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.

13 Apr 2021

Towards high-throughput in situ structural biology using electron cryotomography (Prog. Biophys. Mol. Biol.)

Electron cryotomography is a rapidly evolving method for imaging macromolecules directly within the native environment of cells and tissues. Combined with sub-tomogram averaging, it allows structural and cell biologists to obtain sub-nanometre resolution structures in situ. However, low throughput in cryo-ET sample preparation and data acquisition, as well as difficulties in target localisation and subtomogram averaging image processing, limit its widespread usability. In this review, Bharat, TAM et. al. discuss new advances in the field that address these throughput and technical problems. They focus on recent efforts made to resolve issues in sample thinning, improvement in data collection speed at the microscope, strategies for localisation of macromolecules using correlated light and electron microscopy and advancements made to improve resolution in sub-tomogram averaging. These advances will considerably decrease the amount of time and effort required for cryo-ET and sub-tomogram averaging, ushering in a new era of structural biology where in situ macromolecular structure determination will be routine. 

13 Apr 2021

NMR spectroscopy captures the essential role of dynamics in regulating biomolecular function (Cell)

Biomolecules are in constant motion. To understand how they function, and why malfunctions can cause disease, it is necessary to describe their three-dimensional structures in terms of dynamic conformational ensembles. Here, L. Kay and T.R. Alderson  demonstrate how nuclearmagnetic resonance (NMR) spectroscopy provides an essential, dynamic view of structural biology that captures biomolecular motions at atomic resolution. They focus on examples that emphasize the diversity of biomolecules and biochemical applications that are amenable to NMR, such as elucidating functional dynamics in large molecular machines, characterizing transient conformations implicated in the onset of disease, and obtaining atomic-level descriptions of intrinsically disordered regions that make weak interactions involved in liquid-liquid phase separation. Finally, theydiscuss the pivotal role that NMR has played in driving forward our understanding of the biomolecular dynamics-function paradigm.

29 Mar 2021

A 'Build and Retrieve' methodology to simultaneously solve cryo-EM structures of membrane proteins (Nat. Methods)

Single-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous membrane protein samples hampers the progress of their structural determination. Here, E. W. Yu et. al. develop a bottom-up iterative method, Build and Retrieve (BaR), that enables the identification and determination of cryo-EM structures of a variety of inner and outer membrane proteins, including membrane protein complexes of different sizes and dimensions, from a heterogeneous, impure protein sample. They also use the BaR methodology to elucidate structural information from Escherichia coli K12 crude membrane and raw lysate. The findings demonstrate that it is possible to solve high-resolution structures of a number of relatively small (<100 kDa) and less abundant (<10%) unidentified membrane proteins within a single, heterogeneous sample. Importantly, these results highlight the potential of cryo-EM for systems structural proteomics.

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.