Membrane protein crystallography in the era of modern structural biology

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.

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.

Reflections on the Many Facets of Protein Microcrystallography

2014 ◽  
Vol 67 (12) ◽  
pp. 1793 ◽  
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.

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

2013 ◽  
Vol 69 (11) ◽  
pp. 2257-2265 ◽  
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.

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

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.

scholarly journals Illuminating the Secrets of Crystals - Microcrystal Electron Diffraction in Structural Biology

2018 ◽  
Author(s):  
Rob Barringer ◽  
Thomas Meier
Keyword(s):  
Electron Diffraction ◽  
Structural Biology ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Novel Method ◽  
Practical Level ◽  
Crystallographic Theory

An exploration of the crystallographic theory of the relatively novel method of Microcrystal Electron Diffraction (MicroED), via comparison to X-ray crystallography at the theoretical and practical level as it applies to biological macromolecules. We then attempt to outline the limitations and challenges that the technique currently faces in structural biology, and suggest future areas of study that may improve and optimize the technique.

scholarly journals Identifying, studying and making good use of macromolecular crystals

2014 ◽  
Vol 70 (8) ◽  
pp. 993-1008 ◽  
Author(s):  
Guillermo Calero ◽  
Aina E. Cohen ◽  
Joseph R. Luft ◽  
Janet Newman ◽  
Edward H. Snell
Keyword(s):  
Data Collection ◽  
Visible Light ◽  
Structural Biology ◽  
Protein Structures ◽  
Data Bank ◽  
Potential Outcomes ◽  
Structural Knowledge ◽  
X Ray ◽  
X Ray Crystallography ◽  
Molecular Scale

Structural biology has contributed tremendous knowledge to the understanding of life on the molecular scale. The Protein Data Bank, a depository of this structural knowledge, currently contains over 100 000 protein structures, with the majority stemming from X-ray crystallography. As the name might suggest, crystallography requires crystals. As detectors become more sensitive and X-ray sources more intense, the notion of a crystal is gradually changing from one large enough to embellish expensive jewellery to objects that have external dimensions of the order of the wavelength of visible light. Identifying these crystals is a prerequisite to their study. This paper discusses developments in identifying these crystals during crystallization screening and distinguishing them from other potential outcomes. The practical aspects of ensuring that once a crystal is identified it can then be positioned in the X-ray beam for data collection are also addressed.

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 The search for a structural basis for therapeutic intervention against the SARS coronavirus

2007 ◽  
Vol 64 (1) ◽  
pp. 204-213 ◽  
Author(s):  
Mark Bartlam ◽  
Xiaoyu Xue ◽  
Zihe Rao
Keyword(s):  
Infectious Disease ◽  
Severe Acute Respiratory Syndrome ◽  
Structural Biology ◽  
Therapeutic Intervention ◽  
Protein Structures ◽  
Structural Basis ◽  
X Ray ◽  
X Ray Crystallography ◽  
Main Protease ◽  
Underlying Mechanisms

The 2003 outbreak of severe acute respiratory syndrome (SARS), caused by a previously unknown coronavirus called SARS-CoV, had profound social and economic impacts worldwide. Since then, structure–function studies of SARS-CoV proteins have provided a wealth of information that increases our understanding of the underlying mechanisms of SARS. While no effective therapy is currently available, considerable efforts have been made to develop vaccines and drugs to prevent SARS-CoV infection. In this review, some of the notable achievements made by SARS structural biology projects worldwide are examined and strategies for therapeutic intervention are discussed based on available SARS-CoV protein structures. To date, 12 structures have been determined by X-ray crystallography or NMR from the 28 proteins encoded by SARS-CoV. One key protein, the SARS-CoV main protease (Mpro), has been the focus of considerable structure-based drug discovery efforts. This article highlights the importance of structural biology and shows that structures for drug design can be rapidly determined in the event of an emerging infectious disease.

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