannabinoid receptor.

The human cannabinoid G protein-coupled receptors (GPCRs) CB1 and CB2 mediate the functional responses to the endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG), as well as the widely consumed plant (phyto)cannabinoid Δ9-tetrahydrocannabinol (THC)1. The cannabinoid receptors have been the targets of intensive drug discovery efforts due to the therapeutic potential of modulators for controlling pain2, epilepsy3, obesity4, and other maladies. While much progress has recently been made in understanding the biophysical properties of GPCRs, investigations of the molecular mechanisms of the cannabinoids and their receptors have lacked high-resolution structural data. We used GPCR engineering and lipidic cubic phase (LCP) crystallization to determine the structure of the human CB1 receptor bound to the inhibitor taranabant at 2.6 Å resolution. CB1's extracellular surface, including the highly conserved membrane-proximal amino-terminal (N-terminal) region, is distinct from other lipid-activated GPCRs and forms a critical part of the ligand binding pocket. Docking studies further demonstrate how this same pocket may accommodate the cannabinoid agonist THC. Our CB1 structure provides an atomic framework for studying cannabinoid receptor function, and will aid the design and optimization of cannabinoid system modulators for therapeutic ends.

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

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 µm crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

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

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 µm crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

LCP crystallization and X-ray diffraction analysis of VcmN, a MATE transporter from Vibrio cholerae.

Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane. Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets. Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP). X-ray diffraction data were collected to 2.5 Å resolution from a single crystal obtained in a sandwich plate. The crystal belonged to space group P212121, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. As a result of further LCP crystallization trials, crystals of larger size were obtained using sitting-drop plates. X-ray diffraction data were collected to 2.2 Å resolution from a single crystal obtained in a sitting-drop plate. The crystal belonged to space group P212121, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The present work provides valuable insights into the atomic resolution structure determination of membrane transporters.

Fast two-dimensional grid and transmission X-ray microscopy scanning methods for visualizing and characterizing protein crystals.

A fast continuous grid scan protocol has been incorporated into the Swiss Light Source (SLS) data acquisition and analysis software suite on the macromolecular crystallography (MX) beamlines. Its combination with fast readout single-photon counting hybrid pixel array detectors (PILATUS and EIGER) allows for diffraction-based identification of crystal diffraction hotspots and the location and centering of membrane protein microcrystals in the lipid cubic phase (LCP) in in meso in situ serial crystallography plates and silicon nitride supports. Diffraction-based continuous grid scans with both still and oscillation images are supported. Examples that include a grid scan of a large (50 nl) LCP bolus and analysis of the resulting diffraction images are presented. Scanning transmission X-ray microscopy (STXM) complements and benefits from fast grid scanning. STXM has been demonstrated at the SLS beamline X06SA for near-zero-dose detection of protein crystals mounted on different types of sample supports at room and cryogenic temperatures. Flash-cooled crystals in nylon loops were successfully identified in differential and integrated phase images. Crystals of just 10 µm thickness were visible in integrated phase images using data collected with the EIGER detector. STXM offers a truly low-dose method for locating crystals on solid supports prior to diffraction data collection at both synchrotron microfocusing and free-electron laser X-ray facilities.

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

Membrane proteins (MPs) are important components of cellular membranes and primary drug targets. Rational drug design relies on the exact structural information about the protein, however MPs are difficult to handle and crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization method, which yields well-diffracting but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. Introduction of the X-ray free-electron laser (XFEL) source that produces extremely bright femtosecond pulses enabled room temperature data collection from microcrystals with minimal, if any, radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, the largest and the most important drug target superfamily, yet notoriously difficult for structural determination. In LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of membrane protein microcrystals to the intersection with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can significantly improve the diffraction resolution when only micron-sized crystals are available and when crystal quality is too poor to yield interpretable structural data at conventional synchrotron microfocus beamlines. Here we present the protocol for preparation, characterization and delivery of microcrystals in LCP for LCP-SFX experiments.

Designed Functional Lipidic Biomaterials: Applications in Molecular Recognition, Drug Delivery and Membrane Protein Crystallization

A large library of lipids with novel functionalities was designed and synthesized in order to investigate the effects of lipid structure on the ensuing nanomaterial structure, dynamics, stability and phase behavior. These were assembled within lipidic mesophases and their dispersions, forming biomaterials that were effectively used in areas ranging from molecular recognition, sequestration and enrichment of nucleic acids to biosensing and fuel cells. An exciting new class of biomaterials that are based on cyclopropanated lipids was developed. These form stable lipidic cubic phases (LCPs) at low temperature, opening the way to conduct biophysical and biochemical investigations on temperature sensitive bio-macromolecules. Furthermore, pH-sensitive lipidic matrices for hydrophilic as well as hydrophobic drug incorporation and release were designed, as well as stimuli-responsive lipids that can be incorporated into biomaterials, rendering them stimuli-responsive. Efficient pH- and light-induced binding, release and sequestration of hydrophilic dyes were demonstrated. Significantly, these processes could be activated sequentially, thereby achieving both temporal and dosage control. Finally, a new light responsive, host-guest delivery system was developed, in which single step as well as sequential light-triggered release and retention of an embedded hydrophilic dye upon irradiation with UV and visible light can be affected.

Functionality and stability data of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology.

The presented data provides additional information about the assessment of affinity purified nicotinic acetylcholine receptor (nAChR) rich membrane solubilized with long chain (16 saturated carbons) lysophospholipid with glycerol headgroup (LFG-16). The assessment of stability and functionality of solubilized membrane protein is a critical step prior to further crystallization trails. One of the key factors for this task is the appropriate choice of a detergent that can support nAChR activity and stability comparable to the crude membranes. The stability of the nAChR-LFG-16 complex incorporated into lipid cubic phase (LCP) was monitored for a period of 30 days by means of fluorescence recovery after photobleaching (FRAP) and the functionality was evaluated after its incorporation into Xenopus oocyte by means of the two electrode voltage clamp technique.

Crystal Structure of the Glycophorin A Transmembrane Dimer in Lipidic Cubic Phase.

The mechanisms of assembly and function for many important type I/II (single-pass) transmembrane (TM) receptors are proposed to involve the formation and/or alteration of specific interfaces among their membrane-embedded α-helical TM domains. The application of lipidic cubic phase (LCP) bilayer media for crystallization of single-α-helical TM complexes has the potential to provide valuable structural and mechanistic insights into many such systems. However, the fidelity of the interfaces observed in crowded crystalline arrays has been difficult to establish from the very limited number of such structures determined using X-ray diffraction data. Here we examine this issue using the glycophorin A (GpA) model system, whose homodimeric TM helix interface has been characterized by solution and solid-state NMR and biochemical techniques but never crystallographically. We report that a GpA-TM peptide readily crystallized in a monoolein cubic phase bilayer, yielding a dimeric α-helical structure that is in excellent agreement with previously reported NMR measurements made in several different types of host media. These results provide compelling support for the wider application of LCP techniques to enable X-ray crystallographic analysis of single-pass TM interactions.

Nucleation and Growth of Membrane Protein Crystals In Meso—A Fluorescence Microscopy Study

Since the introduction of in meso crystallization of membrane proteins in lipidic cubic phase (LCP) by Landau and Rosenbusch in 1996, numerous studies attempted to elucidate the mechanism of in meso crystal nucleation and growth. Here we present a fluorescence microscopy study of the crystallization process of the light-driven proton pump bacteriorhodopsin. The crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. These observations suggest an Ostwald ripening mechanism of the in meso crystal growth. Analysis of the microcrystal spatial distribution suggests that microcrystal nucleation occurs predominately at the LCP domain boundaries.

Crystal Structures of SecYEG in Lipidic Cubic Phase Elucidate a Precise Resting and a Peptide-Bound State

The bacterial SecYEG translocon functions as a conserved protein-conducting channel. Conformational transitions of SecYEG allow protein translocation across the membrane without perturbation of membrane permeability. Here, we report the crystal structures of intact SecYEG at 2.7-Å resolution and of peptide-bound SecYEG at 3.6-Å resolution. The higher-resolution structure revealed that the cytoplasmic loop of SecG covers the hourglass-shaped channel, which was confirmed to also occur in the membrane by disulfide bond formation analysis and molecular dynamics simulation. The cytoplasmic loop may be involved in protein translocation. In addition, the previously unknown peptide-bound crystal structure of SecYEG implies that interactions between the cytoplasmic side of SecY and signal peptides are related to lateral gate opening at the first step of protein translocation. These SecYEG structures therefore provide a number of structural insights into the Sec machinery for further study.

Assessment of the functionality and stability of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology

In our previous study we examined the functionality and stability of nicotinic acetylcholine receptor (nAChR)-detergent complexes (nAChR-DCs) from affinity-purified Torpedo californica (Tc) using fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) and planar lipid bilayer (PLB) recordings for phospholipid and cholesterol like detergents. In the present study we enhanced the functional characterization of nAChR-DCs by recording macroscopic ion channel currents in Xenopus oocytes using the two electrode voltage clamp (TEVC). The use of TEVC allows for the recording of macroscopic currents elicited by agonist activation of nAChR-DCs that assemble in the oocyte plasma membrane. Furthermore, we examined the stability of nAChR-DCs, which is obligatory for the nAChR crystallization, using a 30day FRAP assay in LCP for each detergent. The present results indicate a marked difference in the fractional fluorescence recovery (ΔFFR) within the same detergent family during the 30day period assayed. Within the cholesterol analog family, sodium cholate and CHAPSO displayed a minimum ΔFFR and a mobile fraction (MF) over 80%. In contrast, CHAPS and BigCHAP showed a marked decay in both the mobile fraction and diffusion coefficient. nAChR-DCs containing phospholipid analog detergents with an alkylphosphocholine (FC) and lysofoscholine (LFC) of 16 carbon chains (FC-16, LFC-16) were more effective in maintaining a mobile fraction of over 80% compared to their counterparts with shorter acyl chain (C12, C14). The significant differences in macroscopic current amplitudes, activation and desensitization rates among the different nAChR-DCs evaluated in the present study allow to dissect which detergent preserves both, agonist activation and ion channel function. Functionality assays using TEVC demonstrated that LFC16, LFC14, and cholate were the most effective detergents in preserving macroscopic ion channel function, however, the nAChR-cholate complex display a significant delay in the ACh-induce channel activation. In summary, these results suggest that the physical properties of the lipid analog detergents (headgroup and acyl chain length) are the most effective in maintaining both the stability and functionality of the nAChR in the detergent solubilized complex.

Serial femtosecond crystallography of soluble proteins in lipidic cubic phase.

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.

In meso in situ serial X-ray crystallography of soluble and membrane proteins.

The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the β2-adrenoreceptor-Gs protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development.

Lipidic cubic phase serial millisecond crystallography using synchrotron radiation.

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.