Clofazimine broadly inhibits coronaviruses including SARS-CoV-2.

The COVID-19 pandemic is the third outbreak this century of a zoonotic disease caused by a coronavirus, following the emergence of severe acute respiratory syndrome (SARS) in 20031 and Middle East respiratory syndrome (MERS) in 20122. Treatment options for coronaviruses are limited. Here we show that clofazimine-an anti-leprosy drug with a favourable safety profile3-possesses inhibitory activity against several coronaviruses, and can antagonize the replication of SARS-CoV-2 and MERS-CoV in a range of in vitro systems. We found that this molecule, which has been approved by the US Food and Drug Administration, inhibits cell fusion mediated by the viral spike glycoprotein, as well as activity of the viral helicase. Prophylactic or therapeutic administration of clofazimine in a hamster model of SARS-CoV-2 pathogenesis led to reduced viral loads in the lung and viral shedding in faeces, and also alleviated the inflammation associated with viral infection. Combinations of clofazimine and remdesivir exhibited antiviral synergy in vitro and in vivo, and restricted viral shedding from the upper respiratory tract. Clofazimine, which is orally bioavailable and comparatively cheap to manufacture, is an attractive clinical candidate for the treatment of outpatients and-when combined with remdesivir-in therapy for hospitalized patients with COVID-19, particularly in contexts in which costs are an important factor or specialized medical facilities are limited. Our data provide evidence that clofazimine may have a role in the control of the current pandemic of COVID-19 and-possibly more importantly-in dealing with coronavirus diseases that may emerge in the future.

Elf1 promotes Rad26's interaction with lesion-arrested Pol II for transcription-coupled repair.

Transcription-coupled nucleotide excision repair (TC-NER) is a highly conserved DNA repair pathway that removes bulky lesions in the transcribed genome. Cockayne syndrome B protein (CSB), or its yeast ortholog Rad26, has been known for decades to play important roles in the lesion-recognition steps of TC-NER. Another conserved protein ELOF1, or its yeast ortholog Elf1, was recently identified as a core transcription-coupled repair factor. How Rad26 distinguishes between RNA polymerase II (Pol II) stalled at a DNA lesion or other obstacles and what role Elf1 plays in this process remains unknown. Here, we present cryo-EM structures of Pol II-Rad26 complexes stalled at different obstacles that show that Rad26 uses a common mechanism to recognize a stalled Pol II, with additional interactions when Pol II is arrested at a lesion. A cryo-EM structure of lesion-arrested Pol II-Rad26 bound to Elf1 revealed that Elf1 induces further interactions between Rad26 and a lesion-arrested Pol II. Biochemical and genetic data support the importance of the interplay between Elf1 and Rad26 in TC-NER initiation. Together, our results provide important mechanistic insights into how two conserved transcription-coupled repair factors, Rad26/CSB and Elf1/ELOF1, work together at the initial lesion recognition steps of transcription-coupled repair.

RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder.

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im-induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im-induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides-dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im-induced transcriptional arrest.

Molecular Mechanism of RNA Polymerase II Transcriptional Mutagenesis by the Epimerizable DNA Lesion, Fapy·dG.

Oxidative DNA lesions cause significant detrimental effects on a living species. Two major DNA lesions resulting from dG oxidation, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo) and formamidopyrimidine (Fapy·dG), are produced from a common chemical intermediate. Fapy·dG is formed in comparable yields under oxygen-deficient conditions. Replicative bypass of Fapy·dG in human cells is more mutagenic than that of 8-OxodGuo. Despite the biological importance of transcriptional mutagenesis, there are no reports of the effects of Fapy·dG on RNA polymerase II (Pol II) activity. Here we perform comprehensive kinetic studies to investigate the impact of Fapy·dG on three key transcriptional fidelity checkpoint steps by Pol II: insertion, extension, and proofreading steps. The ratios of error-free versus error-prone incorporation opposite Fapy·dG are significantly reduced in comparison with undamaged dG. Similarly, Fapy·dG:A mispair is extended with comparable efficiency as that of the error-free, Fapy·dG:C base pair. The α- and ß-configurational isomers of Fapy·dG have distinct effects on Pol II insertion and extension. Pol II can preferentially cleave error-prone products by proofreading. To further understand the structural basis of transcription processing of Fapy·dG, five different structures were solved, including Fapy·dG template-loading state (apo), error-free cytidine triphosphate (CTP) binding state (prechemistry), error-prone ATP binding state (prechemistry), error-free Fapy·dG:C product state (postchemistry), and error-prone Fapy·dG:A product state (postchemistry), revealing distinctive nucleotide binding and product states. Taken together, our study provides a comprehensive mechanistic framework for better understanding how Fapy·dG lesions impact transcription and subsequent pathological consequences.

Transcriptional processing of an unnatural base pair by eukaryotic RNA polymerase II.

The development of unnatural base pairs (UBPs) has greatly increased the information storage capacity of DNA, allowing for transcription of unnatural RNA by the heterologously expressed T7 RNA polymerase (RNAP) in Escherichia coli. However, little is known about how UBPs are transcribed by cellular RNA polymerases. Here, we investigated how synthetic unnatural nucleotides, NaM and TPT3, are recognized by eukaryotic RNA polymerase II (Pol II) and found that Pol II is able to selectively recognize UBPs with high fidelity when dTPT3 is in the template strand and rNaMTP acts as the nucleotide substrate. Our structural analysis and molecular dynamics simulation provide structural insights into transcriptional processing of UBPs in a stepwise manner. Intriguingly, we identified a novel 3'-RNA binding site after rNaM addition, termed the swing state. These results may pave the way for future studies in the design of transcription and translation strategies in higher organisms with expanded genetic codes.

RNA polymerase II stalls on oxidative DNA damage via a torsion-latch mechanism involving lone pair-π and CH-π interactions.

Oxidation of guanine generates several types of DNA lesions, such as 8-oxoguanine (8OG), 5-guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp). These guanine-derived oxidative DNA lesions interfere with both replication and transcription. However, the molecular mechanism of transcription processing of Gh and Sp remains unknown. In this study, by combining biochemical and structural analysis, we revealed distinct transcriptional processing of these chemically related oxidized lesions: 8OG allows both error-free and error-prone bypass, whereas Gh or Sp causes strong stalling and only allows slow error-prone incorporation of purines. Our structural studies provide snapshots of how polymerase II (Pol II) is stalled by a nonbulky Gh lesion in a stepwise manner, including the initial lesion encounter, ATP binding, ATP incorporation, jammed translocation, and arrested states. We show that while Gh can form hydrogen bonds with adenosine monophosphate (AMP) during incorporation, this base pair hydrogen bonding is not sufficient to hold an ATP substrate in the addition site and is not stable during Pol II translocation after the chemistry step. Intriguingly, we reveal a unique structural reconfiguration of the Gh lesion in which the hydantoin ring rotates ∼90° and is perpendicular to the upstream base pair planes. The perpendicular hydantoin ring of Gh is stabilized by noncanonical lone pair-π and CH-π interactions, as well as hydrogen bonds. As a result, the Gh lesion, as a functional mimic of a 1,2-intrastrand crosslink, occupies canonical -1 and +1 template positions and compromises the loading of the downstream template base. Furthermore, we suggest Gh and Sp lesions are potential targets of transcription-coupled repair.

Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers.

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.

RNA sequencing by direct tagmentation of RNA/DNA hybrids.

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.

Holotomography: Refractive Index as an Intrinsic Imaging Contrast for 3-D Label-Free Live Cell Imaging.

Live cell imaging provides essential information in the investigation of cell biology and related pathophysiology. Refractive index (RI) can serve as intrinsic optical imaging contrast for 3-D label-free and quantitative live cell imaging, and provide invaluable information to understand various dynamics of cells and tissues for the study of numerous fields. Recently significant advances have been made in imaging methods and analysis approaches utilizing RI, which are now being transferred to biological and medical research fields, providing novel approaches to investigate the pathophysiology of cells. To provide insight into how RI can be used as an imaging contrast for imaging of biological specimens, here we provide the basic principle of RI-based imaging techniques and summarize recent progress on applications, ranging from microbiology, hematology, infectious diseases, hematology, and histopathology.

8-Oxo-guanine DNA damage induces transcription errors by escaping two distinct fidelity control checkpoints of RNA polymerase II.

RNA polymerase II (Pol II) has an intrinsic fidelity control mechanism to maintain faithful genetic information transfer during transcription. 8-Oxo-guanine (8OG), a commonly occurring damaged guanine base, promotes misincorporation of adenine into the RNA strand. Recent structural work has shown that adenine can pair with the syn conformation of 8OG directly upstream of the Pol II active site. However, it remains unknown how 8OG is accommodated in the active site as a template base for the incoming ATP. Here, we used molecular dynamics (MD) simulations to investigate two consecutive steps that may contribute to the adenine misincorporation by Pol II. First, the mismatch is located in the active site, contributing to initial incorporation of adenine. Second, the mismatch is in the adjacent upstream position, contributing to extension from the mismatched bp. These results are supported by an in vitro transcription assay, confirming that 8OG can induce adenine misincorporation. Our simulations further suggest that 8OG forms a stable bp with the mismatched adenine in both the active site and the adjacent upstream position. This stability predominantly originates from hydrogen bonding between the mismatched adenine and 8OG in a noncanonical syn conformation. Interestingly, we also found that an unstable bp present directly upstream of the active site, such as adenine paired with 8OG in the canonical anti conformation, largely disrupts the stability of the active site. Our findings have uncovered two main factors contributing to how 8OG induces transcriptional errors and escapes Pol II transcriptional fidelity control checkpoints.

Structural and biochemical analysis of DNA lesion-induced RNA polymerase II arrest.

Transcription, catalyzed by RNA polymerase II (Pol II) in eukaryotes, is the first step in gene expression. RNA Pol II is a 12-subunit enzyme complex regulated by many different transcription factors during transcription initiation, elongation, and termination. During elongation, Pol II encounters various types of obstacles that can cause transcriptional pausing and arrest. Through decades of research on transcriptional pausing, it is widely known that Pol II can distinguish between different types of obstacles by its active site. A major class of obstacles is DNA lesions. While some DNA lesions can cause transient transcriptional pausing, which can be bypassed by Pol II itself or with the help from other elongation factors, bulky DNA damage can cause prolonged transcriptional pausing and arrest, which signals for transcription coupled repair. Using biochemical and structural biology approaches, the outcomes of many different types of DNA lesions, DNA modifications, and DNA binding molecules to transcription were studied. In this mini review, we will describe the in vitro transcription assays with Pol II to investigate the impacts of various DNA lesions on transcriptional outcomes and the crystallization method of lesion-arrested Pol II complex. These methods can provide a general platform for the structural and biochemical analysis of Pol II transcriptional pausing and bypass mechanisms.

Crystal structure and mutational analyses of ribokinase from Arabidopsis thaliana.

Nitrogen remobilization is a key issue in plants. Recent studies in Arabidopsis thaliana have revealed that nucleoside catabolism supplies xanthine, a nitrogen-rich compound, to the purine ring catabolic pathway, which liberates ammonia from xanthine for reassimilation into amino acids. Similarly, pyrimidine nuclosides are degraded and the pyrimidine bases are fully catabolized. During nucleoside hydrolysis, ribose is released, and ATP-dependent ribokinase (RBSK) phosphorylates ribose to ribose-5'-phosphate to allow its entry into central metabolism recycling the sugar carbons from nucleosides. In this study, we report the crystal structure of RBSK from Arapidopsis thaliana (AtRBSK) in three different ligation states: an unliganded state, a ternary complex with ribose and ATP, and a binary complex with ATP in the presence of Mg2+. In the monomeric conformation, AtRBSK is highly homologous to bacterial RBSKs, including the binding sites for a monovalent cation, ribose, and ATP. Its dimeric conformation, however, does not exhibit the noticeable ligand-induced changes that were observed in bacterial orthologs. Only in the presence of Mg2+, ATP in the binary complex adopts a catalytically competent conformation, providing a mode of action for Mg2+ in AtRBSK activity. The structural data combined with activity analyses of mutants allowed assignment of functional roles for the active site residues. Overall, this study provides the first structural characterization of plant RBSK, and experimentally validates a previous hypothetical model concerning the general reaction mechanism of RBSK.

3.1 Šstructure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids.

Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact with the air-water interface, which often leads to detrimental effects such as denaturation or adoption of preferred orientations, ultimately hindering structure determination. Randomly biotinylating the protein of interest (for example, at its primary amines) and then tethering it to a cryo-EM grid coated with two-dimensional crystals of streptavidin (acting as an affinity surface) can prevent the protein from interacting with the air-water interface. Recently, this approach was successfully used to solve a high-resolution structure of a test sample, a bacterial ribosome. However, whether this method can be used for samples where interaction with the air-water interface has been shown to be problematic remains to be determined. Here we report a 3.1 Šstructure of an RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion (Pol II EC(CPD)) solved using streptavidin grids. Our previous attempt to solve this structure using conventional sample preparation methods resulted in a poor quality cryo-EM map due to Pol II EC(CPD)'s adopting a strong preferred orientation. Imaging the same sample on streptavidin grids improved the angular distribution of its view, resulting in a high-resolution structure. This work shows that streptavidin affinity grids can be used to address known challenges posed by the interaction with the air-water interface.

Crystal structure of inositol 1,3,4,5,6-pentakisphosphate 2-kinase from Cryptococcus neoformans.

The fungal pathogen Cryptococcus neoformans is a causative agent of meningoencephalitis in humans. For its pathogenicity, the inositol polyphosphate biosynthetic pathway plays critical roles. Recently, Ipk1 from C. neoformans (CnIpk1) was identified as an inositol 1,3,4,5,6-pentakisphosphate 2-kinase that catalyzes the phosphorylation of IP5 to form IP6, a substrate for subsequent reaction to produce inositol pyrophosphates, such as PP-IP5/IP7. Furthermore, it was shown that deletion of IPK1 significantly reduces the virulence of C. neoformans, indicating that Ipk1 is a major virulence contributor. In this study, we determined a crystal structure of the apo-form of CnIpk1 at 2.35Å resolution, the first structure for a fungal Ipk1, using a single-wavelength anomalous dispersion method. Even with a low sequence similarity of 26-28%, its overall structure resembles two other Ipk1 orthologs from Arabidopsis thaliana (AtIpk1) and Mus musculus (MmIpk1), and the most crucial residues in the active site are conserved. Unlike AtIpk1 and MmIpk1, however, metal-binding sites for structural stabilization and conformational variations are absent in CnIpk1. The binding environments for substrate IP5 could be inferred by the two different binding sites for sulfate ion in CnIpk1. Taken together, these observations suggest structural similarities and discrepancies for fungal Ipk1 among members of the Ipk1 family and provide structural information for the possible development of drug design for treatment of cryptococcosis.

Structural Insights into an Oxalate-producing Serine Hydrolase with an Unusual Oxyanion Hole and Additional Lyase Activity.

In Burkholderia species, the production of oxalate, an acidic molecule, is a key event for bacterial growth in the stationary phase. Oxalate plays a central role in maintaining environmental pH, which counteracts inevitable population-collapsing alkaline toxicity in amino acid-based culture medium. In the phytopathogen Burkholderia glumae, two enzymes are responsible for oxalate production. First, the enzyme oxalate biosynthetic component A (ObcA) catalyzes the formation of a tetrahedral C6-CoA adduct from the substrates acetyl-CoA and oxaloacetate. Then the ObcB enzyme liberates three products from the C6-CoA adduct: oxalate, acetoacetate, and CoA. Interestingly, these two stepwise reactions are catalyzed by a single bifunctional enzyme, Obc1, from Burkholderia thailandensis and Burkholderia pseudomallei Obc1 has an ObcA-like N-terminal domain and shows ObcB activity in its C-terminal domain despite no sequence homology with ObcB. We report the crystal structure of Obc1 in its apo and glycerol-bound form at 2.5 Å and 2.8 Å resolution, respectively. The Obc1 N-terminal domain is essentially identical both in structure and function to that of ObcA. Its C-terminal domain has an α/ß hydrolase fold that has a catalytic triad for oxalate production and a novel oxyanion hole distinct from the canonical HGGG motif in other α/ß hydrolases. Functional analyses through mutagenesis studies suggested that His-934 is an additional catalytic acid/base for its lyase activity and liberates two additional products, acetoacetate and CoA. These results provide structural and functional insights into bacterial oxalogenesis and an example of divergent evolution of the α/ß hydrolase fold, which has both hydrolase and lyase activity.

Structural basis for bacterial quorum sensing-mediated oxalogenesis.

The Burkholderia species utilize acetyl-CoA and oxaloacetate, substrates for citrate synthase in the TCA cycle, to produce oxalic acid in response to bacterial cell to cell communication, called quorum sensing. Quorum sensing-mediated oxalogenesis via a sequential reaction by ObcA and ObcB counteracts the population-collapsing alkaline pH of the stationary growth phase. Thus, the oxalic acid produced plays an essential role as an excreted public good for survival of the group. Here, we report structural and functional analyses of ObcA, revealing mechanistic features distinct from those of citrate synthase. ObcA exhibits a unique fold, in which a (ß/α)8-barrel fold is located in the C-domain with the N-domain inserted into a loop following α1 in the barrel fold. Structural analyses of the complexes with oxaloacetate and with a bisubstrate adduct indicate that each of the oxaloacetate and acetyl-CoA substrates is bound to an independent site near the metal coordination shell in the barrel fold. In catalysis, oxaloacetate serves as a nucleophile by forming an enolate intermediate mediated by Tyr(322) as a general base, which then attacks the thioester carbonyl carbon of acetyl-CoA to yield a tetrahedral adduct between the two substrates. Therefore, ObcA catalyzes its reaction by combining the enolase and acetyltransferase superfamilies, but the presence of the metal coordination shell and the absence of general acid(s) produces an unusual tetrahedral CoA adduct as a stable product. These results provide the structural basis for understanding the first step in oxalogenesis and constitute an example of the functional diversity of an enzyme for survival and adaptation in the environment.

Overcoming resolution attenuation during tilted cryo-EM data collection.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

Structural basis of transcription recognition of a hydrophobic unnatural base pair by T7 RNA polymerase.

Bacteriophage T7 RNA polymerase (T7 RNAP) is widely used for synthesizing RNA molecules with synthetic modifications and unnatural base pairs (UBPs) for a variety of biotechnical and therapeutic applications. However, the molecular basis of transcription recognition of UBPs by T7 RNAP remains poorly understood. Here we focused on a representative UBP, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole 2-carbaldehyde (Pa), and investigated how the hydrophobic Ds-Pa pair is recognized by T7 RNAP. Our kinetic assays revealed that T7 RNAP selectively recognizes the Ds or Pa base in the templates and preferentially incorporates their cognate unnatural base nucleotide substrate (PaTP or DsTP) over natural NTPs. Our structural studies reveal that T7 RNAP recognizes the unnatural substrates at the pre-insertion state in a distinct manner compared to natural substrates. These results provide mechanistic insights into transcription recognition of UBP by T7 RNAP and provide valuable information for designing the next generation of UBPs.

A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase.

Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show that E. coli RNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition.

Overcoming Resolution Attenuation During Tilted Cryo-EM Data Collection.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle for dataset acquisition. These data reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.