Wang Lab @ Tsinghua University

You et al. In situ structure of the red algal phycobilisome-PSII-PSI-LHC megacomplex. Nature, April, 2023

In oxygenic photosynthetic organisms, light energy is captured by antenna systems and transferred to photosystem II (PSII) and photosystem I (PSI) to drive photosynthesis1,2. The antenna systems of red algae consist of soluble phycobilisomes (PBSs) and transmembrane light-harvesting complexes (LHCs)3. Excitation energy transfer pathways from PBS to photosystems remain unclear owing to the lack of structural information. Here we present in situ structures of PBS-PSII-PSI-LHC megacomplexes from the red alga Porphyridium purpureum at near-atomic resolution using cryogenic electron tomography and in situ single-particle analysis4, providing interaction details between PBS, PSII and PSI. The structures reveal several unidentified and incomplete proteins and their roles in the assembly of the megacomplex, as well as a huge and sophisticated pigment network. This work provides a solid structural basis for unravelling the mechanisms of PBS-PSII-PSI-LHC megacomplex assembly, efficient energy transfer from PBS to the two photosystems, and regulation of energy distribution between PSII and PSI.

Zheng et al. Uniform thin ice on ultraflat graphene for high-resolution cryo-EM. Nature Methods, January, 2023

Cryo-electron microscopy (cryo-EM) visualizes the atomic structure of macromolecules that are embedded in vitrified thin ice at their close-to-native state. However, the homogeneity of ice thickness, a key factor to ensure high image quality, is poorly controlled during specimen preparation and has become one of the main challenges for high-resolution cryo-EM. Here we found that the uniformity of thin ice relies on the surface flatness of the supporting film, and developed a method to use ultraflat graphene (UFG) as the support for cryo-EM specimen preparation to achieve better control of vitreous ice thickness. We show that the uniform thin ice on UFG improves the image quality of vitrified specimens. Using such a method we successfully determined the three-dimensional structures of hemoglobin (64 kDa), α-fetoprotein (67 kDa) with no symmetry, and streptavidin (52 kDa) at a resolution of 3.5 Å, 2.6 Å and 2.2 Å, respectively. Furthermore, our results demonstrate the potential of UFG for the fields of cryo-electron tomography and structure-based drug discovery.

Lu et al. Functionalized graphene grids with various charges for single-particle cryo-EM, Nature Communications, November 7, 2022

A major hurdle for single particle cryo-EM in structural determination lies in the specimen preparation impaired by the air-water interface (AWI) and preferential particle-orientation problems. In this work, we develop functionalized graphene grids with various charges via a dediazoniation reaction for cryo-EM specimen preparation. The graphene grids are paraffin-assistant fabricated, which appear with less contaminations compared with those produced by polymer transfer method. By applying onto three different types of macromolecules, we demonstrate that the high-yield charged graphene grids bring macromolecules away from the AWI and enable adjustable particle-orientation distribution for more robust single particle cryo-EM structural determination.

Su et al. Structural insights into dsRNA processing by Drosophila Dicer-2-Loqs-PD, Nature, July, 2022

Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes^1,2. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs)^3,4. ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes^5,6. Here we report the cryo-electron microscopy structures of Dcr-2-Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5′-phosphate-binding pocket. The overall conformation of Dcr-2-Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2-Loqs-PD.

Xu et al. Structural engineering of graphene for high-resolution cryo-electron microscopy, SmartMat., May 26, 2021

The revolutionary improvement of hardware and algorithm in cryogenic electron microscopy (cryo‐EM) has made it a routine method to obtain structures of macromolecules at near‐atomic resolution. Nevertheless, this technique still faces many challenges. The structure‐solving efficiency of cryo‐EM can be significantly reduced by the biomolecules’ denaturation on the air–water interfaces, the preferred orientation, strong background noise from supporting films and particle motion, and so forth. To overcome these problems, nanomaterials with ultrahigh electronic conductivity and ultrathin thickness are explored as promising cryo‐EM specimen supporting films. Herein, we summarize the structural engineering of graphene, for example, surface and interface modification, as supporting films for grids and the application on high‐resolution cryo‐EM and discuss potential future perspectives.

Niu et al. Molecular basis of cross-species ACE2 interactions with SARS-CoV-2-like viruses of pangolin origin, EMBO J.，June, 2021

Pangolins have been suggested as potential reservoir of zoonotic viruses, including SARS-CoV-2 causing the global COVID-19 outbreak. Here, we study the binding of two SARS-CoV-2-like viruses isolated from pangolins, GX/P2V/2017 and GD/1/2019, to human angiotensin converting enzyme 2 (hACE2), the receptor of SARS-CoV-2. We find that the spike protein receptor-binding domain (RBD) of pangolin CoVs binds to hACE2 as efficiently as the SARS-CoV-2 RBD in vitro. Furthermore, incorporation of pangolin CoVs RBDs allows entry of pseudotyped VSV particles into hACE2-expressing cells. A screen for binding of pangolin CoV RBDs to ACE2 orthologs from various species suggests a broader host range than that of SARS-CoV-2. Additionally, cryo-EM structures of GX/P2V/2017 and GD/1/2019 RBDs in complex with hACE2 show their molecular binding in modes similar to SARS-CoV-2 RBD. Introducing the Q498H substitution found in pangolin CoVs into the SARS-CoV-2 RBD expands its binding capacity to ACE2 homologs of mouse, rat and European hedgehog. These findings suggest that these two pangolin CoVs may infect humans, highlighting the necessity of further surveillance of pangolin CoVs.

Liu et al. Reduced graphene oxide membrane as supporting film for high-resolution cryo-EM. Biophysics Reports, July, 2021

Although single-particle cryogenic electron microscopy (cryo-EM) has been applied extensively for elucidating many crucial biological mechanisms at the molecular level, this technique still faces critical challenges, the major one of which is to prepare the high-quality cryo-EM specimen. Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation, novel supporting films including graphene-based two-dimensional materials have been explored in recent years. Here we report a robust and simple method to fabricate EM grids coated with single- or few-layer reduced graphene oxide (RGO) membrane in large batch for high-resolution cryo-EM structural determination. The RGO membrane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide (GO) membrane. Moreover, we found that the RGO supporting film exhibited nice particle-absorption ability, thus avoiding the air–water interface problem. More importantly, we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100-kDa biomolecules at near-atomic resolution, as exemplified by the study of RBD-ACE2 complex and other small protein molecules. We envision that the RGO membranes can be used as a robust graphene-based supporting film in cryo-EM specimen preparation.

Nie et al. Three epitope-distinct human antibodies from RenMab mice neutralize SARS-CoV-2 and cooperatively minimize the escape of mutants. Cell Discov., July 20, 2021

Coronavirus disease 2019 (COVID-19), a pandemic disease caused by the newly emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused more than 3.8 million deaths to date. Neutralizing antibodies are effective therapeutic measures. However, many naturally occurring mutations at the receptor-binding domain (RBD) have emerged, and some of them can evade existing neutralizing antibodies. Here, we utilized RenMab, a novel mouse carrying the entire human antibody variable region, for neutralizing antibody discovery. We obtained several potent RBD-blocking antibodies and categorized them into four distinct groups by epitope mapping. We determined the involved residues of the epitope of three representative antibodies by cryo-electron microscopy (Cryo-EM) studies. Moreover, we performed neutralizing experiments with 50 variant strains with single or combined mutations and found that the mixing of three epitope-distinct antibodies almost eliminated the mutant escape. Our study provides a sound basis for the rational design of fully human antibody cocktails against SARS-CoV-2 and pre-emergent coronaviral threats.

Liu et al. Exon and protein positioning in a pre-catalytic group II intron RNP primed for splicing. Nucleic Acids Research, Epub 2020 Oct 6.

Group II introns are the putative progenitors of nuclear spliceosomal introns and use the same two-step splicing pathway. In the cell, the intron RNA forms a ribonucleoprotein (RNP) complex with the intron-encoded protein (IEP), which is essential for splicing. Although structures of spliced group II intron RNAs and RNP complexes have been characterized, structural insights into the splicing process remain enigmatic due to lack of pre-catalytic structural models. Here, we report two cryo-EM structures of endogenously produced group II intron RNPs trapped in their pre-catalytic state. Comparison of the catalytically activated precursor RNP to its previously reported spliced counterpart allowed identification of key structural rearrangements accompanying splicing, including a remodeled active site and engagement of the exons. Importantly, altered RNA-protein interactions were observed upon splicing among the RNP complexes. Furthermore, analysis of the catalytically inert precursor RNP demonstrated the structural impact of the formation of the active site on RNP architecture. Taken together, our results not only fill a gap in understanding the structural basis of IEP-assisted group II intron splicing, but also provide parallels to evolutionarily related spliceosomal splicing.

On July 1, 2020, Nan Liu (2020 Ph.D. Graduate) was awarded the honorary title of Outstanding Graduate of Beijing.