1 Feb 2021

Structures and distributions of SARS-CoV-2 spike proteins on intact virions (Nature)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy, but the structure and distribution of S on the virion surface remain unknown. Here J. Briggs et.al. applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.

1 Feb 2021

Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate (Science)

Vaccine efforts to combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the current coronavirus disease 2019 (COVID-19) pandemic, are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. A. B. Ward et. al. performed cryo–election microscopy and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax, which is based on a full-length spike protein formulated in polysorbate 80 detergent. Theirstudies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared with published spike ectodomain structures. They also observed interactions between the spike trimers, allowing formation of higher-order spike complexes. This study confirms the structural integrity of the full-length spike protein immunogen and provides a basis for interpreting immune responses to this multivalent nanoparticle immunogen.

1 Feb 2021

Structural biology in the fight against COVID-19 (Nature)

How can structural biology help us understand and combat SARS-CoV-2? Researchers in the field share their experiences and opinions and point to the challenges that lie ahead.

18 Mar 2021

Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides (Nature Communications)

In the cell, DNA is arranged into highly organised and topologically constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, A.L.B. Pyne, A. Noy, S. A. Harris et. al. overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively supercoiled DNA minicircles at base-pair resolution. They observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. They probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. They show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Their results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

12 Mar 2021

Recognition of non-CpG repeats in Alu and ribosomal RNAs by the Z-RNA binding domain of ADAR1 induces A-Z junctions (Nature Communications)

Adenosine-to-inosine (A-to-I) editing of eukaryotic cellular RNAs is essential for protection against auto-immune disorders. Editing is carried out by ADAR1, whose innate immune response-specific cytoplasmic isoform possesses a Z-DNA binding domain (Zα) of unknown function. Zα also binds to CpG repeats in RNA, which are a hallmark of Z-RNA formation. Unexpectedly, Zα has been predicted — and in some cases even shown — to bind to specific regions within mRNA and rRNA devoid of such repeats. Here, Q. Vicens, B. Vögeli et. al. use NMR, circular dichroism, and other biophysical approaches to demonstrate and characterize the binding of Zα to mRNA and rRNA fragments. Their results reveal a broad range of RNA sequences that bind to Zα and adopt Z-RNA conformations. Binding is accompanied by destabilization of neighbouring A-form regions which is similar in character to what has been observed for B-Z- DNA junctions. The binding of Zα to non-CpG sequences is specific, cooperative and occurs with an affinity in the low micromolar range. This work allows them to propose a model for how Zα could influence the RNA binding specificity of ADAR1.

12 Mar 2021

A method for validating the accuracy of NMR protein structures (Nature Communications)

Williamson et. al. present a method that measures the accuracy of NMR protein structures. It compares random coil index [RCI] against local rigidity predicted by mathematical rigidity theory, calculated from NMR structures [FIRST], using a correlation score (which assesses secondary structure), and an RMSD score (which measures overall rigidity). They test its performance using: structures refined in explicit solvent, which are much better than unrefined structures; decoy structures generated for 89 NMR structures; and conventional predictors of accuracy such as number of restraints per residue, restraint violations, energy of structure, ensemble RMSD, Ramachandran distribution, and clashscore. Restraint violations and RMSD are poor measures of accuracy. Comparisons of NMR to crystal structures show that secondary structure is equally accurate, but crystal structures are typically too rigid in loops, whereas NMR structures are typically too floppy overall. They show that the method is a useful addition to existing measures of accuracy.

12 Mar 2021

Solid-state NMR spectroscopy (Nature Reviews Methods Primers)

Solid-state nuclear magnetic resonance (NMR) spectroscopy is an atomic-level method to determine the chemical structure, 3D structure and dynamics of solids and semi-solids. This Primer summarizes the basic principles of NMR spectroscopy as applied to the wide range of solid systems. The nuclear spin interactions and the effects of magnetic fields and radiofrequency pulses on nuclear spins in solid-state NMR are the same as in liquid-state NMR spectroscopy. However, because of the orientation dependence of the nuclear spin interactions in the solid state, the majority of high-resolution solid-state NMR spectra are measured under magic-angle spinning (MAS), which has profound effects on the types of radiofrequency pulse sequences required to extract structural and dynamical information. Mei Hong et. al. describe the most common MAS NMR experiments and data analysis approaches for investigating biological macromolecules, organic materials and inorganic solids. Continuing development of sensitivity-enhancement NMR approaches, including ¹H-detected fast MAS experiments, dynamic nuclear polarization and experiments in ultra-high magnetic fields, is described. They highlight recent applications of solid-state NMR spectroscopy to biological and materials chemistry. The Primer ends with a discussion of current limitations as well as areas of development of solid-state NMR spectroscopy and points to emerging areas of applications of this sophisticated spectroscopy.

11 Mar 2021

Imaging single glycans (Nature)

Imaging of biomolecules guides our understanding of their diverse structures and functions. Real-space imaging at sub-nanometre resolution using cryo-electron microscopy has provided key insights into proteins and their assemblies. Direct molecular imaging of glycans—the predominant biopolymers on Earth, with a plethora of structural and biological functions—has not been possible so far. The inherent glycan complexity and backbone flexibility require single-molecule approaches for real-space imaging. At present, glycan characterization often relies on a combination of mass spectrometry and nuclear magnetic resonance imaging to provide insights into size, sequence, branching and connectivity, and therefore requires structure reconstruction from indirect information. Here S. Rauschenbach, P. H. Seeberger, K. Kern et. al.  show direct imaging of single glycan molecules that are isolated by mass-selective, soft-landing electrospray ion beam deposition and imaged by low-temperature scanning tunnelling microscopy.The sub-nanometre resolution of the technique enables the visualization of glycan connectivity and discrimination between regioisomers. Direct glycan imaging is an important step towards a better understanding of the structure of carbohydrates.

11 Mar 2021

SINGLE: Atomic-resolution structure identification of nanocrystals by graphene liquid cell EM (Science Advances)

Analysis of the three-dimensional (3D) structures of nanocrystals with solution-phase transmission electron microscopy is beginning to reveal their unique physiochemical properties. J. Park, H. Elmlund et. al.developed a “one-particle Brownian 3D reconstruction method” based on imaging of ensembles of colloidal nanocrystals using graphene liquid cell electron microscopy. Projection images of differently rotated nanocrystals are acquired using a direct electron detector with high temporal (<2.5 ms) resolution and analyzed to obtain an ensemble of 3D reconstructions. Here, they introduce computational methods required for successful atomic-resolution 3D reconstruction: (i) tracking of the individual particles throughout the time series, (ii) subtraction of the interfering background of the graphene liquid cell, (iii) identification and rejection of low-quality images, and (iv) tailored strategies for 2D/3D alignment and averaging that differ from those used in biological cryo–electron microscopy. Their developments are made available through the open-source software package SINGLE.

11 Mar 2021

Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.5 angstrom in cells (Nature Methods)

Cryo-electron microscopy (cryo-EM) enables macromolecular structure determination in vitro and inside cells. In addition to aligning individual particles, accurate registration of sample motion and three-dimensional deformation during exposures are crucial for achieving high-resolution reconstructions. Here D. Tegunov, P. Cramer, J. Mahamid et. al. describe M, a software tool that establishes a reference-based, multi-particle refinement framework for cryo-EM data and couples a comprehensive spatial deformation model to in silico correction of electron-optical aberrations. M provides a unified optimization framework for both frame-series and tomographic tilt-series data. They show that tilt-series data can provide the same resolution as frame-series data on a purified protein specimen, indicating that the alignment step no longer limits the resolution obtainable from tomographic data. In combination with Warp and RELION, M resolves to residue level a 70S ribosome bound to an antibiotic inside intact bacterial cells. Their work provides a computational tool that facilitates structural biology in cells.

19 Feb 2021

Computational modeling of dynein motor proteins at work (Chem. Commun.)

Along with various experimental methods, a combination of theoretical and computational methods is essential to explore different length-scale and time-scale processes in the biological system. The functional mechanism of a dynein, an ATP-fueled motor protein, working in a multiprotein complex, involves a wide range of length/time-scale events. It generates mechanical force from chemical energy and moves on microtubules towards the minus end direction while performing a large number of biological processes including ciliary beating, intracellular material transport, and cell division. Like in the cases of other conventional motor proteins, a combination of experimental techniques including X-crystallography, cryo-electron microscopy, and single molecular assay have provided a wealth of information about the mechanochemical cycle of a dynein. Dyneins have a large and complex structural architecture and therefore, computational modeling of different aspects of a dynein is extremely challenging. As the process of dynein movement involves varying length and timescales, it demands, like in experiments, a combination of computational methods covering such a wide range of processes for the comprehensive investigation of the mechanochemical cycle. In this review article, M. Dutta and B.Jama summarize how the use of state-of-the-art computational methods can provide a detailed molecular understanding of the mechanochemical cycle of the dynein. They implemented all-atom molecular dynamics simulations and hybrid quantum-mechanics/molecular-mechanics simulations to explore the ATP hydrolysis mechanisms at the primary ATPase site (AAA1) of dynein. To investigate the large-scale conformational changes They employed coarse-grained structure-based molecular dynamics simulations to capture the domain motions. Here they explored the conformational changes upon binding of ATP at AAA1, nucleotide state-dependent regulation of the mechanochemical cycle, and inter-head coordination by inter-head tension. Additionally, implementing a phenomenological theoretical model we explore the force-dependent detachment rate of a motorhead from the microtubule and the principle of multi-dynein cooperation during cargo transport.