The actin-related protein 2/3 (Arp2/3) complex mediates the formation of branched actin filaments at the leading edge of motile cells and in the comet tails moving certain intracellular pathogens. Crystal structures of the Arp2/3 complex are available, but the architecture of the junction formed by the Arp2/3 complex at the base of the branch was not known. In this study, we use electron tomography to reconstruct the branch junction with sufficient resolution to show how the Arp2/3 complex interacts with the mother filament. Our analysis reveals conformational changes in both the mother filament and Arp2/3 complex upon branch formation. The Arp2 and Arp3 subunits reorganize into a dimer, providing a short-pitch template for elongation of the daughter filament. Two subunits of the mother filament undergo conformational changes that increase stability of the branch. These data provide a rationale for why branch formation requires cooperative interactions among the Arp2/3 complex, nucleation-promoting factors, an actin monomer, and the mother filament.

The 26S proteasome operates at the executive end of the ubiquitinproteasome pathway. Here, we present a cryo-EM structure of the Saccharomyces cerevisiae 26S proteasome at a resolution of 7.4 Å or 6.7 Å (Fourier-Shell Correlation of 0.5 or 0.3, respectively). We used this map in conjunction with molecular dynamics-based flexible fitting to build a near-atomic resolution model of the holocomplex. The quality of the map allowed us to assign α-helices, the predominant secondary structure element of the regulatory particle subunits, throughout the entire map. We were able to determine the architecture of the Rpn8/Rpn11 heterodimer, which had hitherto remained elusive. The MPN domain of Rpn11 is positioned directly above the AAA-ATPase N-ring suggesting that Rpn11 deubiquitylates substrates immediately following commitment and prior to their unfolding by the AAA-ATPase module. The MPN domain of Rpn11 dimerizes with that of Rpn8 and the C-termini of both subunits form long helices, which are integral parts of a coiled-coil module. Together with the C-terminal helices of the six PCI-domain subunits they form a very large coiled-coil bundle, which appears to serve as a flexible anchoring device for all the lid subunits.protein degradation | electron microscopy | deubiquitylating enzyme T he 26S proteasome is a 2.5 MDa molecular machine designed for the controlled degradation of proteins marked for destruction by the covalent attachment of polyubiquitin chains [for reviews see (1-3)]. It is composed of two copies, each of 33 canonical subunits, as well as some proteasome interacting proteins (PIPs). The 26S holocomplex comprises two types of subcomplexes: the cylindrical 20S core particle (CP) harbouring the proteolytic chamber and the two 19S regulatory particles (RPs), which attach to opposite ends of CP cylinder. The RPs have multiple roles in preparing substrates for degradation: They recognize and bind ubiquitylated proteins, they deubiquitylate them followed by their unfolding, and they control the opening of the gate which gives access to the interior of the CP.While the structure of the 20S core complex was determined by X-ray crystallography almost two decades ago (4, 5), the structure of the 26S complex remained recalcitrant to crystallization attempts, presumably due to its conformational and compositional heterogeneity. Recently, the subunit architecture of the holocomplex has been determined by cryo-electron microscopy (EM) single particle analysis (SPA and ref. 6, 7) independently by two groups using different approaches for the assignment of RP subunits. Lander, et al. (6) obtained a 9 Å resolution map (Fouriershell correlation, FSC ¼ 0.5) of the 26S Saccharomyces cerevisiae proteasome and they determined the subunit positions by means of fusion constructs and automated segmentation methods. Lasker, et al. (7) performed an exhaustive computational search of possible subunit configurations within the boundaries of an 8.5 Å map of the 26S proteasome from Schizosaccharomyces pombe scoring possible configurati...

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The rod outer segment (ROS) of photoreceptor cells houses all components necessary for phototransduction, a set of biochemical reactions that amplify and propagate a light signal. Theoretical approaches to quantify this process require precise information about the physical boundaries of the ROS. Dimensions of internal structures within the ROS of mammalian species have yet to be determined with the precision required for quantitative considerations. Cryoelectron tomography was utilized to obtain reliable three-dimensional morphological information about this important structure from murine retina. Vitrification of samples permitted imaging of the ROS in a minimally perturbed manner and the preservation of substructures. Tomograms revealed the characteristic highly organized arrangement of disc membranes stacked on top of one another with a surrounding plasma membrane. Distances among the various membrane components of the ROS were measured to define the space available for phototransduction to occur. Reconstruction of segments of the ROS from single-axis tilt series images provided a glimpse into the three-dimensional architecture of this highly differentiated neuron. The reconstructions revealed spacers that likely maintain the proper distance between adjacent discs and between discs and the plasma membrane. Spacers were found distributed throughout the discs, including regions that are distant from the rim region of discs.

Electron tomograms of intact frozen-hydrated cells are essentially three-dimensional images of the entire proteome of the cell, and they depict the whole network of macromolecular interactions. However, this information is not easily accessible because of the poor signal-to-noise ratio of the tomograms and the crowded nature of the cytoplasm. Here, we describe a template matching algorithm that is capable of detecting and identifying macromolecules in tomographic volumes in a fully automated manner. The algorithm is based on nonlinear cross correlation and incorporates elements of multivariate statistical analysis. Phantom cells, i.e., lipid vesicles filled with macromolecules, provide a realistic experimental scenario for an assessment of the fidelity of this approach. At the current resolution of Ϸ4 nm, macromolecules in the size range of 0.5-1 MDa can be identified with good fidelity.

The structure of the 26S proteasome from Schizosaccharomyces pombe has been determined to a resolution of 9.1 Å by cryoelectron microscopy and single particle analysis. In addition, chemical cross-linking in conjunction with mass spectrometry has been used to identify numerous residue pairs in close proximity to each other, providing an array of spatial restraints. Taken together these data clarify the topology of the AAA-ATPase module in the 19S regulatory particle and its spatial relationship to the α-ring of the 20S core particle. Image classification and variance analysis reveal a belt of high "activity" surrounding the AAA-ATPase module which is tentatively assigned to the reversible association of proteasome interacting proteins and the conformational heterogeneity among the particles. An integrated model is presented which sheds light on the early steps of protein degradation by the 26S complex.deubiquitylating enzymes | macromolecular complex | ubiquitinproteasome pathway | ubiquitin receptor | single particle classification I n eukaryotic cells, most proteins in the cytosol and the nucleus are regulated via the ubiquitin-proteasome pathway and malfunctions of this pathway have been implicated in a wide variety of diseases (1). The 26S proteasome is the most downstream element of this pathway, executing protein degradation (2-4). Unlike constitutively active proteases, the proteasome has the capacity to degrade almost any protein, yet it acts with exquisite specificity. The key stratagem is self-compartmentalization: The active sites of the proteolytic 20S core particles (CPs) are sequestered from the cellular environment in the interior of this barrelshaped subcomplex (5). Proteins destined for degradation are marked by a polyubiquitin chain, a degradation signal that is recognized by the 19S regulatory particles (RPs) that bind to either one or both ends of the CP to form the 26S holocomplex. The RPs (i) recognize the polyubiquitylated substrates, (ii) trim and recycle the polyubiquitin chains, (iii) unfold substrates to be degraded, and (iv) open the gate to the CP and assist in substrate translocation into the interior of the CP. These tasks are performed by a complex machinery involving at least 19 different subunits, 6 AAA-ATPases (Rpt1-6), and 13 non-ATPases (Rpn1-3, Rpn5-13, Rpn15/Sem1p).Although the structure of the CP has been elucidated in great detail by X-ray crystallography (6, 7), the structure of the RP is only dimly understood at present. Best characterized are the AAA-ATPases which form a heterohexameric subcomplex situated at the base of the RP in close proximity to the α-rings of the CP (8, 9). The C-terminal residues of Rpt2 and Rpt5 were shown to be involved in opening the gate in the α-rings, allowing substrates to enter the CP. A similar mechanism has been postulated for proteasome-activating nucleotidase (PAN), the archaeal homohexameric homolog of the eukaryotic AAA-ATPase module (10). Crystal structures of the two major fragments of PAN suggest that the N-and C-terminal domains...

Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1 Å resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP.

Cryo-electron tomography is an emerging imaging technique that has unique potential for molecular cell biology. At the present resolution of 4-5 nm, large supramolecular structures can be studied in unperturbed cellular environments and, in the future, it will become possible to map molecular landscapes inside cells in a more comprehensive manner. 'Visual proteomics' aims to complement and extend mass-spectrometry-based inventories, and to provide a quantitative description of the macromolecular interactions that underlie cellular functions.