Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET.

Gas vesicles (GVs) are gas-filled protein nanostructures employed by several species of bacteria and archaea as flotation devices to enable access to optimal light and nutrients. The unique physical properties of GVs have led to their use as genetically encodable contrast agents for ultrasound and MRI. Currently, however, the structure and assembly mechanism of GVs remain unknown.

Subtomogram averaging for biophysical analysis and supramolecular context.

Recent advances in hardware, software and computing power have led to increasingly ambitious applications of cryo-electron tomography and subtomogram averaging. It is now possible to reveal both structures and biophysical relationships like protein binding partners and small molecule occupancy in these experiments. However, some data processing choices require the user to prioritize structure or biophysical context.

Rubisco forms a lattice inside alpha-carboxysomes.

Despite the importance of microcompartments in prokaryotic biology and bioengineering, structural heterogeneity has prevented a complete understanding of their architecture, ultrastructure, and spatial organization. Here, we employ cryo-electron tomography to image α-carboxysomes, a pseudo-icosahedral microcompartment responsible for carbon fixation. We have solved a high-resolution subtomogram average of the Rubisco cargo inside the carboxysome, and determined the arrangement of the enzyme.

A bacterial membrane sculpting protein with BAR domain-like activity.

Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here, we report a bacterial protein with BAR domain-like activity, BdpA, from MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and influences scaffolding of OMEs into a consistent diameter and curvature.

In situ imaging of bacterial outer membrane projections and associated protein complexes using electron cryo-tomography.

The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab.

Measuring gas vesicle dimensions by electron microscopy.

Gas vesicles (GVs) are cylindrical or spindle-shaped protein nanostructures filled with air and used for flotation by various cyanobacteria, heterotrophic bacteria, and Archaea. Recently, GVs have gained interest in biotechnology applications due to their ability to serve as imaging agents and actuators for ultrasound, magnetic resonance and several optical techniques. The diameter of GVs is a crucial parameter contributing to their mechanical stability, buoyancy function and evolution in host cells, as well as their properties in imaging applications.

In Situ Imaging and Structure Determination of Biomolecular Complexes Using Electron Cryo-Tomography.

Electron cryo-tomography (cryo-ET) is a technique that allows the investigation of intact macromolecular complexes while they are in their cellular milieu. Over the years, cryo-ET has had a huge impact on our understanding of how large biomolecular complexes look like, how they assemble, disassemble, function, and evolve(d). Recent hardware and software developments and combining cryo-ET with other techniques, e.

Single-Molecule FRET of Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) are now widely recognized as playing critical roles in a broad range of cellular functions as well as being implicated in diverse diseases. Their lack of stable secondary structure and tertiary interactions, coupled with their sensitivity to measurement conditions, stymies many traditional structural biology approaches. Single-molecule Förster resonance energy transfer (smFRET) is now widely used to characterize the physicochemical properties of these proteins in isolation and is being increasingly applied to more complex assemblies and experimental environments.

Fluorescence-Based Detection of Membrane Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy.

Correlated light and electron microscopy (CLEM) has become a popular technique for combining the protein-specific labeling of fluorescence with electron microscopy, both at room and cryogenic temperatures. Fluorescence applications at cryo-temperatures have typically been limited to localization of tagged protein oligomers due to known issues of extended triplet state duration, spectral shifts, and reduced photon capture through cryo-CLEM objectives. Here, we consider fluorophore characteristics and behaviors that could enable more extended applications.

Rapid tilt-series acquisition for electron cryotomography.

Using a new Titan Krios stage equipped with a single-axis holder, we developed two methods to accelerate the collection of tilt-series. We demonstrate a continuous-tilting method that can record a tilt-series in seconds, but with loss of details finer than ∼4 nm. We also demonstrate a fast-incremental method that can record a tilt-series several-fold faster than current methods and with similar resolution.

Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA, and actin filaments.

We used fluorescence spectroscopy and EM to determine how binding of ATP, nucleation-promoting factors, actin monomers, and actin filaments changes the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Arp2/3 complex for labeling with fluorescent dyes at either the C termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ET) between the dyes in the presence of the various ligands.

Order-Disorder Transitions in the Cardiac Troponin Complex.

The troponin complex is a molecular switch that ties shifting intracellular calcium concentration to association and dissociation of actin and myosin, effectively allowing excitation-contraction coupling in striated muscle. Although there is a long history of muscle biophysics and structural biology, many of the mechanistic details that enable troponin's function remain incompletely understood. This review summarizes the current structural understanding of the troponin complex on the muscle thin filament, focusing on conformational changes in flexible regions of the troponin I subunit.

Conformation and Dynamics of the Troponin I C-Terminal Domain: Combining Single-Molecule and Computational Approaches for a Disordered Protein Region.

In recent years, single-molecule Förster resonance energy transfer (smFRET) has emerged as a critical and flexible tool in structural biology, particularly in the study of highly dynamic regions and molecular assemblies. The usefulness of smFRET can be further extended by combining it with computational approaches, marrying the coarse-grained experimental data with higher-resolution in silico calculations. Here we use smFRET to determine six pairwise distances within the intrinsically disordered C-terminal domain of the troponin I subunit (TnIC) of the cardiac troponin complex.

Folding upon phosphorylation: translational regulation by a disorder-to-order transition.

4E binding proteins (4E-BPs) play an important role in the regulation of translation by binding to eukaryotic translation initiation factor 4E (eIF4E) and inhibiting assembly of the eIF4F complex. While phosphorylation of 4E-BPs is known to disrupt their binding to eIF4E, the mechanism by which this occurs has been unclear. In a recent study, Forman-Kay and coworkers demonstrate that this mechanism is primarily structure-based: phosphorylation of 4E-BPs results in a disorder-to-order transition, bringing them from their binding-competent disordered state to a folded state incompatible with eIF4E binding.

The architecture of the 12RSS in V(D)J recombination signal and synaptic complexes.

V(D)J recombination is initiated by RAG1 and RAG2, which together with HMGB1 bind to a recombination signal sequence (12RSS or 23RSS) to form the signal complex (SC) and then capture a complementary partner RSS, yielding the paired complex (PC). Little is known regarding the structural changes that accompany the SC to PC transition or the structural features that allow RAG to distinguish its two asymmetric substrates. To address these issues, we analyzed the structure of the 12RSS in the SC and PC using fluorescence resonance energy transfer (FRET) and molecular dynamics modeling.

Skeletal muscle gender dimorphism from proteomics.

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.).

Gender dimorphism in the exercise-naïve murine skeletal muscle proteome.

Skeletal muscle is a plastic tissue with known gender dimorphism, especially at the metabolic level. A proteomic comparison of male and female murine biceps brachii was undertaken, resolving an average of 600 protein spots of MW 15-150 kDa and pI 5-8. Twenty-six unique full-length proteins spanning 11 KOG groups demonstrated statistically significant (p<0.