2D Crystallization Of Membrane Proteins Research Articles

MicroED: conception, practice and future opportunities.

This article documents a keynote seminar presented at the IUCr Congress in Prague, 2021. The cryo-EM method microcrystal electron diffraction is described and put in the context of macromolecular electron crystallography from its origins in 2D crystals of membrane proteins to today's application to 3D crystals a millionth the size of that needed for X-ray crystallography. Milestones in method development and applications are described with an outlook to the future.

Microfabricated silicon chip as lipid membrane sample holder for serial protein crystallography

Abstract Rapid progress in protein crystallography techniques at X-ray free electron lasers (X-FELs) requires the development of new methods for sample delivery. Recording dynamic changes in protein structures is one of the aims. To assess protein dynamics, sample holders for crystallography need to become active devices, providing possibilities to induce in situ changes of the protein conformation during the experiments. We propose an integrated device for crystallographic studies on 2D crystals of membrane proteins. The conceptual device can support physiological conditions and provide a platform for electrophysiological measurements, as well as for electrical stimulation during the diffraction experiments at X-FELs. This allows for triggering conformational changes in membrane proteins, ultimately permitting to take series of crystallographic snapshots of the dynamic behavior of proteins. The device integrates a microfabricated silicon chip between two microfluidic transport layers. We demonstrate the fabrication of the device, describe its key components and show the method of sample loading and lipid bilayer formation with the use of the microfluidic delivery system. Electrochemical impedance spectroscopy (EIS) has been used to characterize the properties of lipid bilayers formed in the microfabricated silicon nitride support. Minimizing the sample consumption, mimicking physiological conditions during crystallographic data collection and an interface for electrochemical characterization and addressing protein 2D crystals are the key properties of the proposed device.

The CRACAM Robot: Two-Dimensional Crystallization of Membrane Protein.

Membrane proteins are key cellular components that perform essential functions. They are major therapeutic targets. Electron crystallography can provide structural experimental information at atomic scale for membrane proteins forming two-dimensional (2D) crystals. There are two different methods to produce 2D crystals of membrane proteins. (1) either directly in the bulk of the solution (2) or under a lipid monolayer at the air-water interface. This extra lipid monolayer helps to pre-orient the proteins in order to facilitate the growth of 2D crystals. We present here these two methods for 2D crystallization of membrane proteins implemented in a fully automated robot called CRACAM. These methods require small volume of low concentration of proteins, making it possible to explore more conditions with the same amount of protein. These automated methods outperform traditional 2D crystallization approaches in terms of accuracy, flexibility, and throughput.

Preparation of single-layered enzyme 2D crystals for electron crystallography

Electron crystallography allows for a wide range of membrane proteins to be studied once conditions for two-dimensional (2D) crystallization have been identified. Two-dimensional crystallization is most frequently achieved via the dialysis approach, where the detergent-solubilized membrane protein is reconstituted into a lipid bilayer [1]. Vesicles, planar-tubular crystals, and sheets are the three most common 2D crystal morphologies. Vesicle and planar-tubular morphologies are observed for the largest percentage of 2D crystals of membrane proteins. Upon negative stain as well as electron cryo-microscopy (cryo-EM) grid preparation, each planar-tubular and vesicle 2D crystal will result in two ordered bilayers that can be analyzed separately by image processing. If any of these morphologies, however, contains a larger number of stacked crystals, data of tilted crystal stacks in particular can currently not be analyzed. Sheets constitute the most desirable morphology, allowing for the preparation of very flat samples for cryo-EM [2]. This is at present the only type of morphology that may be amenable to collection and analysis of electron diffraction data of highly ordered samples [3]. We could reproducibly induce single-layered sheet formation in the large majority of 2D crystals of two different enzyme samples and are working towards a general protocol applicable to other membrane protein 2D crystals.

GRecon: A Method for the Lipid Reconstitution of Membrane Proteins

Membrane proteins account for about 20–30 % of the protein-encoding genes in the genomes of all living organisms.2 Their fundamental importance in human health and disease is underlined by the fact that many drugs in current use act on them.3 Functional and structural studies of membrane proteins are therefore of increasing importance but more difficult and demanding than studies with soluble proteins, because membrane proteins function in the lipid bilayer of cell membranes. For in vitro studies, the proteins are first extracted from the membrane by detergent solubilization and then purified in detergent solution. Sensitive membrane proteins are often unstable in detergent solution, but quite stable once they are reconstituted into a lipid bilayer. Moreover, many membrane proteins require a lipid environment for activity. The reconstitution process, in which detergent is replaced by lipid, must be carefully controlled, as otherwise the proteins tend to denature and aggregate.4

GRecon: A Method for the Lipid Reconstitution of Membrane Proteins

GRecon: A Method for the Lipid Reconstitution of Membrane Proteins

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions. By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size. While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions. By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size. While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Two-dimensional crystallization conditions of human leukotriene C4 synthase requiring adjustment of a particularly large combination of specific parameters

Human leukotriene C(4) synthase (LTC(4)S) forms highly ordered two-dimensional (2D) crystals under specific reconstitution conditions. It was found that control of a larger number of parameters than is usually observed for 2D crystallization of membrane proteins was necessary to induce crystal formation of LTC(4)S. Here, we describe the parameters that were optimized to yield large and well-ordered 2D crystals of LTC(4)S. Careful fractioning of eluates during the protein purification was essential for obtaining crystals. While the lipid-to-protein ratio was critical in obtaining order, four parameters were decisive in inducing growth of crystals that were up to several microns in size. To obtain a favorable diameter, salt, temperature, glycerol, and initial detergent concentration had to be controlled with great care. Interestingly, several crystal forms could be grown, namely the plane group symmetries of p2, p3, p312, and two different unit cell sizes of plane group symmetry p321.

Controlled 2D crystallization of membrane proteins using methyl-β-cyclodextrin

High-resolution structural data of membrane proteins can be obtained by studying 2D crystals by electron crystallography. Finding the right conditions to produce these crystals is one of the major bottlenecks encountered in 2D crystallography. Many reviews address 2D crystallization techniques in attempts to provide guidelines for crystallographers. Several techniques including new approaches to remove detergent like the biobeads technique and the development of dedicated devices have been described (dialysis and dilution machines). In addition, 2D crystallization at interfaces has been studied, the most prominent method being the 2D crystallization at the lipid monolayer. A new approach based on detergent complexation by cyclodextrins is presented in this paper. To prove the ability of cyclodextrins to remove detergent from ternary mixtures (lipid, detergent and protein) in order to get 2D crystals, this method has been tested with OmpF, a typical β-barrel protein, and with SoPIP2;1, a typical α-helical protein. Experiments over different time ranges were performed to analyze the kinetic effects of detergent removal with cyclodextrins on the formation of 2D crystals. The quality of the produced crystals was assessed with negative stain electron microscopy, cryo-electron microscopy and diffraction. Both proteins yielded crystals comparable in quality to previous crystallization reports.

Two-dimensional crystallization of the ryanodine receptor Ca 2+ release channel on lipid membranes

The ryanodine receptor (RyR) is the largest known membrane protein with a total molecular mass of 2.3 × 10 3 kDa. Well ordered, two-dimensional (2D) crystals are an essential prerequisite to enable RyR structure determination by electron crystallography. Conventionally, the 2D crystallization of membrane proteins is based on a ‘trial-and-error’ strategy, which is both time-consuming and chance-directed. By adopting a new strategy that utilizes protein sequence information and predicted transmembrane topology, we successfully crystallized the RyR on positively charged lipid membranes. Image processing of negatively stained crystals reveals that they are well ordered, with diffraction spots of IQ ⩽ 4 extending to ∼20 Å, the resolution attainable in negative stain. The RyR crystals obtained on the charged lipid membrane have characteristics consistent with 2D arrays that have been observed in native sarcoplasmic reticulum of muscle tissues. These crystals provide ideal materials to enable structural analysis of RyR by high-resolution electron crystallography. Moreover, the reconstituted native-like 2D array provides an ideal model system to gain structural insights into the mechanism of RyR-mediated Ca 2+ signaling processes, in which the intrinsic ability of RyR oligomers to organize into a 2D array plays a crucial role.

2D Crystallization of Membrane Proteins: Rationales and Examples

The difficulty in crystallizing channel proteins in three dimensions limits the use of X-ray crystallography in solving their structures. In contrast, the amphiphilic character of integral membrane proteins promotes their integration into artificial lipid bilayers. Protein–protein interactions may lead to ordering of the proteins within the lipid bilayer into two-dimensional crystals that are amenable to structural studies by electron crystallography and atomic force microscopy. While reconstitution of membrane proteins with lipids is readily achieved, the mechanisms for crystal formation during or after reconstitution are not well understood. The nature of the detergent and lipid as well as pH and counterions is known to influence the crystal type and quality. Protein–protein interactions may also promote crystal stacking and aggregation of the sheet-like crystals, posing problems in data collection. Although highly promising, the number of well-studied examples is still too small to draw conclusions that would be applicable to any membrane protein of interest. Here we discuss parameters influencing the outcome of two-dimensional crystallization trials using prominent examples of channel protein crystals and highlight areas where further improvements to crystallization protocols can be made.