G protein-coupled receptors play a major role in transmembrane signalling in higher organisms and many are important drug targets. We report the 2.7 Å resolution crystal structure of a β
1
...-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey receptor had been selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilised by two disulphide bonds and a sodium ion. Cyanopindolol binding to the β
1
-adrenergic receptor and carazolol binding to the β
2
-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β
2
-adrenergic receptor, directly interacts via a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.
The β1-adrenoceptor (β1AR) is a G protein-coupled receptor (GPCR) that is activated by the endogenous agonists adrenaline and noradrenaline. We have determined the structure of an ultra-thermostable ...β1AR mutant bound to the weak partial agonist cyanopindolol to 2.1 Å resolution. High-quality crystals (100 μm plates) were grown in lipidic cubic phase without the assistance of a T4 lysozyme or BRIL fusion in cytoplasmic loop 3, which is commonly employed for GPCR crystallisation. An intramembrane Na+ ion was identified co-ordinated to Asp872.50, Ser1283.39 and 3 water molecules, which is part of a more extensive network of water molecules in a cavity formed between transmembrane helices 1, 2, 3, 6 and 7. Remarkably, this water network and Na+ ion is highly conserved between β1AR and the adenosine A2A receptor (rmsd of 0.3 Å), despite an overall rmsd of 2.4 Å for all Cα atoms and only 23% amino acid identity in the transmembrane regions. The affinity of agonist binding and nanobody Nb80 binding to β1AR is unaffected by Na+ ions, but the stability of the receptor is decreased by 7.5°C in the absence of Na+. Mutation of amino acid side chains that are involved in the co-ordination of either Na+ or water molecules in the network decreases the stability of β1AR by 5-10°C. The data suggest that the intramembrane Na+ and associated water network stabilise the ligand-free state of β1AR, but still permits the receptor to form the activated state which involves the collapse of the Na+ binding pocket on agonist binding.
Overview of the CCP4 suite and current developments Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D. ...
Acta crystallographica. Section D, Biological crystallography.,
April 2011, Letnik:
67, Številka:
4
Journal Article
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The CCP4 (Collaborative Computational Project, Number 4) software suite is a collection of programs and associated data and software libraries which can be used for macromolecular structure ...determination by X‐ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims. The programs are from a wide variety of sources but are connected by a common infrastructure provided by standard file formats, data objects and graphical interfaces. Structure solution by macromolecular crystallography is becoming increasingly automated and the CCP4 suite includes several automation pipelines. After giving a brief description of the evolution of CCP4 over the last 30 years, an overview of the current suite is given. While detailed descriptions are given in the accompanying articles, here it is shown how the individual programs contribute to a complete software package.
X-ray crystallography is the predominant source of structural information for biological macromolecules, providing fundamental insights into biological function. The availability of robust and ...user-friendly software to process the collected X-ray diffraction images makes the technique accessible to a wider range of scientists. iMosflm/MOSFLM (http://www.mrc-lmb.cam.ac.uk/harry/imosflm) is a software package designed to achieve this goal. The graphical user interface (GUI) version of MOSFLM (called iMosflm) is designed to guide inexperienced users through the steps of data integration, while retaining powerful features for more experienced users. Images from almost all commercially available X-ray detectors can be handled using this software. Although the program uses only 2D profile fitting, it can readily integrate data collected in the 'fine phi-slicing' mode (in which the rotation angle per image is less than the crystal mosaic spread by a factor of at least 2), which is commonly used with modern very fast readout detectors. The GUI provides real-time feedback on the success of the indexing step and the progress of data processing. This feedback includes the ability to monitor detector and crystal parameter refinement and to display the average spot shape in different regions of the detector. Data scaling and merging tasks can be initiated directly from the interface. Using this protocol, a data set of 360 images with ∼2,000 reflections per image can be processed in ∼4 min.
MicroED data collection and processing Hattne, Johan; Reyes, Francis E.; Nannenga, Brent L. ...
Acta crystallographica. Section A, Foundations and advances,
July 2015, 2015-Jul, 2015-07-01, 20150701, Letnik:
71, Številka:
4
Journal Article
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MicroED, a method at the intersection of X‐ray crystallography and electron cryo‐microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to ...determine the atomic structures of several proteins from sub‐micron‐sized, three‐dimensional crystals. A major limiting factor in X‐ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult‐to‐crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high‐resolution, three‐dimensional models by MicroED, and presents some of its unique challenges.
The structures of F₁-ATPase from bovine heart mitochondria inhibited with the dietary phytopolyphenol, resveratrol, and with the related polyphenols quercetin and piceatannol have been determined at ...2.3-, 2.4- and 2.7-Å resolution, respectively. The inhibitors bind to a common site in the inside surface of an annulus made from loops in the three α- and three β-subunits beneath the "crown" of β-strands in their N-terminal domains. This region of F₁-ATPase forms a bearing to allow the rotation of the tip of the γ-subunit inside the annulus during catalysis. The binding site is a hydrophobic pocket between the C-terminal tip of the γ-subunit and the βTP subunit, and the inhibitors are bound via H-bonds mostly to their hydroxyl moieties mediated by bound water molecules and by hydrophobic interactions. There are no equivalent sites between the γ-subunit and either the βDP or the βE subunit. The inhibitors probably prevent both the synthetic and hydrolytic activities of the enzyme by blocking both senses of rotation of the γ-subunit. The beneficial effects of dietary resveratrol may derive in part by preventing mitochondrial ATP synthesis in tumor cells, thereby inducing apoptosis.
The adenosine A2A receptor (A(2A)R) plays a key role in transmembrane signaling mediated by the endogenous agonist adenosine. Here, we describe the crystal structure of human A2AR thermostabilized in ...an active-like conformation bound to the selective agonist 2-p-(2-carboxyethyl)phenylethyl-amino-5'-N-ethylcarboxamido adenosine (CGS21680) at a resolution of 2.6 Å. Comparison of A(2A)R structures bound to either CGS21680, 5'-N-ethylcarboxamido adenosine (NECA), UK432097 6-(2,2-diphenylethylamino)-9-(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-tetrahydrofuran-2-yl-N-2-1-(2-pyridyl)-4-piperidylcarbamoylaminoethylpurine-2-carboxamide, or adenosine shows that the adenosine moiety of the ligands binds to the receptor in an identical fashion. However, an extension in CGS21680 compared with adenosine, the (2-carboxyethyl)phenylethylamino group, binds in an extended vestibule formed from transmembrane regions 2 and 7 (TM2 and TM7) and extracellular loops 2 and 3 (EL2 and EL3). The (2-carboxyethyl)phenylethylamino group makes van der Waals contacts with side chains of amino acid residues Glu169(EL2), His264(EL3), Leu267(7.32), and Ile274(7.39), and the amine group forms a hydrogen bond with the side chain of Ser67(2.65). Of these residues, only Ile274(7.39) is absolutely conserved across the human adenosine receptor subfamily. The major difference between the structures of A(2A)R bound to either adenosine or CGS21680 is that the binding pocket narrows at the extracellular surface when CGS21680 is bound, due to an inward tilt of TM2 in that region. This conformation is stabilized by hydrogen bonds formed by the side chain of Ser67(2.65) to CGS21680, either directly or via an ordered water molecule. Mutation of amino acid residues Ser67(2.65), Glu169(EL2), and His264(EL3), and analysis of receptor activation either in the presence or absence of ligands implicates this region in modulating the level of basal activity of A(2A)R.
Autoindexing diffraction images with iMosflm Powell, Harold R.; Johnson, Owen; Leslie, Andrew G. W.
Acta crystallographica. Section D, Biological crystallography.,
July 2013, Letnik:
69, Številka:
7
Journal Article
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An overview of autoindexing diffraction images based on one‐dimensional fast Fourier transforms is presented. The implementation of the algorithm in the Mosflm/iMosflm program suite is described with ...a discussion of practical issues that may arise and ways of assessing the success or failure of the procedure. Recent developments allow indexing of images that show multiple lattices, and several examples demonstrate the success of this approach in real cases.
The molecular description of the mechanism of F ₁–ATPase is based mainly on high-resolution structures of the enzyme from mitochondria, coupled with direct observations of rotation in bacterial ...enzymes. During hydrolysis of ATP, the rotor turns counterclockwise (as viewed from the membrane domain of the intact enzyme) in 120° steps. Because the rotor is asymmetric, at any moment the three catalytic sites are at different points in the catalytic cycle. In a “ground-state” structure of the bovine enzyme, one site (β E) is devoid of nucleotide and represents a state that has released the products of ATP hydrolysis. A second site (β TP) has bound the substrate, magnesium. ATP, in a precatalytic state, and in the third site (β DP), the substrate is about to undergo hydrolysis. Three successive 120° turns of the rotor interconvert the sites through these three states, hydrolyzing three ATP molecules, releasing the products and leaving the enzyme with two bound nucleotides. A transition-state analog structure, F ₁–TS, displays intermediate states between those observed in the ground state. For example, in the β DP-site of F ₁–TS, the terminal phosphate of an ATP molecule is undergoing in-line nucleophilic attack by a water molecule. As described here, we have captured another intermediate in the catalytic cycle, which helps to define the order of substrate release. In this structure, the β E-site is occupied by the product ADP, but without a magnesium ion or phosphate, providing evidence that the nucleotide is the last of the products of ATP hydrolysis to be released.
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