Lindsay A. Baker

Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

Mitochondrial ATP synthase is responsible for the synthesis of ATP, a universal energy currency in cells. Whereas X-ray crystallography has revealed the structure of the soluble region of the complex and the membrane-intrinsic c-subunits, little is known about the structure of the six other proteins (a, b, f, A6L, e, and g) that comprise the membrane-bound region of the complex in animal mitochondria. Here, we present the structure of intact bovine mitochondrial ATP synthase at ∼18 Å resolution by electron cryomicroscopy of single particles in amorphous ice. The map reveals that the a-subunit and c8-ring of the complex interact with a small contact area and that the b-subunit spans the membrane without contacting the c8-ring. The e- and g-subunits extend from the a-subunit density distal to the c8-ring. The map was calculated from images of a preparation of the enzyme solubilized with the detergent dodecyl maltoside, which is visible in electron cryomicroscopy maps. The structure shows that the micelle surrounding the complex is curved. The observed bend in the micelle of the detergent-solubilized complex is consistent with previous electron tomography experiments and suggests that monomers of ATP synthase are sufficient to produce curvature in lipid bilayers.

The Polydispersity of αB-Crystallin Is Rationalized by an Interconverting Polyhedral Architecture

We report structural models for the most abundant oligomers populated by the polydisperse molecular chaperone αB-crystallin. Subunit connectivity is determined by using restraints obtained from nuclear magnetic resonance spectroscopy and mass spectrometry measurements, enabling the construction of various oligomeric models. These candidate structures are filtered according to their correspondence with ion-mobility spectrometry data and cross-validated by using electron microscopy. The ensuing best-fit structures reveal the polyhedral architecture of αB-crystallin oligomers, and provide a rationale for their polydispersity and facile interconversion.

Advancing faculty development in medical education: a systematic review.

Purpose To (1) provide a detailed account of the nature and scope of faculty development (FD) programs in medical education, (2) assess the quality of FD studies, and (3) identify in what areas and through what means future research can purposefully build on existing knowledge. Method The authors searched MEDLINE, CINAHL, and ERIC for articles reporting evaluations of FD initiatives published between 1989 and 2010. They applied standard systematic review procedures for sifting abstracts, scrutinizing full texts, and abstracting data, including program characteristics, evaluation methods, and outcomes. They used a modified Kirkpatrick model to guide their data abstraction. Results The authors included 22 articles reporting on 21 studies in their review. The most common program characteristics included a series/longitudinal format, intended for individuals, and offered to physicians only. Although the most common aim was to improve teaching effectiveness, several programs had multiple aims, including scholarship and leadership. Program evaluation focused on quantitative approaches. A number of studies employed longitudinal designs and included some follow-up component. Surveys were the most popular data collection method, participants the most common data source, and self-reported behavior changes the most commonly reported outcome. Conclusions Although the authors’ findings showed some recent expansion in the scope of the FD literature, they also highlighted areas that require further focus and growth. Future research should employ more rigorous evaluation methods, explore the role of interprofessional teams and communities of practice in the workplace, and address how different organizational and contextual factors shape the success of FD programs.

The resolution dependence of optimal exposures in liquid nitrogen temperature electron cryomicroscopy of catalase crystals

Electron beam damage is the fundamental limit to resolution in electron cryomicroscopy (cryo-EM) of frozen, hydrated specimens. Radiation damage increases with the number of electrons used to obtain an image and affects information at higher spatial frequencies before low-resolution information. For the experimentalist, a balance exists between electron exposures sufficient to obtain a useful signal-to-noise ratio (SNR) in images and exposures that limit the damage to structural features. In single particle cryo-EM this balance is particularly delicate: low-resolution features must be imaged with a sufficient SNR to allow image alignment so that high-resolution features recorded below the noise level can be recovered by averaging independent images. By measuring the fading of Fourier components from images obtained at 200 kV of thin crystals of catalase embedded in ice, we have determined the electron exposures that will maximize the SNR at resolutions between 86 and 2.9A. These data allow for a rational choice of exposure for single particle cryo-EM. For example, for 20A resolution, the SNR is maximized at approximately 20e(-)/A(2), whereas for 3A resolution, it is maximized at approximately 10 e(-)/A(2). We illustrate the effects of exposure in single particle cryo-EM with data collected at approximately 12-15 and approximately 24-30 e(-)/A(2).

RADIATION DAMAGE IN ELECTRON CRYOMICROSCOPY

In an electron microscope, the electron beam used to determine the structures of biological tissues, cells, and molecules destroys the specimen as the image is acquired. This destruction occurs before a statistically well-defined image can be obtained and is consequently the fundamental limit to resolution in biological electron cryomicroscopy (cryo-EM). Damage from the destructive interaction of electrons with frozen-hydrated specimens occurs in three stages: primary damage, as electrons ionize the sample, break bonds, and produce secondary electrons and free radicals; secondary damage, as the secondary electrons and free radicals migrate through the specimen and cause further chemical reactions; and tertiary damage, as hydrogen gas is evolved within the sample, causing gross morphological changes to the specimen. The deleterious effects of radiation are minimized in cryo-EM by limiting the exposure of the specimen to incident electrons and cooling the sample to reduce secondary damage. This review emphasizes practical considerations for minimizing radiation damage, including measurement of electron exposure, estimation of absorbed doses of energy, selection of microscope voltage and specimen temperature, and selection of electron exposure to optimize images.

Cryo-EM structure of the yeast ATP synthase.

We have used electron cryomicroscopy of single particles to determine the structure of the ATP synthase from Saccharomyces cerevisiae. The resulting map at 24 A resolution can accommodate atomic models of the F(1)-c(10) subcomplex, the peripheral stalk subcomplex, and the N-terminal domain of the oligomycin sensitivity conferral protein. The map is similar to an earlier electron cryomicroscopy structure of bovine mitochondrial ATP synthase but with important differences. It resolves the internal structure of the membrane region of the complex, especially the membrane embedded subunits b, c, and a. Comparison of the yeast ATP synthase map, which lacks density from the dimer-specific subunits e and g, with a map of the bovine enzyme that included e and g indicates where these subunits are located in the intact complex. This new map has allowed construction of a model of subunit arrangement in the F(O) motor of ATP synthase that dictates how dimerization of the complex via subunits e and g might occur.

The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 A crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophage SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.

Understanding the needs of department chairs in academic medicine.

Purpose The challenges for senior academic leadership in medicine are significant and becoming increasingly complex. Adapting to the rapidly changing environment of health care and medical education requires strong leadership and management skills. This article provides empirical evidence about the intricate needs of department chairs to provide insight into the design of support and development opportunities. Method In an exploratory case study, 21 of 25 (84%) department chairs within a faculty of medicine at a large Canadian university participated in semistructured interviews from December 2009 to February 2010. The authors conducted an inductive thematic analysis and identified a coding structure through an iterative process of relating and grouping of emerging themes. Results These participants were initially often insufficiently prepared for the demands of their roles. They identified a specific set of needs. They required cultural and structural awareness to navigate their hospital and university landscapes. A comprehensive network of support was necessary for eliciting advice and exchanging information, strategy, and emotional support. They identified a critical need for infrastructure growth and development. Finally, they stressed that they needed improvement in both effective interpersonal and influence skills in order to meet their mandate. Conclusions Given the complexities and emotional burden of their role, it is necessary for chairs to have a range of supports and capabilities to succeed in their roles. Their leadership effectiveness can be enhanced by providing transitional processes and supports, development, and mentoring as well as facilitating the development of communities of peers.

Phages have adapted the same protein fold to fulfill multiple functions in virion assembly

Evolutionary relationships may exist among very diverse groups of proteins even though they perform different functions and display little sequence similarity. The tailed bacteriophages present a uniquely amenable system for identifying such groups because of their huge diversity yet conserved genome structures. In this work, we used structural, functional, and genomic context comparisons to conclude that the head–tail connector protein and tail tube protein of bacteriophage λ diverged from a common ancestral protein. Further comparisons of tertiary and quaternary structures indicate that the baseplate hub and tail terminator proteins of bacteriophage may also be part of this same family. We propose that all of these proteins evolved from a single ancestral tail tube protein fold, and that gene duplication followed by differentiation led to the specialized roles of these proteins seen in bacteriophages today. Although this type of evolutionary mechanism has been proposed for other systems, our work provides an evolutionary mechanism for a group of proteins with different functions that bear no sequence similarity. Our data also indicate that the addition of a structural element at the N terminus of the λ head–tail connector protein endows it with a distinctive protein interaction capability compared with many of its putative homologues.

Angle determination for side views in single particle electron microscopy

In order to build a first model in single particle electron microscopy the relative angular orientation of each image of a protein complex must be determined. These orientations can be described by three Eulerian angles. Images of complexes that present the same view can be aligned in two-dimensions and averaged in order to increase their signal-to-noise ratio. Based on these averaged images, several standard approaches exist for determining Euler angles for randomly oriented projection images. The common lines and angular reconstitution methods work well for particles with symmetry while the random conical tilting and related orthogonal tilt reconstruction methods work in most cases but require the acquisition of tilt pairs of images. For the situation where views of particles can be identified that are rotations about a single axis parallel to the grid, an alternative algorithm to determine the orientations of class averages without the need to acquire tilt pairs can be applied. This type of view of a complex is usually called a side view. This paper describes the detailed workings and characterization of an algorithm, named rotational analysis, which uses real-space fiducial markers derived from the averages themselves to determine the Euler angles for side views. We demonstrate how this algorithm works in practice by applying it to a data set of images of affinity-purified bovine mitochondrial ATP synthase.