Structure of the plant plastid-encoded RNA polymerase.

Chloroplast genes encoding photosynthesis-associated proteins are predominantly transcribed by the plastid-encoded RNA polymerase (PEP). PEP is a multi-subunit complex composed of plastid-encoded subunits similar to bacterial RNA polymerases (RNAPs) stably bound to a set of nuclear-encoded PEP-associated proteins (PAPs). PAPs are essential to PEP activity and chloroplast biogenesis, but their roles are poorly defined. Here, we present cryoelectron microscopy (cryo-EM) structures of native 21-subunit PEP and a PEP transcription elongation complex from white mustard (Sinapis alba). We identify that PAPs encase the core polymerase, forming extensive interactions that likely promote complex assembly and stability. During elongation, PAPs interact with DNA downstream of the transcription bubble and with the nascent mRNA. The models reveal details of the superoxide dismutase, lysine methyltransferase, thioredoxin, and amino acid ligase enzymes that are subunits of PEP. Collectively, these data provide a foundation for the mechanistic understanding of chloroplast transcription and its role in plant growth and adaptation.

Columnar structure of human telomeric chromatin.

Telomeres, the ends of eukaryotic chromosomes, play pivotal parts in ageing and cancer and are targets of DNA damage and the DNA damage response1-5. Little is known about the structure of telomeric chromatin at the molecular level. Here we used negative stain electron microscopy and single-molecule magnetic tweezers to characterize 3-kbp-long telomeric chromatin fibres. We also obtained the cryogenic electron microscopy structure of the condensed telomeric tetranucleosome and its dinucleosome unit. The structure displayed close stacking of nucleosomes with a columnar arrangement, and an unusually short nucleosome repeat  length that comprised about 132 bp DNA wound in a continuous superhelix around histone octamers. This columnar structure is primarily stabilized by the H2A carboxy-terminal and histone amino-terminal tails in a synergistic manner. The columnar conformation results in exposure of the DNA helix, which may make it susceptible to both DNA damage and the DNA damage response. The conformation also exists in an alternative open state, in which one nucleosome is unstacked and flipped out, which exposes the acidic patch of the histone surface. The structural features revealed in this work suggest mechanisms by which protein factors involved in telomere maintenance can access telomeric chromatin in its compact form.

Correlation between the binding affinity and the conformational entropy of nanobody SARS-CoV-2 spike protein complexes.

Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure-activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein-nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.

Application of super-resolution and correlative double sampling in cryo-electron microscopy.

Developments in cryo-EM have allowed atomic or near-atomic resolution structure determination to become routine in single particle analysis (SPA). However, near-atomic resolution structures determined using cryo-electron tomography and sub-tomogram averaging (cryo-ET STA) are much less routine. In this paper, we show that collecting cryo-ET STA data using the same conditions as SPA, with both correlated double sampling (CDS) and the super-resolution mode, allowed apoferritin to be reconstructed out to the physical Nyquist frequency of the images. Even with just two tilt series, STA yields an apoferritin map at 2.9 Å resolution. These results highlight the exciting potential of cryo-ET STA in the future of protein structure determination. While processing SPA data recorded in super-resolution mode may yield structures surpassing the physical Nyquist limit, processing cryo-ET STA data in the super-resolution mode gave no additional resolution benefit. We further show that collecting SPA data in the super-resolution mode, with CDS activated, reduces the estimated B-factor, leading to a reduction in the number of particles required to reach a target resolution without compromising the data size on disk and the area imaged in SerialEM. However, collecting SPA data in CDS does reduce throughput, given that a similar resolution structure, with a slightly larger B-factor, is achievable with optimised parameters for speed in EPU (without CDS).

3.9 Å structure of the nucleosome core particle determined by phase-plate cryo-EM.

The Volta phase plate is a recently developed electron cryo-microscopy (cryo-EM) device that enables contrast enhancement of biological samples. Here we have evaluated the potential of combining phase-plate imaging and single particle analysis to determine the structure of a small protein-DNA complex. To test the method, we made use of a 200 kDa Nucleosome Core Particle (NCP) reconstituted with 601 DNA for which a high-resolution X-ray crystal structure is known. We find that the phase plate provides a significant contrast enhancement that permits individual NCPs and DNA to be clearly identified in amorphous ice. The refined structure from 26,060 particles has an overall resolution of 3.9 Å and the density map exhibits structural features consistent with the estimated resolution, including clear density for amino acid side chains and DNA features such as the phosphate backbone. Our results demonstrate that phase-plate cryo-EM promises to become an important method to determine novel near-atomic resolution structures of small and challenging samples, such as nucleosomes in complex with nucleosome-binding factors.

Exploring TRF2-Dependent DNA Distortion Through Single-DNA Manipulation Studies.

TRF2 is a component of shelterin, a telomere-specific protein complex that protects the ends of mammalian chromosomes from DNA damage signaling and improper repair. TRF2 functions as a homodimer and its interaction with telomeric DNA has been studied, but its full-length DNA-binding properties are unknown. This study examines TRF2's interaction with single-DNA strands and focuses on the conformation of the TRF2-DNA complex and TRF2's preference for DNA chirality. The results show that TRF2-DNA can switch between extended and compact conformations, indicating multiple DNA-binding modes, and TRF2's binding does not have a strong preference for DNA supercoiling chirality when DNA is under low tension. Instead, TRF2 induces DNA bending under tension. Furthermore, both the N-terminal domain of TRF2 and the Myb domain enhance its affinity for the telomere sequence, highlighting the crucial role of multivalent DNA binding in enhancing its affinity and specificity for telomere sequence. These discoveries offer unique insights into TRF2's interaction with telomeric DNA.

Linker histone defines structure and self-association behaviour of the 177 bp human chromatosome.

Linker histones play essential roles in the regulation and maintenance of the dynamic chromatin structure of higher eukaryotes. The influence of human histone H1.0 on the nucleosome structure and biophysical properties of the resulting chromatosome were investigated and compared with the 177-bp nucleosome using Cryo-EM and SAXS. The 4.5 Å Cryo-EM chromatosome structure showed that the linker histone binds at the nucleosome dyad interacting with both linker DNA arms but in a tilted manner leaning towards one of the linker sides. The chromatosome is laterally compacted and rigid in the dyad and linker DNA area, in comparison with the nucleosome where linker DNA region is more flexible and displays structural variability. In solution, the chromatosomes appear slightly larger than the nucleosomes, with the volume increase compared to the bound linker histone, according to solution SAXS measurements. SAXS X-ray diffraction characterisation of Mg-precipitated samples showed that the different shapes of the 177 chromatosome enabled the formation of a highly ordered lamello-columnar phase when precipitated by Mg2+, indicating the influence of linker histone on the nucleosome stacking. The biological significance of linker histone, therefore, may be affected by the change in the polyelectrolyte and DNA conformation properties of the chromatosomes, in comparison to nucleosomes.

Author Correction: Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2.

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.