The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryomicroscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years 1,2 . However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 Å resolution cryo-EM reconstruction for a prototypical human membrane protein, the β3 GABAA receptor homopentamer 3 . Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apo-ferritin, our strategy led to a 1.2 Å resolution .

The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryomicroscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years 1,2 . However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 Å resolution cryo-EM reconstruction for a prototypical human membrane protein, the β3 GABAA receptor homopentamer 3 . Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apo-ferritin, our strategy led to a 1.2 Å resolution reconstruction that, for the first time, offers a genuine atomic resolution view of a protein molecule using single particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualised in difference maps, allowing a direct analysis of hydrogen bonding networks. Combination of the technological advances described here with further approaches to accelerate data acquisition and improve sample quality provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery. Adrian Koh, Toby Darling and Jake Grimmett for support with computing; Garbi Lezcano Singla, Erik Franken for support with the EER format; and Gerald van Hoften and Gerard Hosmar for support with the Falcon-4 camera.

Electron transfer rates to P700' have been determined in wildtype and three interposon mutants (psaE-, ndhf-, and psaE-ndhf-) of Synechococcus sp. PCC 7002. All three mutants grew significantly more slowly than wild type at low light intensities, and each failed to grow photoheterotrophically in the presence of 3-(3,4-dichloropheny1)-1,l-dimethylurea (DCMU) and a metabolizable carbon source. l h e kinetics of P700+ reduction were similar in the wild-type and mutant whole cells in the absence of DCMU. In the presence of DCMU, the P700+ reduction rate in the psaE mutant was significantly slower than in the wild type. In the presence of DCMU and potassium cyanide, added to inhibit the outflow of electrons through cytochrome oxidase, P700+ reduction rates increased for both the psaE-and ndhF-strains. The reduction rates for these two mutants were nonetheless slower than that observed for the wild-type strain. l h e further addition of methyl viologen caused the rate of P700+ reduction in the wild type to become as slow as that for the psaE mutant in the absence of methyl viologen. Civen the ability of methyl viologen to intercept electrons from the acceptor side of photosystem I, this response reveals a lesion in cyclic electron flow in the psaE mutant. In the presence of DCMU, the rate of P700' reduction in the psaE ndhf double mutant was very slow and nearly identical with that for the wild-type strain in the presence of 2,4-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a condition under which physiological electron donation to P700+ should be completely inhibited. These results suggest that NdhF-and PsaE-dependent electron donation to P700+ occurs only via plastoquinone and/or cytochrome b6/f and indicate that there are three major electron sources for P700+ reduction in this cyanobacterium. We conclude that, although PsaE is not required for linear electron flow to NADP+, it is an essential component in the cyclic electron transport pathway around photosystem 1.

Isoniazid is a mainstay of antibiotic therapy for the treatment of tuberculosis, but its molecular mechanism of action is unclear. Previous investigators have hypothesized that isoniazid is a prodrug that requires in vivo activation by KatG, the catalase-peroxidase of Mycobacterium tuberculosis, and that resistance to isoniazid strongly correlates with deletions or point mutations in KatG. One such mutation, KatG(S315T), is found in approximately 50% of clinical isolates exhibiting isoniazid resistance. In this work, 1H nuclear magnetic resonance T1 relaxation measurements indicate that KatG and KatG(S315T) each bind isoniazid at a position approximately 12 A from the active site heme iron. Electron paramagnetic resonance spectroscopy revealed heterogeneous populations of high-spin ferric heme in both wild-type KatG and KatG(S315T) with the ratios of each species differing between the two enzymes. Small changes in the proportions of these high-spin species upon addition of isoniazid support the finding that isoniazid binds near the heme periphery of both enzymes. Titration of wild-type KatG with isoniazid resulted in the appearance of a "type I" substrate-induced difference spectrum analogous to those seen upon substrate binding to the cytochromes P450. The difference spectrum may result from an isoniazid-induced change in a portion of the KatG heme iron from 6- to 5-coordinate. Titration of KatG(S315T) with isoniazid failed to produce a measurable difference spectrum indicating an altered active site configuration. These results suggest that KatG(S315T) confers resistance to isoniazid through subtle changes in the isoniazid binding site.

Calcineurin belongs to a family of serine/threonine protein phosphatases that contain active site dinuclear metal cofactors. Bacteriophage protein phosphatase is also considered to be a member of this family based on sequence comparisons (Lohse, D. L., Denu, J. M., and Dixon, J. E. (1995) Structure 3, 987-990). Using EPR spectroscopy, we demonstrate that protein phosphatase accommodates a dinuclear metal center. Calcineurin and protein phosphatase likewise contain a conserved histidine that is not a metal ligand but is within 5 Å of either metal in calcineurin. In this study the conserved histidine in calcineurin was mutated to glutamine and the mutant protein analyzed by EPR spectroscopy and kinetic methods. Parallel studies with an analogous protein phosphatase mutant were also carried out. Kinetic studies using paranitrophenyl phosphate as substrate showed a decrease in k cat of 460-and 590-fold for the calcineurin and protein phosphatase mutants, respectively, compared with the wild type enzymes. With a phosphopeptide substrate, mutagenesis of the conserved histidine resulted in a decrease in k cat of 1,300-fold for calcineurin. With the analogous protein phosphatase mutant, k cat decreased 530-fold compared with wild type protein phosphatase using phenyl phosphate as a substrate. EPR studies of the iron-reconstituted enzymes indicated that although both mutant enzymes can accommodate a dinuclear metal center, spectroscopic differences compared with wild type proteins suggest a perturbation of the ligand environment, possibly by disruption of a hydrogen bond between the histidine and a metal-coordinated solvent molecule.Calcineurin, also known as protein phosphatase 2B, consists of a 58-kDa catalytic subunit, calcineurin A, and a 19-kDa regulatory subunit, calcineurin B. It is a serine/threonine protein phosphatase whose activity is regulated by Ca 2ϩ /calmodulin. Calcineurin is the target of the immunosuppressant drugs cyclosporin A and FK506 (1, 2). These drugs bind to intracellular proteins, termed immunophilins; cyclophilin is the binding protein for cyclosporin A, and FK506 binds to the FK506-binding proteins. The complex of immunosuppressant drug and immunophilin in turn binds to and inhibits the phosphatase activity of calcineurin. Calcineurin inhibition prevents the transcriptional activation of the interleukin 2 gene in helper T cells, leading to suppression of the immune response.Calcineurin is a member of the class of serine/threonine protein phosphatases, whose members include protein phosphatases 1 (PP1) 1 and 2A (PP2A), phosphatases essential for a number of signal transduction pathways in eukaryotic cells (3, 4). Another protein phosphatase from bacteriophage , PP, also belongs to this family (5). In addition, a number of less characterized enzymes containing the "phosphoesterase" consensus motif of this family, DXH(X) n GDXXD(X) n GNHD/E, have been identified via protein sequence comparisons (6, 7). It has been hypothesized that this motif provides a scaffold for an active site dinuclear meta...