scholarly journals Abstract P-30: Structure of the 80S Ribosome From Candida Albicans Revealed by Integrative Structural Biology Approach

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S25-S25
Author(s):  
Yuriy Zgadzay ◽  
Olga Kolosova ◽  
Artem Stetsenko ◽  
Konstantin Usachev ◽  
Shamil Validov ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Candida Albicans ◽  
Structural Biology ◽  
Single Particle ◽  
Bacterial Flora ◽  
World Population ◽  
80S Ribosome ◽  
X Ray ◽  
X Ray Crystallography ◽  
Resolution Structure

Background: The fungus Candida albicans is one of the most common fungal human pathogens, causing numerous mucocutaneous infections. About 80% of the world population is colonized with this pathogen, and in many cases, it does not reveal its pathogenic properties. However in the events when the suppression of normal bacterial flora occurs, e.g. as a consequence of chemotherapy or antibiotics treatment, multi-resistant strains of Candida albicans can rapidly colonize the host organism and prevent the normal flora recovery, which in turn can trigger many other diseases especially in the case of immunocompromised patients. Methods: We used an integrated structural biology approach based on the single-particle cryo-EM reconstruction and macromolecule X-ray crystallography. Results: One of the most promising targets for antibiotic action is a protein synthesis apparatus and ribosomes in the cell. In this study, we characterized structurally the protein synthesis machinery of Candida albicans using the state-of-the-art techniques of single-particle Cryo-electron microscopy and macromolecular X-ray crystallography. Conclusion: We obtained the 2.4 Å resolution structure of the 80S ribosome from Candida albicans and the 4.2 Å resolution structure of the vacant C. albicans ribosome by X-ray crystallography. We believe that this study will shed light on mechanisms of antimicrobial resistance in C. albicans and improve candidiasis treatment in the future.

scholarly journals On the explicit use of experimental images in high resolution cryo-EM refinement

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 665
Author(s):  
Jacqueline Cherfils ◽  
Jorge Navaza
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structural Biology ◽  
Single Particle ◽  
Atomic Resolution ◽  
Full Potential ◽  
X Ray ◽  
X Ray Crystallography ◽  
Atomic Models

Single particle cryogenic electron microscopy (cryo-EM) is transforming structural biology by enabling the analysis of difficult macromolecular specimens, such as membrane proteins or large complexes with flexible elements, at near atomic resolution with an accuracy close to that of X-ray crystallography. As the technique continues to improve, it is important to assess and exploit its full potential to produce the most possible reliable atomic models. Here we propose to use the experimental images as the data for refinement and validation, instead of the reconstructed maps as currently used. This procedure, which is in spirit quite similar to that used in X-ray crystallography where the data include experimental phases, should contribute to improve the quality of the cryo-EM atomic models.

scholarly journals On the explicit use of experimental images in high resolution cryo-EM refinement

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 665
Author(s):  
Jacqueline Cherfils ◽  
Jorge Navaza
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Membrane Proteins ◽  
Structural Biology ◽  
Single Particle ◽  
Atomic Resolution ◽  
Full Potential ◽  
X Ray ◽  
X Ray Crystallography

Single particle cryogenic electron microscopy (cryo-EM) is transforming structural biology by enabling the analysis of difficult macromolecular specimens, such as membrane proteins or large complexes with flexible elements, at near atomic resolution with an accuracy close to that of X-ray crystallography. As the technique continues to improve, it is important to assess and exploit its full potential to produce the most possible reliable atomic models. Here we propose to use the experimental images as the data for refinement and validation, instead of the reconstructed maps as currently used. This procedure, which is in spirit quite similar to that used in X-ray crystallography where the data include experimental phases, should contribute to improve the quality of the models.

A Solid-State NMR Approach to Structure Determination of Membrane-Associated Peptides and Proteins

1997 ◽  
Author(s):  
S.J. Opella ◽  
L.E. Chirlian
Keyword(s):  
Nmr Spectroscopy ◽  
Membrane Proteins ◽  
Structural Biology ◽  
Experimental Studies ◽  
Globular Proteins ◽  
Biological Properties ◽  
Cellular Functions ◽  
X Ray ◽  
X Ray Crystallography ◽  
Form And Function

Structural biology relies on detailed descriptions of the three-dimensional structures of peptides, proteins, and other biopolymers to explain the form and function of biological systems ranging in complexity from individual molecules to entire organisms. NMR spectroscopy and X-ray crystallography, in combination with several types of calculations, provide the required structural information. In recent years, the structures of several hundred proteins have been determined by one or both of these experimental methods. However, since the protein molecules must either reorient rapidly in samples for multidimensional solution NMR spectroscopy or form high quality single crystals in samples for X-ray crystallography, nearly all of the structures determined up to now have been of the soluble, globular proteins that are found in the cytoplasm and periplasmof cells and fortuitously have these favorable properties. Since only a minority of biological properties are expressed by globular proteins, and proteins, in general, have evolved in order to express specific functions rather than act as samples for experimental studies, there are other classes of proteins whose structures are currently unknown but are of keen interest in structural biology. More than half of all proteins appear to be associated with membranes, and many cellular functions are expressed by proteins in other types of supramolecular complexes with nucleic acids, carbohydrates, or other proteins. The interest in the structures of membrane proteins, structural proteins, and proteins in complexes provides many opportunities for the further development and application of NMR spectroscopy. Our understanding of polypeptides associated with lipids in membranes, in particular, is primitive, especially compared to that for globular proteins. This is largely a consequence of the experimental difficulties encountered in their study by conventional NMR and X-ray approaches. Fortunately, the principal features of two major classes of membrane proteins have been identified from studies of several tractable examples. Bacteriorhodopsin (Henderson et al., 1990), the subunits of the photosynthetic reaction center (Deisenhofer et al., 1985), and filamentous bacteriophage coat proteins (Shon et al., 1991; McDonnell et al., 1993) have all been shown to have long transmembrane hydrophobic helices, shorter amphipathic bridging helices in the plane of the bilayers, both structured and mobile loops connecting the helices, and mobile N- and C-terminal regions.

scholarly journals Integrative Approaches in Structural Biology: A More Complete Picture from the Combination of Individual Techniques

Biomolecules ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 370 ◽  
Author(s):  
Linda Cerofolini ◽  
Marco Fragai ◽  
Enrico Ravera ◽  
Christoph A. Diebolder ◽  
Ludovic Renault ◽  
...  
Keyword(s):  
Structural Biology ◽  
Catalytic Mechanism ◽  
X Ray ◽  
X Ray Crystallography ◽  
X Ray Scattering ◽  
Protein Model ◽  
Pros And Cons ◽  
Transient Interactions ◽  
Single Technique ◽  
Nmr Restraints

With the recent technological and computational advancements, structural biology has begun to tackle more and more difficult questions, including complex biochemical pathways and transient interactions among macromolecules. This has demonstrated that, to approach the complexity of biology, one single technique is largely insufficient and unable to yield thorough answers, whereas integrated approaches have been more and more adopted with successful results. Traditional structural techniques (X-ray crystallography and Nuclear Magnetic Resonance (NMR)) and the emerging ones (cryo-electron microscopy (cryo-EM), Small Angle X-ray Scattering (SAXS)), together with molecular modeling, have pros and cons which very nicely complement one another. In this review, three examples of synergistic approaches chosen from our previous research will be revisited. The first shows how the joint use of both solution and solid-state NMR (SSNMR), X-ray crystallography, and cryo-EM is crucial to elucidate the structure of polyethylene glycol (PEG)ylated asparaginase, which would not be obtainable through any of the techniques taken alone. The second deals with the integrated use of NMR, X-ray crystallography, and SAXS in order to elucidate the catalytic mechanism of an enzyme that is based on the flexibility of the enzyme itself. The third one shows how it is possible to put together experimental data from X-ray crystallography and NMR restraints in order to refine a protein model in order to obtain a structure which simultaneously satisfies both experimental datasets and is therefore closer to the ‘real structure’.

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.

Uncovering the structures of modular polyketide synthases

2015 ◽  
Vol 32 (3) ◽  
pp. 436-453 ◽  
Author(s):  
Kira J. Weissman
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Structural Characterization ◽  
Small Angle ◽  
Single Particle ◽  
Polyketide Synthases ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Microscopy

This review covers a breakthrough in the structural biology of the gigantic modular polyketide synthases (PKS): the structural characterization of intact modules by single-particle cryo-electron microscopy and small-angle X-ray scattering.

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