Three-dimensional crystallization of membrane proteins

1988 ◽  
Vol 21 (4) ◽  
pp. 429-477 ◽  
Author(s):  
W. Kühlbrandt
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Membrane Protein Complexes ◽  
Essential Prerequisite

As recently as 10 years ago, the prospect of solving the structure of any membrane protein by X-ray crystallography seemed remote. Since then, the threedimensional (3-D) structures of two membrane protein complexes, the bacterial photosynthetic reaction centres of Rhodopseudomonas viridis (Deisenhofer et al. 1984, 1985) and of Rhodobacter sphaeroides (Allen et al. 1986, 1987 a, 6; Chang et al. 1986) have been determined at high resolution. This astonishing progress would not have been possible without the pioneering work of Michel and Garavito who first succeeded in growing 3-D crystals of the membrane proteins bacteriorhodopsin (Michel & Oesterhelt, 1980) and matrix porin (Garavito & Rosenbusch, 1980). X-ray crystallography is still the only routine method for determining the 3-D structures of biological macromolecules at high resolution and well-ordered 3-D crystals of sufficient size are the essential prerequisite.

scholarly journals Mass spectrometry of membrane protein complexes

2019 ◽  
Vol 400 (7) ◽  
pp. 813-829 ◽  
Author(s):  
Julian Bender ◽  
Carla Schmidt
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Membrane Protein Complexes ◽  
Hydrophobic Nature ◽  
Alternative Approaches ◽  
Lipid Environment ◽  
And Function

Abstract Membrane proteins are key players in the cell. Due to their hydrophobic nature they require solubilising agents such as detergents or membrane mimetics during purification and, consequently, are challenging targets in structural biology. In addition, their natural lipid environment is crucial for their structure and function further hampering their analysis. Alternative approaches are therefore required when the analysis by conventional techniques proves difficult. In this review, we highlight the broad application of mass spectrometry (MS) for the characterisation of membrane proteins and their interactions with lipids. We show that MS unambiguously identifies the protein and lipid components of membrane protein complexes, unravels their three-dimensional arrangements and further provides clues of protein-lipid interactions.

Cryo-Electron Microscopy: Moving Beyond X-Ray Crystal Structures for Drug Receptors and Drug Development

2020 ◽  
Vol 60 (1) ◽  
pp. 51-71 ◽  
Author(s):  
Javier García-Nafría ◽  
Christopher G. Tate
Keyword(s):  
Membrane Proteins ◽  
G Protein ◽  
Rational Design ◽  
Protein Complexes ◽  
Host Cells ◽  
X Ray ◽  
X Ray Crystallography ◽  
Receptor Modulation ◽  
Lipid Environment ◽  
Drug Receptors

Electron cryo-microscopy (cryo-EM) has revolutionized structure determination of membrane proteins and holds great potential for structure-based drug discovery. Here we discuss the potential of cryo-EM in the rational design of therapeutics for membrane proteins compared to X-ray crystallography. We also detail recent progress in the field of drug receptors, focusing on cryo-EM of two protein families with established therapeutic value, the γ-aminobutyric acid A receptors (GABAARs) and G protein–coupled receptors (GPCRs). GABAARs are pentameric ion channels, and cryo-EM structures of physiological heteromeric receptors in a lipid environment have uncovered the molecular basis of receptor modulation by drugs such as diazepam. The structures of ten GPCR–G protein complexes from three different classes of GPCRs have now been determined by cryo-EM. These structures give detailed insights into molecular interactions with drugs, GPCR–G protein selectivity, how accessory membrane proteins alter receptor–ligand pharmacology, and the mechanism by which HIV uses GPCRs to enter host cells.

scholarly journals The Resolution in X-ray Crystallography and Single-Particle Cryogenic Electron Microscopy

Crystals ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 580
Author(s):  
Victor R.A. Dubach ◽  
Albert Guskov
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particle Analysis ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Fourier Transform

X-ray crystallography and single-particle analysis cryogenic electron microscopy are essential techniques for uncovering the three-dimensional structures of biological macromolecules. Both techniques rely on the Fourier transform to calculate experimental maps. However, one of the crucial parameters, resolution, is rather broadly defined. Here, the methods to determine the resolution in X-ray crystallography and single-particle analysis are summarized. In X-ray crystallography, it is becoming increasingly more common to include reflections discarded previously by traditionally used standards, allowing for the inclusion of incomplete and anisotropic reflections into the refinement process. In general, the resolution is the smallest lattice spacing given by Bragg’s law for a particular set of X-ray diffraction intensities; however, typically the resolution is truncated by the user during the data processing based on certain parameters and later it is used during refinement. However, at which resolution to perform such a truncation is not always clear and this makes it very confusing for the novices entering the structural biology field. Furthermore, it is argued that the effective resolution should be also reported as it is a more descriptive measure accounting for anisotropy and incompleteness of the data. In single particle cryo-EM, the situation is not much better, as multiple ways exist to determine the resolution, such as Fourier shell correlation, spectral signal-to-noise ratio and the Fourier neighbor correlation. The most widely accepted is the Fourier shell correlation using a threshold of 0.143 to define the resolution (so-called “gold-standard”), although it is still debated whether this is the correct threshold. Besides, the resolution obtained from the Fourier shell correlation is an estimate of varying resolution across the density map. In reality, the interpretability of the map is more important than the numerical value of the resolution.

scholarly journals Topography Prediction of Helical Transmembrane Proteins by a New Modification of the Sliding Window Method

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Maria N. Simakova ◽  
Nikolai N. Simakov
Keyword(s):  
Membrane Proteins ◽  
Transmembrane Domain ◽  
Domain Boundary ◽  
Three Dimensional ◽  
Transmembrane Proteins ◽  
Sliding Window ◽  
Dimensional Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Window Method

Protein functions are specified by its three-dimensional structure, which is usually obtained by X-ray crystallography. Due to difficulty of handling membrane proteins experimentally to date the structure has only been determined for a very limited part of membrane proteins (<4%). Nevertheless, investigation of structure and functions of membrane proteins is important for medicine and pharmacology and, therefore, is of significant interest. Methods of computer modeling based on the data on the primary protein structure or the symbolic amino acid sequence have become an actual alternative to the experimental method of X-ray crystallography for investigating the structure of membrane proteins. Here we presented the results of the study of 35 transmembrane proteins, mainly GPCRs, using the novel method of cascade averaging of hydrophobicity function within the limits of a sliding window. The proposed method allowed revealing 139 transmembrane domains out of 140 (or 99.3%) identified by other methods. Also 236 transmembrane domain boundary positions out of 280 (or 84%) were predicted correctly by the proposed method with deviation from the predictions made by other methods that does not exceed the detection error of this method.

scholarly journals YidC family members are involved in the membrane insertion, lateral integration, folding, and assembly of membrane proteins

2004 ◽  
Vol 166 (6) ◽  
pp. 769-774 ◽  
Author(s):  
Ross E. Dalbey ◽  
Andreas Kuhn
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Family Members ◽  
Membrane Insertion ◽  
Conducting Channel ◽  
Energy Devices ◽  
Domains Of Life ◽  
Membrane Protein Complexes ◽  
Sensor Proteins

Members of the YidC family exist in all three domains of life, where they control the assembly of a large variety of membrane protein complexes that function as transporters, energy devices, or sensor proteins. Recent studies in bacteria have shown that YidC functions on its own as a membrane protein insertase independent of the Sec protein–conducting channel. YidC can also assist in the lateral integration and folding of membrane proteins that insert into the membrane via the Sec pathway.

scholarly journals Evidence for Membrane Complex Assembly in Nanoelectrospray Generated Lipid Bilayers

2019 ◽  
Author(s):  
Matthias Wilm
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayers ◽  
Layered Structure ◽  
Protein Complexes ◽  
Complex Assembly ◽  
Membrane Complex ◽  
Detergent Extraction ◽  
Membrane Protein Complexes

1.AbstractNanoelectrospray can be used to generate a layered structure consisting of bipolar lipids, detergent-solubilized membrane proteins, and glycerol that self-assembles upon detergent extraction into one extended layer of a protein containing membrane. This manuscript presents the first evidence that this method might allow membrane protein complexes to assemble in this process.

scholarly journals New approach for membrane protein reconstitution into peptidiscs and basis for their adaptability to different proteins

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Gabriella Angiulli ◽  
Harveer Singh Dhupar ◽  
Hiroshi Suzuki ◽  
Irvinder Singh Wason ◽  
Franck Duong Van Hoa ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Structural Basis ◽  
New Approach ◽  
Functional Studies ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Membrane Protein Complexes ◽  
Membrane Protein Reconstitution

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present ‘on-gradient’ reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional ‘on-bead’ approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.

Stoichiometry determination of macromolecular membrane protein complexes

2017 ◽  
Vol 398 (2) ◽  
pp. 155-164 ◽  
Author(s):  
Susann Zilkenat ◽  
Iwan Grin ◽  
Samuel Wagner
Keyword(s):  
High Resolution ◽  
Membrane Protein ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Transmembrane Protein ◽  
Protein Structures ◽  
Membrane Protein Complexes ◽  
Improved Methods ◽  
Structure Calculations

Abstract Gaining knowledge of the structural makeup of protein complexes is critical to advance our understanding of their formation and functions. This task is particularly challenging for transmembrane protein complexes, and grows ever more imposing with increasing size of these large macromolecular structures. The last 10 years have seen a steep increase in solved high-resolution membrane protein structures due to both new and improved methods in the field, but still most structures of large transmembrane complexes remain elusive. An important first step towards the structure elucidation of these difficult complexes is the determination of their stoichiometry, which we discuss in this review. Knowing the stoichiometry of complex components not only answers unresolved structural questions and is relevant for understanding the molecular mechanisms of macromolecular machines but also supports further attempts to obtain high-resolution structures by providing constraints for structure calculations.

Sign in / Sign up

Export Citation Format

Share Document