X-ray crystallographic studies of protein–ligand interactions

2003 ◽  
Vol 31 (5) ◽  
pp. 973-979 ◽  
Author(s):  
R.A. Palmer ◽  
H. Niwa
Keyword(s):  
Binding Site ◽  
Protein Structures ◽  
Three Dimensions ◽  
Small Samples ◽  
Mistletoe Lectin ◽  
Surface Binding ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sugar Binding ◽  
Wide Range

X-ray crystallography enables details of covalent and non-covalent interactions to be analysed quantitatively in three dimensions, thus providing the basis for the understanding of binding of ligands to proteins as well as modes of action such as cell-surface binding. This article is concerned with current methods employed for the X-ray analysis of protein structures complexed with ligands. It deals mainly with ‘what can be done’ in current research, rather than providing details of ‘how to do it’. In recent years significant advances have been made in a variety of techniques: growing protein crystals from very small samples by scanning a wide range of conditions; X-ray intensity data collection and measurement through the use of charge-coupled devices and high-intensity, versatile synchrotron sources; cryo-crystallography which both stabilizes the crystals and provides improved data; methods for analysing and interpreting the structures, dependent, at least in part, on both structural and sequence databases; and improvements in hardware and software. To illustrate the type of results achievable two examples involving protein–sugar interactions are discussed: (i) SNAII (the lectin Sambucus nigra agglutinin-II from elder) N-terminal sugar-binding site where terminal sugar units in a glycosylation chain from a symmetry-related molecule bind and (ii) MLI (mistletoe lectin I) C-terminal sugar-binding site with lactose.

Ulrich Wolfgang Arndt. 23 April 1924 — 24 March 2006

2014 ◽  
Vol 60 ◽  
pp. 39-55
Author(s):  
R. A. Crowther ◽  
A. G. W. Leslie
Keyword(s):  
Protein Structures ◽  
Three Dimensional ◽  
Direct Methods ◽  
Biological Molecules ◽  
Data Generation ◽  
Data Set ◽  
X Ray ◽  
X Ray Crystallography ◽  
Whole Process ◽  
Wide Range

Ulrich (Uli) Arndt was a physicist and engineer whose contributions to the development of a wide range of instrumentation for X-ray crystallography played an important part in our ability to solve the atomic structure of large biological molecules. Such detailed information about protein structures has for the past 50 years underpinned the huge advances in the field of molecular biology. His innovations spanned all aspects of data generation and collection, from improvements in X-ray tubes, through novel designs for diffractometers and cameras to film scanners and more direct methods of X-ray detection. When he started in the field, the intensities of individual X-ray reflections were often estimated by eye from films. By the end of his career the whole process of collecting from a crystal a three-dimensional data set, possibly comprising hundreds of thousands of measurements, was fully automated and very rapid.

scholarly journals X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Lan Guan ◽  
Parameswaran Hariharan
Keyword(s):  
Molecular Recognition ◽  
Binding Pocket ◽  
Cation Binding ◽  
Recognition Mechanism ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sugar Binding ◽  
Sugar Specificity ◽  
Major Facilitator ◽  
Mfs Transporters

AbstractMajor facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-d-galactoside or dodecyl-β-d-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.

scholarly journals Engineered occluded apo-intermediate of LacY

2018 ◽  
Vol 115 (50) ◽  
pp. 12716-12721 ◽  
Author(s):  
Irina Smirnova ◽  
Vladimir Kasho ◽  
H. Ronald Kaback
Keyword(s):  
Binding Site ◽  
Lactose Permease ◽  
Binding Studies ◽  
X Ray Crystallography ◽  
Sugar Binding ◽  
Transport Cycle ◽  
Open Structures ◽  
Unrestricted Access ◽  
Tight Association ◽  
Alternating Access

The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.

scholarly journals Beyond History: The List of The Most Well Studied Human Protein Structures

2020 ◽  
Author(s):  
Zhenlu Li ◽  
Matthias Buck
Keyword(s):  
Protein Structures ◽  
Protein Sequences ◽  
Human Protein ◽  
Current Status ◽  
Protein Database ◽  
X Ray ◽  
X Ray Crystallography ◽  
Protein Biophysics ◽  
The Relationship ◽  
Past Trend

Of 20,000 or so canonical human protein sequences, as of July 2020, 6,747 proteins have had their full or partial medium to high-resolution structures determined by x-ray crystallography or other methods. Which of these proteins dominate the protein database (the PDB) and why? In this paper, we list the 272 top protein structures based on the number of their PDB depositions. This set of proteins accounts for more than 40% of all available human PDB entries and represent past trend and current status for protein science. We briefly discuss the relationship which some of the prominent protein structures have with protein biophysics research and mention their relevance to human diseases. The information may inspire researchers who are new to protein science, but it also provides a year 2020 snap-shot for the state of protein science.

Catalyst-Free Synthesis of Diastereomerically Pure 3-Sulfonyl­azetidin-2-ones via Microwave-Assisted Tandem Wolff Rearrangement–Staudinger Cycloaddition

Synthesis ◽  
2020 ◽  
Author(s):  
Mikhail Krasavin ◽  
Judith Synofzik ◽  
Olga Bakulina ◽  
Olga Balabas ◽  
Dmitry Dar’in
Keyword(s):  
Drug Design ◽  
Single Crystal ◽  
Wolff Rearrangement ◽  
X Ray ◽  
Design And Optimization ◽  
X Ray Crystallography ◽  
Microwave Assisted ◽  
Catalyst Free ◽  
Relative Stereochemistry ◽  
Wide Range

A wide range of α-diazo-β-ketosulfones have been applied to thermally promoted tandem Wolff rearrangement – Staudinger [2+2] cycloaddition with imines to give polysubstituted β-lactam sulfones. Dia­stereomerically pure syn-diastereomers were obtained in good yields and the relative stereochemistry was confirmed by single-crystal X-ray crystallography. These findings significantly expand the scope of this transformation, in contrast to substantial limitations reported previously. Moreover, this methodology enables flexible exploration of new substitution patterns around the privileged β-lactam core for drug design and optimization.

scholarly journals Development of a shutterless continuous rotation method using an X-ray CMOS detector for protein crystallography

2009 ◽  
Vol 42 (6) ◽  
pp. 1165-1175 ◽  
Author(s):  
Kazuya Hasegawa ◽  
Kunio Hirata ◽  
Tetsuya Shimizu ◽  
Nobutaka Shimizu ◽  
Takaaki Hikima ◽  
...  
Keyword(s):  
Data Collection ◽  
Dynamic Range ◽  
Protein Structures ◽  
Protein Crystallography ◽  
New Method ◽  
Oxide Semiconductor ◽  
X Ray ◽  
Rotation Method ◽  
Continuous Rotation ◽  
Wide Range

A new shutterless continuous rotation method using an X-ray complementary metal-oxide semiconductor (CMOS) detector has been developed for high-speed, precise data collection in protein crystallography. The principle of operation and the basic performance of the X-ray CMOS detector (Hamamatsu Photonics KK C10158DK) have been shown to be appropriate to the shutterless continuous rotation method. The data quality of the continuous rotation method is comparable to that of the conventional oscillation method using a CCD detector and, furthermore, the combination with fine φ slicing improves the data accuracy without increasing the data-collection time. The new method is more sensitive to diffraction intensity because of the narrow dynamic range of the CMOS detector. However, the strong diffraction spots were found to be precisely measured by recording them on successive multiple images by selecting an adequate rotation step. The new method has been used to successfully determine three protein structures by multi- and single-wavelength anomalous diffraction phasing and has thereby been proved applicable in protein crystallography. The apparatus and method may become a powerful tool at synchrotron protein crystallography beamlines with important potential across a wide range of X-ray wavelengths.

scholarly journals Approach of Serial Crystallography

Crystals ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 854
Author(s):  
Ki Hyun Nam
Keyword(s):  
Radiation Damage ◽  
Room Temperature ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Past ◽  
Wide Range ◽  
Experimental Limitation ◽  
Serial Crystallography ◽  
Collection Strategies

Radiation damage and cryogenic sample environment are an experimental limitation observed in the traditional X-ray crystallography technique. However, the serial crystallography (SX) technique not only helps to determine structures at room temperature with minimal radiation damage, but it is also a useful tool for profound understanding of macromolecules. Moreover, it is a new tool for time-resolved studies. Over the past 10 years, various sample delivery techniques and data collection strategies have been developed in the SX field. It also has a wide range of applications in instruments ranging from the X-ray free electron laser (XFEL) facility to synchrotrons. The importance of the various approaches in terms of the experimental techniques and a brief review of the research carried out in the field of SX has been highlighted in this editorial.

scholarly journals A specific oligosaccharide-binding site in the alternansucrase catalytic domain mediates alternan elongation

2020 ◽  
Vol 295 (28) ◽  
pp. 9474-9489 ◽  
Author(s):  
Manon Molina ◽  
Claire Moulis ◽  
Nelly Monties ◽  
David Guieysse ◽  
Sandrine Morel ◽  
...  
Keyword(s):  
Binding Site ◽  
Molar Mass ◽  
Enzyme Stability ◽  
Catalytic Site ◽  
Glycoside Hydrolase Family ◽  
Surface Binding ◽  
High Molar Mass ◽  
Sugar Binding ◽  
Binding Pockets ◽  
Domain V

Microbial α-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) from Leuconostoc citreum NRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASRΔ2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln700 and Tyr717) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains.

scholarly journals Role of Computational Methods in Going beyond X-ray Crystallography to Explore Protein Structure and Dynamics

2018 ◽  
Vol 19 (11) ◽  
pp. 3401 ◽  
Author(s):  
Ashutosh Srivastava ◽  
Tetsuro Nagai ◽  
Arpita Srivastava ◽  
Osamu Miyashita ◽  
Florence Tama
Keyword(s):  
Protein Dynamics ◽  
Computational Methods ◽  
Protein Structures ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Insight Into

Protein structural biology came a long way since the determination of the first three-dimensional structure of myoglobin about six decades ago. Across this period, X-ray crystallography was the most important experimental method for gaining atomic-resolution insight into protein structures. However, as the role of dynamics gained importance in the function of proteins, the limitations of X-ray crystallography in not being able to capture dynamics came to the forefront. Computational methods proved to be immensely successful in understanding protein dynamics in solution, and they continue to improve in terms of both the scale and the types of systems that can be studied. In this review, we briefly discuss the limitations of X-ray crystallography in studying protein dynamics, and then provide an overview of different computational methods that are instrumental in understanding the dynamics of proteins and biomacromolecular complexes.

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