Structural basis of TFIIH activation for nucleotide excision repair.

Nucleotide excision repair (NER) is the major DNA repair pathway that removes UV-induced and bulky DNA lesions. There is currently no structure of NER intermediates, which form around the large multisubunit transcription factor IIH (TFIIH). Here we report the cryo-EM structure of an NER intermediate containing TFIIH and the NER factor XPA. Compared to its transcription conformation, the TFIIH structure is rearranged such that its ATPase subunits XPB and XPD bind double- and single-stranded DNA, consistent with their translocase and helicase activities, respectively. XPA releases the inhibitory kinase module of TFIIH, displaces a 'plug' element from the DNA-binding pore in XPD, and together with the NER factor XPG stimulates XPD activity. Our results explain how TFIIH is switched from a transcription to a repair factor, and provide the basis for a mechanistic analysis of the NER pathway

Structure of replicating SARS-CoV-2 polymerase

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes1–3. Here we present the cryo-electron microscopic structure of the SARS-CoV-2 RdRp in active form, mimicking the replicating enzyme. The structure comprises the viral proteins nsp12, nsp8, and nsp7, and over two turns of RNA template-product duplex. The active site cleft of nsp12 binds the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the second turn of RNA. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged ‘sliding poles’. These sliding poles can account for the known processivity of the RdRp that is required for replicating the long coronavirus genome3. Our results enable a detailed analysis of the inhibitory mechanisms that underlie the antiviral activity of substances such as remdesivir, a drug for the treatment of coronavirus disease 2019 (COVID-19)4

Liquid-crystalline phase transitions in lipid droplets are related to cellular states and specific organelle association.

Lipid droplets (LDs) are ubiquitous organelles comprising a central hub for cellular lipid metabolism and trafficking. This role is tightly associated with their interactions with several cellular organelles. Here, we provide a systematic and quantitative structural description of LDs in their native state in HeLa cells enabled by cellular cryoelectron microscopy. LDs consist of a hydrophobic neutral lipid mixture of triacylglycerols (TAG) and cholesteryl esters (CE), surrounded by a single monolayer of phospholipids. We show that under normal culture conditions, LDs are amorphous and that they transition into a smectic liquid-crystalline phase surrounding an amorphous core at physiological temperature under certain cell-cycle stages or metabolic scenarios. Following determination of the crystal lattice spacing of 3.5 nm and of a phase transition temperature below 43 degrees C, we attributed the liquid-crystalline phase to CE. We suggest that under mitotic arrest and starvation, relative CE levels increase, presumably due to the consumption of TAG metabolites for membrane synthesis and mitochondrial respiration, respectively, supported by direct visualization of LD-mitochondrial membrane contact sites. We hypothesize that the structural phase transition may have a major impact on the accessibility of lipids in LDs to enzymes or lipid transporters. These may become restricted in the smectic phase, affecting the exchange rate of lipids with surrounding membranes and lead to a different surface occupancy of LD-associated proteins. Therefore, the composition and the resulting internal structure of LDs is expected to play a key role in their function as hubs of cellular lipid flux

The structure of a dimeric form of SARS-CoV-2 polymerase

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs

Architecture of the RNA polymerase II-Paf1C-TFIIS transcription elongation complex.

The conserved polymerase-associated factor 1 complex (Paf1C) plays multiple roles in chromatin transcription and genomic regulation. Paf1C comprises the five subunits Paf1, Leo1, Ctr9, Cdc73 and Rtf1, and binds to the RNA polymerase II (Pol II) transcription elongation complex (EC). Here we report the reconstitution of Paf1C from Saccharomyces cerevisiae, and a structural analysis of Paf1C bound to a Pol II EC containing the elongation factor TFIIS. Cryo-electron microscopy and crosslinking data reveal that Paf1C is highly mobile and extends over the outer Pol II surface from the Rpb2 to the Rpb3 subunit. The Paf1-Leo1 heterodimer and Cdc73 form opposite ends of Paf1C, whereas Ctr9 bridges between them. Consistent with the structural observations, the initiation factor TFIIF impairs Paf1C binding to Pol II, whereas the elongation factor TFIIS enhances it. We further show that Paf1C is globally required for normal mRNA transcription in yeast. These results provide a three-dimensional framework for further analysis of Paf1C function in transcription through chromatin

Real-time cryo-electron microscopy data preprocessing with Warp.

The acquisition of cryo-electron microscopy (cryo-EM) data from biological specimens must be tightly coupled to data preprocessing to ensure the best data quality and microscope usage. Here we describe Warp, a software that automates all preprocessing steps of cryo-EM data acquisition and enables real-time evaluation. Warp corrects micrographs for global and local motion, estimates the local defocus and monitors key parameters for each recorded micrograph or tomographic tilt series in real time. The software further includes deep-learning-based models for accurate particle picking and image denoising. The output from Warp can be fed into established programs for particle classification and 3D-map refinement. Our benchmarks show improvement in the nominal resolution, which went from 3.9 Å to 3.2 Å, of a published cryo-EM data set for influenza virus hemagglutinin. Warp is easy to install from http://github.com/cramerlab/warp and computationally inexpensive, and has an intuitive, streamlined user interface

Using the Volta phase plate with defocus for cryo-EM single particle analysis.

Previously, we reported an in-focus data acquisition method for cryo-EM single-particle analysis with the Volta phase plate (Danev and Baumeister, 2016). Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also removes the resolution limit inherent to the in-focus method thus allowing 3D reconstructions with resolutions better than 3 angstrom

Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.5 Å in cells

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 we 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. We 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. Our work provides a computational tool that facilitates structural biology in cells

Structures of transcription pre-initiation complex with TFIIH and Mediator.

For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC-core Mediator complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the 'E-bridge' helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC-core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II

Structure of SWI/SNF chromatin remodeller RSC bound to a nucleosome

Chromatin-remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted, transcriptionally active promoter regions (NDRs)1,2. In the yeast Saccharomyces cerevisiae, the essential SWI/SNF complex RSC3 contains 16 subunits, including the ATP-dependent DNA translocase Sth14,5. RSC removes nucleosomes from promoter regions6,7 and positions the specialized +1 and −1 nucleosomes that flank NDRs8,9. Here we present the cryo-electron microscopy structure of RSC in complex with a nucleosome substrate. The structure reveals that RSC forms five protein modules and suggests key features of the remodelling mechanism. The body module serves as a scaffold for the four flexible modules that we call DNA-interacting, ATPase, arm and actin-related protein (ARP) modules. The DNA-interacting module binds extra-nucleosomal DNA and is involved in the recognition of promoter DNA elements8,10,11 that influence RSC functionality12. The ATPase and arm modules sandwich the nucleosome disc with the Snf2 ATP-coupling (SnAC) domain and the finger helix, respectively. The translocase motor of the ATPase module engages with the edge of the nucleosome at superhelical location +2. The mobile ARP module may modulate translocase–nucleosome interactions to regulate RSC activity5. The RSC–nucleosome structure provides a basis for understanding NDR formation and the structure and function of human SWI/SNF complexes that are frequently mutated in cancer13