8 Feb 2021

A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent (Nature Struct.& Mol. Biol)

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a membrane-bound carboxypeptidase that forms a dimer and serves as the cellular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 is also a key negative regulator of the renin–angiotensin sys- tem that modulates vascular functions. B. Chen et al. report here the properties of a trimeric ACE2 ectodomain variant, engineered using a structure-based approach. The trimeric ACE2 variant has a binding affinity of ~60 pM for the spike protein of SARS-CoV-2 (compared with 77 nM for monomeric ACE2 and 12–22 nM for dimeric ACE2 constructs), and its peptidase activity and the abil- ity to block activation of angiotensin II receptor type 1 in the renin–angiotensin system are preserved. Moreover, the engineered ACE2 potently inhibits SARS-CoV-2 infection in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.

8 Feb 2021

Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM (Science Advances)

The recent outbreaks of SARS-CoV-2 pose a global health emergency. The SARS-CoV-2 trimeric spike (S) glyco- protein interacts with the human ACE2 receptor to mediate viral entry into host cells. Z. Huang, Y. Cong et al. report the cryo-EM structures of a tightly closed SARS-CoV-2 S trimer with packed fusion peptide and an ACE2-bound S trimer at 2.7- and 3.8-Å resolution, respectively. Accompanying ACE2 binding to the up receptor-binding domain (RBD), the associated ACE2-RBD exhibits continuous swing motions. Notably, the SARS-CoV-2 S trimer appears much more sensitive to the ACE2 receptor than the SARS-CoV S trimer regarding receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of SARS-CoV-2. They defined the RBD T470-T478 loop and Y505 as viral determinants for specific recognition of SARS-CoV-2 RBD by ACE2. Their findings depict the mechanism of ACE2-induced S trimer conformational transitions from the ground prefusion state toward the postfusion state, facilitating development of anti–SARS-CoV-2 vaccines and therapeutics.

8 Feb 2021

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis (Cell)

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcrip- tion complex (RTC). Here, Z. Rao, Z. Lou et al. present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. They determine the atomic cryo-EM structure of an extended RTC assembled by nsp7- nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. They also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 50 extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

29 Mar 2021

Unravelling the structural complexity of glycolipids with cryogenic infrared spectroscopy (Nat. Commun.)

Glycolipids are complex glycoconjugates composed of a glycan headgroup and a lipid moiety. Their modular biosynthesis creates a vast amount of diverse and often isomeric structures, which fulfill highly specific biological functions. To date, no gold-standard analytical technique can provide a comprehensive structural elucidation of complex glycolipids, and insufficient tools for isomer distinction can lead to wrong assignments. Herein K. Pagel et. al. use cryogenic gas-phase infrared spectroscopy to systematically investigate different kinds of isomerism in immunologically relevant glycolipids. They show that all structural features, including isomeric glycan headgroups, anomeric configurations and different lipid moieties, can be unambiguously resolved by diagnostic spectroscopic fingerprints in a narrow spectral range. The results allow for the characterization of isomeric glycolipid mixtures and biological applications.

29 Mar 2021

Current limitations to high-resolution structure determination by single-particle cryoEM (Q. Rev. Biophys.)

CryoEM has become the method of choice for determining the structure of large macromolecular complexes in multiple conformations, at resolutions where unambiguous atomic models can be built. Two effects that have limited progress in single-particle cryoEM are (i) beam- induced movement during image acquisition and (ii) protein adsorption and denaturation at the air-water interface during specimen preparation. While beam-induced movement now appears to have been resolved by all-gold specimen support grids with very small holes, surface effects at the air-water interface are a persistent problem. Strategies to overcome these effects include the use of alternative support films and new techniques for specimen deposition. E. D’Imprima and W. Kühlbrandt examine the future potential of recording perfect images of biological samples for routine structure determination at atomic resolution.

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.