The Diamond Light Source and the challenges ahead for structural biology: some informal remarks

The remarkable advances in structural biology in the past three decades have led to the determination of increasingly complex structures that lie at the heart of many important biological processes. Many of these advances have been made possible by the use of X-ray crystallography using synchrotron radiation. In this short article, some of the challenges and prospects that lie ahead will be summarized.

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From lows to highs: using low-resolution models to phase X-ray data

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913022336 ◽

2013 ◽

Vol 69 (11) ◽

pp. 2257-2265 ◽

Cited By ~ 9

Author(s):

David I. Stuart ◽

Nicola G. A. Abrescia

Keyword(s):

Structural Biology ◽

General Concept ◽

Molecular Replacement ◽

X Ray ◽

X Ray Crystallography ◽

X Ray Scattering ◽

Phase Extension ◽

Intact Virus ◽

Structural Methods

The study of virus structures has contributed to methodological advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here, the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an ∼13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.

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Structure determination of GPCRs: cryo-EM compared with X-ray crystallography

Biochemical Society Transactions ◽

10.1042/bst20210431 ◽

2021 ◽

Author(s):

Javier García-Nafría ◽

Christopher G. Tate

Keyword(s):

Structural Biology ◽

Structure Determination ◽

Drug Targets ◽

Cellular Systems ◽

Single Family ◽

Structure Based Drug Design ◽

X Ray ◽

Surface Receptors ◽

X Ray Crystallography

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.

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Structural Biology of Influenza Hemagglutinin: An Amaranthine Adventure

Viruses ◽

10.3390/v12091053 ◽

2020 ◽

Vol 12 (9) ◽

pp. 1053

Author(s):

Nicholas C. Wu ◽

Ian A. Wilson

Keyword(s):

Membrane Fusion ◽

Structural Biology ◽

Receptor Binding ◽

Viral Entry ◽

Antigenic Drift ◽

Current Status ◽

X Ray ◽

X Ray Crystallography ◽

The Past ◽

Influenza Hemagglutinin

Hemagglutinin (HA) glycoprotein is an important focus of influenza research due to its role in antigenic drift and shift, as well as its receptor binding and membrane fusion functions, which are indispensable for viral entry. Over the past four decades, X-ray crystallography has greatly facilitated our understanding of HA receptor binding, membrane fusion, and antigenicity. The recent advances in cryo-EM have further deepened our comprehension of HA biology. Since influenza HA constantly evolves in natural circulating strains, there are always new questions to be answered. The incessant accumulation of knowledge on the structural biology of HA over several decades has also facilitated the design and development of novel therapeutics and vaccines. This review describes the current status of the field of HA structural biology, how we got here, and what the next steps might be.

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Membrane protein crystallography in the era of modern structural biology

Biochemical Society Transactions ◽

10.1042/bst20200066 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2505-2524

Author(s):

Tristan O. C. Kwan ◽

Danny Axford ◽

Isabel Moraes

Keyword(s):

Structural Biology ◽

Molecular Level ◽

Protein Structures ◽

Three Dimensional ◽

Biological Macromolecules ◽

X Ray ◽

X Ray Crystallography ◽

Cellular Processes ◽

Biochemical Level

The aim of structural biology has been always the study of biological macromolecules structures and their mechanistic behaviour at molecular level. To achieve its goal, multiple biophysical methods and approaches have become part of the structural biology toolbox. Considered as one of the pillars of structural biology, X-ray crystallography has been the most successful method for solving three-dimensional protein structures at atomic level to date. It is however limited by the success in obtaining well-ordered protein crystals that diffract at high resolution. This is especially true for challenging targets such as membrane proteins (MPs). Understanding structure-function relationships of MPs at the biochemical level is vital for medicine and drug discovery as they play critical roles in many cellular processes. Though difficult, structure determination of MPs by X-ray crystallography has significantly improved in the last two decades, mainly due to many relevant technological and methodological developments. Today, numerous MP crystal structures have been solved, revealing many of their mechanisms of action. Yet the field of structural biology has also been through significant technological breakthroughs in recent years, particularly in the fields of single particle electron microscopy (cryo-EM) and X-ray free electron lasers (XFELs). Here we summarise the most important advancements in the field of MP crystallography and the significance of these developments in the present era of modern structural biology.

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LA RIVOLUZIONE DELLA CRIO-MICROSCOPIA ELETTRONICA NELLO STUDIO STRUTTURALE DI PROTEINE E ACIDI NUCLEICI

Istituto Lombardo - Accademia di Scienze e Lettere - Rendiconti di Scienze ◽

10.4081/scie.2017.638 ◽

2020 ◽

Author(s):

Martino Bolognesi

Keyword(s):

Electron Microscopy ◽

Amino Acids ◽

Structural Biology ◽

Molecular Structures ◽

X Ray ◽

X Ray Crystallography ◽

The Past ◽

Cryo Electron Microscopy ◽

Resolution Level ◽

Nello Studio

Observing the fine details of molecular structures (e.g. in proteins and in nucleic acids) has been a central part of Structural Biology over the past 50 years. The recent advent of single particle cryo-electron microscopy brought a revolution in this field, that previously relied on X-ray crystallography and nuclear magnetic resonance. It is now possible to explore the structures of large subcellular assemblies, such as the ribosome, resolving details on the scale of amino acids and nucleotides, in favorable cases reaching the 2 Å resolution level.

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Reflections on the Many Facets of Protein Microcrystallography

Australian Journal of Chemistry ◽

10.1071/ch14455 ◽

2014 ◽

Vol 67 (12) ◽

pp. 1793 ◽

Cited By ~ 5

Author(s):

Marion Boudes ◽

Damià Garriga ◽

Fasséli Coulibaly

Keyword(s):

Structural Biology ◽

State Of The Art ◽

Data Bank ◽

Biological Macromolecules ◽

Structure Based Drug Design ◽

X Ray ◽

X Ray Crystallography ◽

The Many ◽

The Impact

The use of X-ray crystallography for the structure determination of biological macromolecules has experienced a steady expansion over the last 20 years with the Protein Data Bank growing from <1000 deposited structures in 1992 to >100 000 in 2014. The large number of structures determined each year not only reflects the impact of X-ray crystallography on many disciplines in the biological and medical fields but also its accessibility to non-expert laboratories. Thus protein crystallography is now largely a mainstream research technique and is routinely integrated in high-throughput pipelines such as structural genomics projects and structure-based drug design. Yet, significant frontiers remain that continuously require methodological developments. In particular, membrane proteins, large assemblies, and proteins from scarce natural sources still represent challenging targets for which obtaining the large diffracting crystals required for classical crystallography is often difficult. These limitations have fostered the emergence of microcrystallography, novel approaches in structural biology that collectively aim at determining structures from the smallest crystals. Here, we review the state of the art of macromolecular microcrystallography and recent progress achieved in this field.

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Quantitative investigation of linear arbitrary polarization in an APPLE-II undulator

Journal of Synchrotron Radiation ◽

10.1107/s1600577518001960 ◽

2018 ◽

Vol 25 (2) ◽

pp. 378-384

Author(s):

Matthew Hand ◽

Hongchang Wang ◽

Francesco Maccherozzi ◽

Marco Apollonio ◽

Jingtao Zhu ◽

...

Keyword(s):

Electron Beam ◽

Synchrotron Radiation ◽

Light Source ◽

Polarization State ◽

Measured Data ◽

Beam Polarization ◽

Quantitative Investigation ◽

X Ray ◽

The World

Insertion devices are utilized at synchrotron radiation facilities around the world for their capability to provide a high-brilliance X-ray beam. APPLE-II type undulators are especially important for their capacity to switch between a variety of photon beam polarization states. A high-precision soft X-ray polarimeter has been used to investigate the polarization calibration of an APPLE-II undulator (period length λu= 64 mm) installed on beamline I06 at Diamond Light Source. Systematic measurement of the beam polarization state at a range of linear arbitrary angles has been compared with the expected result for a given set of undulator gap and row phase parameters calculated from theory. Determination of the corresponding Stokes–Poincaré parameters from the measured data reveals a discrepancy between the two. The limited number of energy/polarization combinations included in the undulator calibration tables necessitates the use of interpolated values for the missing points which is expected to contribute to the discrepancy. However, by modifying the orbit of the electron beam through the undulator by at least 160 µm it has been found that for certain linear polarizations the discrepancies can be corrected. Overall, it is suggested that complete correction of the Stokes–Poincaré parameters for all linear angles would require alteration of both these aspects.

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Determining structures of biomacromolecular complexes from ambiguous NMR restraints and cryoEM data

10.1101/2020.03.27.990309 ◽

2020 ◽

Author(s):

Reto Walser ◽

Alexander G. Milbradt

Keyword(s):

Structural Biology ◽

Boundary Region ◽

Model System ◽

Global Information ◽

X Ray ◽

X Ray Crystallography ◽

Nmr Data ◽

Nmr Restraints ◽

Integrated Structural Biology

AbstractIntegrated structural biology aims at combining different techniques to tackle challenging systems. Where individual techniques are not delivering structures of suitable quality, harnessing the strengths of various methods can often overcome this problem. X-ray crystallography and NMR have been the two most widely applied structural biology disciplines. In recent years cryoelectron microscopy (cryoEM) has become ever more powerful and is now capable of providing structures at resolutions comparable to those common in X-ray crystallography. Unfortunately, both NMR and cryoEM have inherent limitations on the system under study. However, the two techniques can be considered somewhat complementary as NMR has an upper and cryoEM a lower molecular weight (MW) limit. Here, we present a joint NMR and cryoEM methodology for the determination of biomacromolecular structures at the boundary region between the MW limits of the two techniques. The method relies on measuring chemical shift perturbations, which is the most readily accessible NMR parameter for characterizing the interaction of biomacromolecular complexes. Low-resolution cryoEM information yields global information on the shape of the complex and is used for complementing the local NMR data. We have successfully applied this method to the model system histidine-containing phosphoprotein (HPr) in complex with the glucose-specific acceptor protein IIAGlc from Escherichia coli.

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