Solution structure and biochemical characterization of a spare part protein that restores activity to an oxygen-damaged glycyl radical enzyme.

Glycyl radical enzymes (GREs) utilize a glycyl radical cofactor to carry out a diverse array of chemically challenging enzymatic reactions in anaerobic bacteria. Although the glycyl radical is a powerful catalyst, it is also oxygen sensitive such that oxygen exposure causes cleavage of the GRE at the site of the radical. This oxygen sensitivity presents a challenge to facultative anaerobes dwelling in areas prone to oxygen exposure. Once GREs are irreversibly oxygen damaged, cells either need to make new GREs or somehow repair the damaged one. One particular GRE, pyruvate formate lyase (PFL), can be repaired through the binding of a 14.3 kDa protein, termed YfiD, which is constitutively expressed in E. coli. Herein, we have solved a solution structure of this 'spare part' protein using nuclear magnetic resonance spectroscopy. These data, coupled with data from circular dichroism, indicate that YfiD has an inherently flexible N-terminal region (residues 1-60) that is followed by a C-terminal region (residues 72-127) that has high similarity to the glycyl radical domain of PFL. Reconstitution of PFL activity requires that YfiD binds within the core of the PFL barrel fold; however, modeling suggests that oxygen-damaged, i.e. cleaved, PFL cannot fully accommodate YfiD. We further report that a PFL variant that mimics the oxygen-damaged enzyme is highly susceptible to proteolysis, yielding additionally truncated forms of PFL. One such PFL variant of ~ 77 kDa makes an ideal scaffold for the accommodation of YfiD. A molecular model for the rescue of PFL activity by YfiD is presented.

Biophysical Examination of the Calcium-Modulated Nickel-Binding Properties of Human Calprotectin Reveals Conformational Change in the EF-Hand Domains and His3Asp Site.

Calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is a host-defense protein that sequesters nutrient transition metals from microbes. Each S100A8/S100A9 heterodimer contains four EF-hand domains and two transition-metal-binding sites. We investigate the effect of Ca(II) ions on the structure and Ni(II)-binding properties of human CP. By employing energy dispersive X-ray (EDX) spectroscopy, we evaluate the metal content of Ni(II)-bound CP-Ser [oligomer of S100A8(C42S) and S100A9(C3S)] crystals obtained in the absence and presence of Ca(II). We present a 2.1 Å resolution crystal structure of Ni(II)-bound CP-Ser and compare this structure to a reported Ni(II)- and Ca(II)-bound CP-Ser structure [Nakashige, T. G., et al. (2017) J. Am. Chem. Soc. 139, 8828-8836]. This analysis reveals conformational changes associated with coordination of Ca(II) to the EF-hands of S100A9 and that Ca(II) binding affects the coordination number and geometry of the Ni(II) ion bound to the His3Asp site. In contrast, negligible differences are observed for the Ni(II)-His6 site in the absence and presence of Ca(II). Biochemical studies show that, whereas the His6 site has a thermodynamic preference for Ni(II) over Zn(II), the His3Asp site selects for Zn(II) over Ni(II), and relatively rapid metal exchange occurs at this site. These observations inform the working model for how CP withholds nutrient metals in the extracellular space.

Metalloprotein Crystallography: More than a Structure.

Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25-50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the availability of advanced detectors, and the incorporation of spectroscopic equipment at a number of synchrotron beamlines, have yielded exciting developments in metalloprotein structure determination. Here we will present results on the advantageous uses of metals in metalloprotein crystallography, including using metallocofactors to obtain phasing information, using K-edge X-ray absorption spectroscopy to identify metals coordinated in metalloprotein crystals, and using UV-vis spectroscopy on crystals to probe the enzymatic activity of the crystallized protein.

Effects of protein structure on iron-polypeptide vibrational dynamic coupling in cytochrome c.

Cytochrome c (Cyt c) has a heme covalently bound to the polypeptide via a Cys-X-X-Cys-His (CXXCH) linker that is located in the interface region for protein-protein interactions. To determine whether the polypeptide matrix influences iron vibrational dynamics, nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on (57)Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c-552, and variants M13V, M13V/K22M, and A7F, which have structural modifications that alter the composition or environment of the CXXCH pentapeptide loop. Simulations of the NRVS data indicate that the 150-325 cm(-1) region is dominated by NHis-Fe-SMet axial ligand and polypeptide motions, while the 325-400 cm(-1) region shows dominant contributions from ν(Fe-NPyr) (Pyr = pyrrole) and other heme-based modes. Diagnostic spectral signatures that directly relate to structural features of the heme active site are identified using a quantum chemistry-centered normal coordinate analysis (QCC-NCA). In particular, spectral features that directly correlate with CXXCH loop stiffness, the strength of the Fe-His interaction, and the degree of heme distortion are identified. Cumulative results from our investigation suggest that compared to the wild type (wt), variants M13V and M13V/K22M have a more rigid CXXCH pentapeptide segment, a stronger Fe-NHis interaction, and a more ruffled heme. Conversely, the A7F variant has a more planar heme and a weaker Fe-NHis bond. These results are correlated to the observed changes in reduction potential between wt protein and the variants studied here. Implications of these results for Cyt c biogenesis and electron transfer are also discussed.

Manganese binding properties of human calprotectin under conditions of high and low calcium: X-ray crystallographic and advanced electron paramagnetic resonance spectroscopic analysis.

The antimicrobial protein calprotectin (CP), a hetero-oligomer of the S100 family members S100A8 and S100A9, is the only identified mammalian Mn(II)-sequestering protein. Human CP uses Ca(II) ions to tune its Mn(II) affinity at a biologically unprecedented hexahistidine site that forms at the S100A8/S100A9 interface, and the molecular basis for this phenomenon requires elucidation. Herein, we investigate the remarkable Mn(II) coordination chemistry of human CP using X-ray crystallography as well as continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies. An X-ray crystallographic structure of Mn(II)-CP containing one Mn(II), two Ca(II), and two Na(I) ions per CP heterodimer is reported. The CW EPR spectrum of Ca(II)- and Mn(II)-bound CP prepared with a 10:0.9:1 Ca(II):Mn(II):CP ratio is characterized by an unusually low zero-field splitting of 485 MHz (E/D = 0.30) for the S = 5/2 Mn(II) ion, consistent with the high symmetry of the His6 binding site observed crystallographically. Results from electron spin-echo envelope modulation and electron-nuclear double resonance experiments reveal that the six Mn(II)-coordinating histidine residues of Ca(II)- and Mn(II)-bound CP are spectroscopically equivalent. The observed (15)N (I = 1/2) hyperfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (A = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the binding affinity of CP for Mn(II) drops by two to three orders of magnitude and coincides with Mn(II) binding at the His6 site as well as other sites. This study demonstrates the role of Ca(II) in enabling high-affinity and specific binding of Mn(II) to the His6 site of human calprotectin.

Heme-protein vibrational couplings in cytochrome c provide a dynamic link that connects the heme-iron and the protein surface.

The active site of cytochrome c (Cyt c) consists of a heme covalently linked to a pentapeptide segment (Cys-X-X-Cys-His), which provides a link between the heme and the protein surface, where the redox partners of Cyt c bind. To elucidate the vibrational properties of heme c, nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on (57)Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c(552), including (13)C(8)-heme-, (13)C(5)(15)N-Met-, and (13)C(15)N-polypeptide (pp)-labeled samples, revealing heme-based vibrational modes in the 200- to 450-cm(-1) spectral region. Simulations of the NRVS spectra of H. thermophilus cytochrome c(552) allowed for a complete assignment of the Fe vibrational spectrum of the protein-bound heme, as well as the quantitative determination of the amount of mixing between local heme vibrations and pp modes from the Cys-X-X-Cys-His motif. These results provide the basis to propose that heme-pp vibrational dynamic couplings play a role in electron transfer (ET) by coupling vibrations of the heme directly to vibrations of the pp at the protein-protein interface. This could allow for the direct transduction of the thermal (vibrational) energy from the protein surface to the heme that is released on protein/protein complex formation, or it could modulate the heme vibrations in the protein/protein complex to minimize reorganization energy. Both mechanisms lower energy barriers for ET. Notably, the conformation of the distal Met side chain is fine-tuned in the protein to localize heme-pp mixed vibrations within the 250- to 400-cm(-1) spectral region. These findings point to a particular orientation of the distal Met that maximizes ET.

Two cases of intranasal naloxone self-administration in opioid overdose.

BACKGROUND: Overdose is a leading cause of death for former prisoners, exacting its greatest toll during the first 2 weeks post release. Protective effects have been observed with training individuals at high risk of overdose and prescribing them naloxone, an opioid antagonist that reverses the effects of the opioid-induced respiratory depression that causes death. CASES: The authors report 2 people with opiate use histories who self-administered intranasal naloxone to treat their own heroin overdoses following release from prison. Patient A is a 34-year-old male, who reported having experienced an overdose on heroin the day after he was released from incarceration. Patient B is a 29-year-old female, who reported an overdose on her first injection of heroin, 17 days post release from incarceration. Both patients self-administered the medication but were assisted at some point during the injury by a witness whom they had personally instructed in how to prepare and administer the medication. Neither patient experienced withdrawal symptoms following exposure to naloxone. DISCUSSION: Self-administration of naloxone should not be a goal of overdose death prevention training. A safer, more reliable approach is to prescribe naloxone to at-risk patients and train and also equip members of their household and social or drug-using networks in overdose prevention and response.

Applying 3D ED/MicroED workflows toward the next frontiers.

We report on the latest advancements in Microcrystal Electron Diffraction (3D ED/MicroED), as discussed during a symposium at the National Center for CryoEM Access and Training housed at the New York Structural Biology Center. This snapshot describes cutting-edge developments in various facets of the field and identifies potential avenues for continued progress. Key sections discuss instrumentation access, research applications for small molecules and biomacromolecules, data collection hardware and software, data reduction software, and finally reporting and validation. 3D ED/MicroED is still early in its wide adoption by the structural science community with ample opportunities for expansion, growth, and innovation.

The influence of heme ruffling on spin densities in ferricytochromes c probed by heme core 13C NMR.

The heme in cytochromes c undergoes a conserved out-of-plane distortion known as ruffling. For cytochromes c from the bacteria Hydrogenobacter thermophilus and Pseudomonas aeruginosa , NMR and EPR spectra have been shown to be sensitive to the extent of heme ruffling and to provide insights into the effect of ruffling on the electronic structure. Through the use of mutants of each of these cytochromes that differ in the amount of heme ruffling, NMR characterization of the low-spin (S = ½) ferric proteins has confirmed and refined the developing understanding of how ruffling influences the spin distribution on heme. The chemical shifts of the core heme carbons were obtained through site-specific labeling of the heme via biosynthetic incorporation of (13)C-labeled 5-aminolevulinic acid derivatives. Analysis of the contact shifts of these core heme carbons allowed Fermi contact spin densities to be estimated and changes upon ruffling to be evaluated. The results allow a deconvolution of the contributions to heme hyperfine shifts and a test of the influence of heme ruffling on the electronic structure and hyperfine shifts. The data indicate that as heme ruffling increases, the spin densities on the ß-pyrrole carbons decrease while the spin densities on the α-pyrrole carbons and meso carbons increase. Furthermore, increased ruffling is associated with stronger bonding to the heme axial His ligand.

Collaboration or coercion? Partnering to divert prescription opioid medications.

Diversion of prescription opioids is a widespread problem in the USA. While "doctor shopping" and pill brokering are well-described types of medication diversion, we sought to understand the social dynamic of diversion of prescription opioids and identify other diversion methods. Using qualitative data collected as part of a 12-week Rapid Assessment and Response study of prescription opioid overdose and abuse (the RARx Study) conducted in three communities in two New England states, we reviewed and thematically coded 195 interviews. Diversion took many forms: doctor shopping, pill brokering, and, most commonly, siphoning from the family medicine chest. Partnering-of patients with other "patients," of patients with "caregivers"- to obtain prescription opioids was also described. Motivations for partnering indicated doing so out of fear of violence, for financial benefit, or in exchange for transportation or other services. Partnering for prescription opioids exhibited a range of power differentials, from collaboration to coercion, and tended to involve vulnerable populations such as the elderly, disabled, or destitute. Increased awareness among health providers of the ease of access and diversion of prescription opioids is needed to promote patient safety and prevent interpersonal violence.

Barriers to medical provider support for prescription naloxone as overdose antidote for lay responders.

Poisonings are the leading cause of adult injury death in the United States. Over 12 weeks in 2011, 143 key informant interviews were conducted using a structured interview guide in three study sites in New England. This analysis focuses on the 24 interviews with emergency department providers, substance use treatment providers, pain specialists, and generalist/family medicine practitioners. Using an iterative coding process, we analyzed statements regarding support and concern about naloxone prescription for pain patients and drug users. The study's implications and limitations are discussed and future research suggested. The Centers for Disease Control and Prevention funded this study.

How does use of a prescription monitoring program change pharmacy practice?

OBJECTIVES: To assess differences in prescription monitoring program (PMP) use between two states with different PMP accessibility (Connecticut [CT] and Rhode Island [RI]), to explore use of PMPs in pharmacy practice, and to examine associations between PMP use and pharmacists' responses to suspected diversion or "doctor shopping." DESIGN: Descriptive nonexperimental study. SETTING: CT and RI from March through August 2011. PARTICIPANTS: Licensed pharmacists in CT and RI. INTERVENTION: Anonymous surveys e-mailed to pharmacists MAIN OUTCOME MEASURES: PMP use, use of patient reports in pharmacy practice, and responses to suspected doctor shopping or diversion. RESULTS: Responses from 294 pharmacists were received (CT: 198; RI: 96). PMP users were more likely to use the PMP to detect drug abuse (CT: 79%; RI: 21.9%; P < 0.01) and doctor shopping (67%; 7%; P < 0.01). When faced with suspicious medication use behavior, PMP users were less likely than nonusers to discuss their concerns with the patient (adjusted odds ratio 0.48 [95% CI 0.25-0.92]) but as likely to contact the provider (0.86 [0.21-3.47]), refer the patient back to the prescriber (1.50 [0.79-2.86]), and refuse to fill the prescription (0.63 [0.30-1.30]). PMP users were less likely to state they were out of stock of the drug (0.27 [0.12-0.60]) compared with nonusers. Pharmacists reported high interest in attending continuing education on safe dispensing (72.8%). CONCLUSION: Pharmacists are important participants in the effort to address prescription drug misuse and abuse. Current PMP use with prevailing systems had limited influence on pharmacy practice. Findings point to future research and needed practice and education innovations to improve patient safety and safer opioid dispensing for pharmacists.

20 years of crystal hits: progress and promise in ultrahigh-throughput crystallization screening.

Diffraction-based structural methods contribute a large fraction of the biomolecular structural models available, providing a critical understanding of macromolecular architecture. These methods require crystallization of the target molecule, which remains a primary bottleneck in crystal-based structure determination. The National High-Throughput Crystallization Center at Hauptman-Woodward Medical Research Institute has focused on overcoming obstacles to crystallization through a combination of robotics-enabled high-throughput screening and advanced imaging to increase the success of finding crystallization conditions. This paper will describe the lessons learned from over 20 years of operation of our high-throughput crystallization services. The current experimental pipelines, instrumentation, imaging capabilities and software for image viewing and crystal scoring are detailed. New developments in the field and opportunities for further improvements in biomolecular crystallization are reflected on.

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography.

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

Life after the ban: an assessment of US syringe exchange programs' attitudes about and early experiences with federal funding.

OBJECTIVES: We aimed to determine whether syringe exchange programs (SEPs) currently receive or anticipate pursuing federal funding and barriers to funding applications following the recent removal of the long-standing ban on using federal funds for SEPs. METHODS: We conducted a telephone-administered cross-sectional survey of US SEPs. Descriptive statistics summarized responses; bivariate analyses examined differences in pursuing funding and experiencing barriers by program characteristics. RESULTS: Of the 187 SEPs (92.1%) that responded, 90.9% were legally authorized. Three received federal funds and 116 intended to pursue federal funding. Perceived federal funding barriers were common and included availability and accessibility of funds, legal requirements such as written police support, resource capacity to apply and comply with funding regulations, local political and structural organization, and concern around altering program culture. Programs without legal authorization, health department affiliation, large distribution, or comprehensive planning reported more federal funding barriers. CONCLUSIONS: Policy implementation gaps appear to render federal support primarily symbolic. In practice, funding opportunities may not be available to all SEPs. Increased technical assistance and legal reform could improve access to federal funds, especially for SEPs with smaller capacity and tenuous local support.

How does use of a prescription monitoring program change medical practice?

OBJECTIVES: The objectives of this study were to test for differences in prescription monitoring program (PMP) use between two states, Connecticut (CT) and Rhode Island (RI), with a different PMP accessibility; to explore use of PMP reports in clinical practice; and to examine associations between PMP use and clinician's responses to suspected diversion or "doctor shopping" (i.e., multiple prescriptions from multiple providers). DESIGN, SETTING, SUBJECTS: From March to August 2011, anonymous surveys were emailed to providers licensed to prescribe Schedule II medications in CT (N = 16,924) and RI (N = 5,567). OUTCOME MEASURES: PMP use, use of patient reports in clinical practice, responses to suspected doctor shopping, or diversion. RESULTS: Responses from 1,385 prescribers were received: 998 in CT and 375 in RI. PMP use was greater in CT, where an electronic PMP is available (43.9% vs 16.3%, χ(2) = 85.2, P < 0.0001). PMP patient reports were used to screen for drug abuse (36.2% CT vs 10.0% RI, χ(2) = 60.9, P < 0.0001) and detect doctor shopping (43.9% CT vs 18.5% RI, χ(2) = 68.3, P < 0.0001). Adjusting for potential confounders, responses by PMP users to suspicious medication use behavior were more likely to entail clinical response (i.e., refer to another provider odds ratio, OR, 1.75 [95% confidence interval, CI, 1.10, 2.80]; screen for drug abuse OR 1.93 [1.39, 2.68]; revisit pain/treatment agreement OR 1.97 [1.45, 2.67]; conduct urine screen OR 1.82 [1.29, 2.57]; refer to substance abuse treatment OR 1.30 [0.96, 1.75]) rather than legal intervention (OR 0.45 [0.21, 0.94]) or inaction (OR 0.09 [0.01, 0.70]). CONCLUSIONS: Prescribers' use of an electronic PMP may influence medical practice, especially opioid abuse detection, and is associated with clinical responses to suspected doctor shopping or diversion.

Computational identification of a systemic antibiotic for gram-negative bacteria.

Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the outer membrane, is an attractive target due to its surface location. Darobactins produced by Photorhabdus, a nematode gut microbiome symbiont, target BamA. We reasoned that a computational search for genes only distantly related to the darobactin operon may lead to novel compounds. Following this clue, we identified dynobactin A, a novel peptide antibiotic from Photorhabdus australis containing two unlinked rings. Dynobactin is structurally unrelated to darobactins, but also targets BamA. Based on a BamA-dynobactin co-crystal structure and a BAM-complex-dynobactin cryo-EM structure, we show that dynobactin binds to the BamA lateral gate, uniquely protruding into its ß-barrel lumen. Dynobactin showed efficacy in a mouse systemic Escherichia coli infection. This study demonstrates the utility of computational approaches to antibiotic discovery and suggests that dynobactin is a promising lead for drug development.

Temperature dependent equilibrium native to unfolded protein dynamics and properties observed with IR absorption and 2D IR vibrational echo experiments.

Dynamic and structural properties of carbonmonoxy (CO)-coordinated cytochrome c(552) from Hydrogenobacter thermophilus (Ht-M61A) at different temperatures under thermal equilibrium conditions were studied with infrared absorption spectroscopy and ultrafast two-dimensional infrared (2D IR) vibrational echo experiments using the heme-bound CO as the vibrational probe. Depending on the temperature, the stretching mode of CO shows two distinct bands corresponding to the native and unfolded proteins. As the temperature is increased from low temperature, a new absorption band for the unfolded protein grows in and the native band decreases in amplitude. Both the temperature-dependent circular dichroism and the IR absorption area ratio R(A)(T), defined as the ratio of the area under the unfolded band to the sum of the areas of the native and unfolded bands, suggest a two-state transition from the native to the unfolded protein. However, it is found that the absorption spectrum of the unfolded protein increases its inhomogeneous line width and the center frequency shifts as the temperature is increased. The changes in line width and center frequency demonstrate that the unfolding does not follow simple two-state behavior. The temperature-dependent 2D IR vibrational echo experiments show that the fast dynamics of the native protein are virtually temperature independent. In contrast, the fast dynamics of the unfolded protein are slower than those of the native protein, and the unfolded protein fast dynamics and at least a portion of the slower dynamics of the unfolded protein change significantly, becoming faster as the temperature is raised. The temperature dependence of the absorption spectrum and the changes in dynamics measured with the 2D IR experiments confirm that the unfolded ensemble of conformers continuously changes its nature as unfolding proceeds, in contrast to the native state, which displays a temperature-independent distribution of structures.

Structural biology in the time of COVID-19: perspectives on methods and milestones.

The global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has wreaked unprecedented havoc on global society, in terms of a huge loss of life and burden of morbidity, economic upheaval and social disruption. Yet the sheer magnitude and uniqueness of this event has also spawned a massive mobilization of effort in the scientific community to investigate the virus, to develop therapeutics and vaccines, and to understand the public health impacts. Structural biology has been at the center of these efforts, and so it is advantageous to take an opportunity to reflect on the status of structural science vis-à-vis its role in the fight against COVID-19, to register the unprecedented response and to contemplate the role of structural biology in addressing future outbreak threats. As the one-year anniversary of the World Health Organization declaration that COVID-19 is a pandemic has just passed, over 1000 structures of SARS-CoV-2 biomolecules have been deposited in the Worldwide Protein Data Bank (PDB). It is rare to obtain a snapshot of such intense effort in the structural biology arena and is of special interest as the 50th anniversary of the PDB is celebrated in 2021. It is additionally timely as it overlaps with a period that has been termed the 'resolution revolution' in cryoelectron microscopy (CryoEM). CryoEM has recently become capable of producing biomolecular structures at similar resolutions to those traditionally associated with macromolecular X-ray crystallo-graphy. Examining SARS-CoV-2 protein structures that have been deposited in the PDB since the virus was first identified allows a unique window into the power of structural biology and a snapshot of the advantages of the different techniques available, as well as insight into the complementarity of the structural methods.

Contributions to cytochrome c inner- and outer-sphere reorganization energy.

Cytochromes c are small water-soluble proteins that catalyze electron transfer in metabolism and energy conversion processes. Hydrogenobacter thermophilus cytochrome c 552 presents a curious case in displaying fluxionality of its heme axial methionine ligand; this behavior is altered by single point mutation of the Q64 residue to N64 or V64, which fixes the ligand in a single configuration. The reorganization energy (λ) of these cytochrome c 552 variants is experimentally determined using a combination of rotating disc electrochemistry, chronoamperometry and cyclic voltammetry. The differences between the λ determined from these complementary techniques helps to deconvolute the contribution of the active site and its immediate environment to the overall λ (λ Total). The experimentally determined λ values in conjunction with DFT calculations indicate that the differences in λ among the protein variants are mainly due to the differences in contributions from the protein environment and not just inner-sphere λ. DFT calculations indicate that the position of residue 64, responsible for the orientation of the axial methionine, determines the geometric relaxation of the redox active molecular orbital (RAMO). The orientation of the RAMO with respect to the heme is key to determining electron transfer coupling (H AB) which results in higher ET rates in the wild-type protein relative to the Q64V mutant despite a 150 mV higher λ Total in the former.