The acquisition of cryo-electron microscopy (cryo-EM) data from biological specimen must be tightly coupled to data pre-processing to ensure best data quality and microscope usage. Here we provide Warp, a software for real-time evaluation and pre-processing of cryo-EM data during their acquisition. Warp corrects micrographs for global and local motion, estimates the local defocus with the use of novel algorithms, 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 from 3.9 Å to 3.2 Å through fully automated processing of a published cryo-EM data set for influenza virus hemagglutinin. Warp is easy to install, computationally inexpensive, and has an intuitive and streamlined user interface. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes 1-3. Here we present a cryo-electron microscopy structure of the SARS-CoV-2 RdRp in an active form that mimics the replicating enzyme. The structure comprises the viral proteins non-structural protein 12 (nsp12), nsp8 and nsp7, and more than two turns of RNA template-product duplex. The active-site cleft of nsp12 binds to 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 RdRp that is required for replicating the long genome of coronaviruses 3. 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. Coronaviruses are positive-strand RNA viruses that pose a major health risk 1 : SARS-CoV-2 has caused a pandemic of the disease known as COVID-19 5,6. Coronaviruses use an RdRp complex for the replication of their genome and for the transcription of their genes 2,3. This RdRp complex is the target of nucleoside analogue inhibitors-in particular, remdesivir 7,8. Remdesivir inhibits the RdRp of multiple coronaviruses 9,10 , and shows antiviral activity in cell culture and animal models 11. Remdesivir is currently being tested in the clinic in many countries 12 and has recently been approved for emergency treatment of patients with COVID-19 in the United States 4. The RdRp of SARS-CoV-2 is composed of a catalytic subunit known as nsp12 13 as well as two accessory subunits, nsp8 and nsp7 3,14. The structure of this RdRp has recently been reported 15 ; it is highly similar to the RdRp of SARS-CoV 16 , a zoonotic coronavirus that spread into the human population in 2002 1. The nsp12 subunit contains an N-terminal nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain, an interface domain and a C-terminal RdRp domain 15,16. The RdRp domain resembles a right hand, comprising the fingers, palm and thumb subdomains 15,16 that are found in all single-subunit polymerases. Subunits nsp7 and nsp8 bind to the thumb, and an additional copy of nsp8 binds to the fingers domain 15,16. Structural information is also available for nsp8-nsp7 complexes 17,18. To obtain the structure of the SARS-CoV-2 RdRp in its active form, we prepared recombinant nsp12, nsp8 and nsp7 (Fig. 1a, Methods). When added to a minimal RNA hairpin substrate (Fig. 1b), the purified proteins gave rise to RNA-dependent RNA extension activity, which depended on nsp8 and nsp7 (Fig. 1c). We assembled and purified a stable RdRp-RNA complex with the use of a self-annealing RNA, and collected single-particle cryo-electron microscopy (cryo-EM) data (Ex...

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

For transcription initiation, RNA polymerase (Pol) II assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). We report cryo-EM structures of the yeast PIC and PIC-core Mediator (cMed) complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal TFIIH and suggest how the TFIIH modules ‘core’ and ‘kinase’ function in promoter opening and Pol II phosphorylation, respectively. The TFIIH core subunit Ssl2 (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 (human MAT1) anchors the kinase Kin28 (human Cdk7) that is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC-cMed complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain (CTD) of Pol II.

The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.