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Biochemical Society Transactions Nov 2021G protein-coupled receptors (GPCRs) are the largest single family of cell surface receptors encoded by the human genome and they play pivotal roles in co-ordinating... (Review)
Review
G protein-coupled receptors (GPCRs) are the largest single family of cell surface receptors encoded by the human genome and they play pivotal roles in co-ordinating cellular systems throughout the human body, making them ideal drug targets. Structural biology has played a key role in defining how receptors are activated and signal through G proteins and β-arrestins. The application of structure-based drug design (SBDD) is now yielding novel compounds targeting GPCRs. There is thus significant interest from both academia and the pharmaceutical industry in the structural biology of GPCRs as currently only about one quarter of human non-odorant receptors have had their structure determined. Initially, all the structures were determined by X-ray crystallography, but recent advances in electron cryo-microscopy (cryo-EM) now make GPCRs tractable targets for single-particle cryo-EM with comparable resolution to X-ray crystallography. So far this year, 78% of the 99 GPCR structures deposited in the PDB (Jan-Jul 2021) were determined by cryo-EM. Cryo-EM has also opened up new possibilities in GPCR structural biology, such as determining structures of GPCRs embedded in a lipid nanodisc and multiple GPCR conformations from a single preparation. However, X-ray crystallography still has a number of advantages, particularly in the speed of determining many structures of the same receptor bound to different ligands, an essential prerequisite for effective SBDD. We will discuss the relative merits of cryo-EM and X-ray crystallography for the structure determination of GPCRs and the future potential of both techniques.
Topics: Cryoelectron Microscopy; Crystallography, X-Ray; Humans; Ligands; Protein Conformation; Receptors, G-Protein-Coupled
PubMed: 34581758
DOI: 10.1042/BST20210431 -
Journal of Visualized Experiments : JoVE Jan 2011Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of...
Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography. To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999). Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop. Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.
Topics: Crystallization; Crystallography, X-Ray; Diffusion; Proteins
PubMed: 21304455
DOI: 10.3791/2285 -
Molecules (Basel, Switzerland) Feb 2020With the advent of structural biology in the drug discovery process, medicinal chemists gained the opportunity to use detailed structural information in order to... (Review)
Review
With the advent of structural biology in the drug discovery process, medicinal chemists gained the opportunity to use detailed structural information in order to progress screening hits into leads or drug candidates. X-ray crystallography has proven to be an invaluable tool in this respect, as it is able to provide exquisitely comprehensive structural information about the interaction of a ligand with a pharmacological target. As fragment-based drug discovery emerged in the recent years, X-ray crystallography has also become a powerful screening technology, able to provide structural information on complexes involving low-molecular weight compounds, despite weak binding affinities. Given the low numbers of compounds needed in a fragment library, compared to the hundreds of thousand usually present in drug-like compound libraries, it now becomes feasible to screen a whole fragment library using X-ray crystallography, providing a wealth of structural details that will fuel the fragment to drug process. Here, we review theoretical and practical aspects as well as the pros and cons of using X-ray crystallography in the drug discovery process.
Topics: Crystallography, X-Ray; Drug Discovery; Drug Evaluation, Preclinical; Humans; Ligands; Proteins; Small Molecule Libraries
PubMed: 32106588
DOI: 10.3390/molecules25051030 -
The FEBS Journal Oct 2021The motto of this Virtual Issue of The FEBS Journal is a paraphrase of the statement made in 1897 by Mark Twain, which is usually quoted as 'Reports of my death have...
The motto of this Virtual Issue of The FEBS Journal is a paraphrase of the statement made in 1897 by Mark Twain, which is usually quoted as 'Reports of my death have been greatly exaggerated'. With the incredible progress in the utilization of cryo-EM for the determination of high-resolution macromolecular structures that led to the award of the Nobel Prize in Chemistry to Jacques Dubochet, Joachim Frank, and Richard Henderson in 2017, it became a common assumption that crystallography was dead. However, as this Virtual Issue should show very clearly, that is emphatically not the case. To put the current relative importance of different technologies of determination of macromolecular structures into perspective, 78% of structures deposited in the Protein Data Bank since January 2020 were still determined by X-ray crystallography. The reasons why that is the case will be clear after reading the papers gathered here.
Topics: Animals; Crystallography, X-Ray; Humans; Models, Molecular; Molecular Structure; Protein Conformation; Proteins
PubMed: 33759375
DOI: 10.1111/febs.15822 -
Protein Science : a Publication of the... Jan 2017With the ability to resolve structures of macromolecules at atomic resolution, X-ray crystallography has been the most powerful tool in modern structural biology. At the... (Review)
Review
With the ability to resolve structures of macromolecules at atomic resolution, X-ray crystallography has been the most powerful tool in modern structural biology. At the same time, recent technical improvements have triggered a resolution revolution in the single particle cryo-EM method. While the two methods are different in many respects, from sample preparation to structure determination, they both have the power to solve macromolecular structures at atomic resolution. It is important to understand the unique advantages and caveats of the two methods in solving structures and to appreciate the complementary nature of the two methods in structural biology. In this review we provide some examples, and discuss how X-ray crystallography and cryo-EM can be combined in deciphering structures of macromolecules for our full understanding of their biological mechanisms.
Topics: Cryoelectron Microscopy; Crystallography, X-Ray
PubMed: 27543495
DOI: 10.1002/pro.3022 -
Acta Crystallographica. Section D,... Feb 2010Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still...
Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
Topics: Algorithms; Crystallography, X-Ray; Models, Molecular; Software Design
PubMed: 20124702
DOI: 10.1107/S0907444909052925 -
Scientific Reports Nov 2023The optimized synthesis of [5-oxo-4,4-diphenylimidazolidin-2-ylidene]cyanamide, which is known as 2-cyanoguanidinophenytoin (CNG-DPH) (3), and...
The optimized synthesis of [5-oxo-4,4-diphenylimidazolidin-2-ylidene]cyanamide, which is known as 2-cyanoguanidinophenytoin (CNG-DPH) (3), and (imidazo[4,5-d]imidazole-2,5-diylidine)dicyanamide (4) has been reported in the present work. Furthermore, new Mannich bases derived from CNG-DPH were synthesized via its reaction with formaldehyde and using the corresponding amines, piperidine (base 5), and morpholine (base 6). Also, the antimicrobial activity and X-ray crystal structures for CNG-DPH and their Mannich bases were studied. The bases 3 and 6 crystallized in a monoclinic system; the crystal structure of 3 containing four molecules in the unit cell with a P2/c space group. The unit cell of 6 has eight molecules with a C2/c space group. The inter and intra hydrogen bond contacts packed and stabilized both of the structures. The morpholine ring of base 6 demonstrated a distinctive chair configuration. Mannich bases 5 and 6 showed promising antimicrobial effects. base 4 has a greater percentage for in vitro cytotoxicity (IC) against normal cells, whereas 3 has the lowest ratio.
Topics: Mannich Bases; Crystallography, X-Ray; Anti-Infective Agents; Morpholines
PubMed: 37945617
DOI: 10.1038/s41598-023-45533-1 -
Journal of the American Chemical Society Feb 2020Haliclonadiamine and papuamine are bis-indane marine natural products isolated from the marine sponge sp. Their relative structures were previously reported to differ...
Haliclonadiamine and papuamine are bis-indane marine natural products isolated from the marine sponge sp. Their relative structures were previously reported to differ by inversion at only one of their eight shared stereocenters. Here X-ray crystallography shows the opposite to be true: papuamine has a 1,3,8,9,14,15,20,22 configuration, while haliclonadiamine has a 1,3,8,9,14,15,20,22 configuration. Paradoxically the ECD of each structure displays a negative Cotton effect. X-ray crystallography reveals the two structures adopt similar conformations of their 13-membered macrocyclic core that comprises a configurationally relevant diene. B97x-D/Def2-TZVPP-(MeOH)-calculated ECD supports the diene configuration with the macrocycle dominating the ECD Cotton effect for haliclonadiamine and papuamine. Additional crystallographic and chiroptical analyses of three sponge samples from geographically distant locations indicate this pair of natural products always exists as a configurationally related couple. The co-discovery of a biosynthetic precursor, halichondriamine C, present in these same Haliclona samples must be considered when discussing any biosynthetic pathway. Taken together, this work justifies a reassignment of haliclonadiamine's structure and opens the question of how this complex stereochemical relationship between haliclonadiamine and palauamine arises biosynthetically.
Topics: Alkaloids; Crystallography, X-Ray; Magnetic Resonance Spectroscopy; Molecular Structure; Optics and Photonics
PubMed: 31986017
DOI: 10.1021/jacs.9b12926 -
Protein Science : a Publication of the... Jan 2022The DIALS software for the processing of X-ray diffraction data is presented, with an emphasis on how the suite may be used as a toolkit for data processing. The...
The DIALS software for the processing of X-ray diffraction data is presented, with an emphasis on how the suite may be used as a toolkit for data processing. The description starts with an overview of the history and intent of the toolkit, usage as an automated system, command-line use, and ultimately how new tools can be written using the API to perform bespoke analysis. Consideration is also made to the application of DIALS to techniques outside of macromolecular X-ray crystallography.
Topics: Crystallography, X-Ray; Electronic Data Processing; Software
PubMed: 34747533
DOI: 10.1002/pro.4224 -
Channels (Austin, Tex.) Jan 2018Transient receptor potential (TRP) ion channels are molecular sensors of a large variety of stimuli including temperature, mechanical stress, voltage, small molecules... (Review)
Review
Transient receptor potential (TRP) ion channels are molecular sensors of a large variety of stimuli including temperature, mechanical stress, voltage, small molecules including capsaicin and menthol, and lipids such as phosphatidylinositol 4,5-bisphosphate (PIP). Since the same TRP channels may respond to different physical and chemical stimuli, they can serve as signal integrators. Many TRP channels are calcium permeable and contribute to Ca homeostasis and signaling. Although the TRP channel family was discovered decades ago, only recently have the structures of many of these channels been solved, largely by cryo-electron microscopy (cryo-EM). Complimentary to cryo-EM, X-ray crystallography provides unique tools to unambiguously identify specific atoms and can be used to study ion binding in channel pores. In this review we describe crystallographic studies of the TRP channel TRPV6. The methodology used in these studies may serve as a template for future structural analyses of different types of TRP and other ion channels.
Topics: Animals; Crystallography, X-Ray; Humans; Models, Molecular; Transient Receptor Potential Channels
PubMed: 29589513
DOI: 10.1080/19336950.2018.1457898