Home › Research › Structural Studies of Macromolecular Assemblies
Our Scientists
Structural Studies of Macromolecular Assemblies
Research Summary
Eva Nogales's lab is dedicated to gaining mechanistic insight into crucial molecular processes in the life of the eukaryotic cell. Her lab's main research areas are the dynamic self-assembly of the cytoskeleton during its essential functions in cell division and the molecular machines involved in macromolecular synthesis and degradation within the central dogma. The unifying principle in their work is the emphasis on studying macromolecular assemblies as whole units of molecular function by direct visualization of their architecture, functional states, and regulatory interactions. To gain a molecular understanding of their systems of interest, they use electron microscopy (EM) and image analysis, complemented with biochemical and biophysical assays.
Cytoskeleton and Cell Division Microtubule dynamics and interactions with kinetochore complexes. A long-standing interest of my lab is the process of microtubule dynamic instability. We have characterized tubulin polymers that we propose mimic the structural intermediates in the processes of microtubule assembly and disassembly. We are particularly interested in the role of the nucleotide state in microtubule polymerization and structure.
Microtubule dynamic instability is essential during mitosis, and a number of cellular factors, including structural kinetochore components, interact with the dynamic ends of microtubules and are able to modify the behavior of these polymers. Most importantly, outer kinetochore complexes appear to make use of the unique structures at microtubule ends to harvest the energy of the nucleotide-coupled assembly and disassembly processes to carry out cellular work.
Figure 1: Overall architecture of the Ndc80 kinetochore complex (left). Cryo-EM structure of the microtubule-binding head of the Ndc80 complex bound to a microtubule with a tubulin monomer repeat (right).
Adapted from Alushin, G., Ramey, V.H., Pasqualato, S., Ball, D.A., Grigorieff, N., Musacchio, A., and Nogales, E. 2010. Nature 467:805–810.
Figure 2: 3D model of the budding yeast kinetochore bound to a depolymerizing microtubule. The cryo-EM structure of the Dam1 complex is shown in blue, the microtubule is green, the nucleosome is dark purple, inner kinetochore complexes are shown in light purple, the Mtw1 complex is pink, the C-terminus of Spc105 is orange, Ndc80p is light blue, Nuf2p is gold, Spc24 is green, Spc25 is red, Ndc80p’s unstructured N-terminus is magenta and Ndc80’s coiled-coil is grey.
Adapted from Alushin, G. and Nogales, E. 2011. Current Opininon in Structural Biology 21:661-669.
Figure 3: EM images and schematic diagrams of the yeast septin rod, and paired filament assemblies. The yeast hetero-octameric septin complex is a linear rod with the subunits arrayed in the order and with the interfaces indicated. The rod is nonpolar because it has a two-fold axis of rotational symmetry running left to right between and orthogonal to the central pair of Cdc10 septins. The C-terminal extensions of the two Cdc3 and Cdc12 pairs project from the same face of the rod and presumably associate to form parallel coiled coils that are essential for rod stability. Filaments form via end-on-end assembly of the rods mediated by Cdc11-Cdc11 interaction through an N-C interface.
Adapted from Bertin, A., McMurray, M.A., Grob, P., Park, S.S., Garcia, G. 3rd, Patanwala, I., Ng, H.L., Alber, T., Thorner, J., and Nogales, E. 2008. Proceedings of the National Academy of Sciences USA 105:8274–8279.
Figure 4: Projection image of a section of the Saccharomyces cerevisiae bud neck, showing septin filaments abut the cell membrane (left). Segmented tomogram showing septin filaments running both parallel and perpendicular to the mother-bud axis (right).
Adapted from Bertin, A., McMurray, M.A., Pierson, J., Thai, L., McDonald, K.L., Zerh, E.A., Peters, P., Garcia III, G., Thorner, J., and Nogales, E. 2012. Molecular Biology of the Cell 23:423–432.
Figure 5: Architecture of ORC. A: 3D reconstruction of Drosophila ORC (origin recognition complex) with five AAA+ domains from the ATP-DnaA helical filament (colored in alternating red and orange) docked into the toroidal core of the complex. B: Model of ORC with five AAA+ domains (green and red) and four winged-helix domains (WHDs, yellow).
Adapted from Clarey, M.G., Erzberger, J.P., Grob, P., Leschziner, A.E., Berger, J.M., Nogales, E., and Botchan, M. 2006. Nature Structural and Molecular Biology 13:684–690.
Figure 6: Top, models of open and closed conformations of the yeast chromatin remodeler RSC. Bottom, model of RSC binding to a nucleosome based on shape complementarity.
Adapted from Leschziner, A.E., Saha, A., Wittmeyer, J., Zhang, Y., Bustamante, C., Cairns, B.R., and Nogales, E. 2007 Mar 13 [Epub ahead of print].Proceedings of the National Academy of Sciences USA.
Figure 7: Pseudoatomic model of most of the yeast Rrp44-exosome obtained by docking the crystal structures of its components into the 3D EM reconstruction. The Rrp44-exosome architecture suggests an active site sequestration mechanism for strict control of 3' exoribonuclease activity in the Rrp44-exosome complex.
Figure 8: Subnanometer cryo-EM structure of Cascade, showing six CasC subunits in alternating gray and blue, two CasB subunits in yellow, CasA in purple, CascD in orange, CasE in magenta, and crRNA in green.
Adapted from Wiedenheft, B., Lander, G.C., Zhou, K., Jore, M.M., Brouns, S.J.J., van der Oost, J., Doudna, J.A., and Nogales, E. 2011. Nature 477:486-489.
Figure 9: Cryo-EM structure of the yeast proteasome. The different subunits in the 19S regulatory particle are color-coded.
Adapted from Lander, G.C., Estrin, E., Matyskiela, M.E., Bashore, C., Nogales, E., and Martin, A. 2012. Nature482:186–191.
In collaboration with David Drubin and Georjana Barnes (University of California, Berkeley) and Stefan Westermann (Research Institute of Molecular Pathology, Vienna, Austria), we have shown that the 10-subunit Dam1 kinetochore complex assembles into rings around microtubules that move processively with microtubule ends, thus coupling microtubule depolymerization to directional movement. Our cryo-EM studies of Dam1 rings around microtubules have allowed us to describe the structure and organization of the Dam1 ring and have shed light on the nature of Dam1 oligomerization and on the interaction of the Dam1 complex with the microtubule surface.
Computer animation of the disassembly and reassembly of tubulin into microtubules, illustrating the existence of structural intermediates and their relationship to the nucleotide state. Produced for HHMI by Stylus Visuals, Kensington, California.
The Ndc80 complex, the microtubule-interacting component of the highly conserved KMN kinetochore network (KNL-1/Mis12 complex/Ndc80), is a long dumbbell-shaped molecule containing a dramatic kink within its 560-Å coiled-coil rod that is likely essential for correct kinetochore geometry. Our subnanometer structure of Ndc80 bound to the microtubule shows that the complex binds with a monomeric tubulin repeat at the intra- and interdimer interfaces, generating a minimal "toeprint" involving amino acids highly conserved between α- and β-tubulins. The binding is sensitive to tubulin conformation and has a stabilizing effect on microtubules. Binding to the microtubule is coupled to a self-interaction of the Ndc80 complexes along protofilaments that readily explains the cooperativity of the process. We are interested in the regulation of Ndc80 by Aurora B and on how Ndc80 interacts with other KMN components and, in yeast, with the Dam1 complex. Our intention is to gather enough information on these systems to generate a comprehensive model of shared molecular mechanisms and distinct properties that together result in the robust and regulated interaction of chromosome and spindle during mitosis.
Septinassemblyand membrane interactions. Septins are highly conserved GTPases that form filaments in vivo and in vitro and are essential in a variety of membrane-remodeling processes, most notably cytokinesis. We are particularly interested in the combinatorial character of septin-septin interactions and the interplay between membrane interaction and septin self-assembly. In collaboration with Jeremy Thorner (UC Berkeley), we have shown how the four essential mitotic yeast septins (Cdc3, Cdc10, Cdc11, and Cdc12) and the nonessential Shs1 come together in functional complexes and how they interact within filaments. We have described a marked enhancement in assembly that is totally dependent on and specific to PIP2 (phosphatidylinositol-4,5-bisphosphate), a signaling lipid known to be localized at the bud neck and to be essential to the cytokinetic process. Our recent characterization of the bud neck ultrastructure has shown the presence of coexisting septin filaments running both parallel and perpendicular to the bud axis. The relationship between in vivo phenotypes and septin assembly, organization, and interaction with different proteins is a major interest in our lab.
Molecular Machines of the Central Dogma DNA replication initiation. In collaboration with Michael Botchan (UC Berkeley), we have defined the architecture of the Drosophila origin recognition complex (ORC), proposed a model of subunit organization and DNA binding, and investigated the effect of nucleotide and phosphorylation state on ORC structure. We are interested in the structural characterization of the full replication preinitiation complex (in collaboration with Michael Botchan and James Berger [UC Berkeley]). We have used single-particle EM to determine the structure of the Drosophila melanogaster Mcm2–7 as well as the full CMG helicase complex (Cdc45, MCM, GINS), leading to a model of how the Mcm2–7 helicase is activated by the binding of GINS and Cdc45.
Transcription regulation. Our studies of challenging, large protein complexes involved in human transcription regulation have included the structural characterization of the RSC chromatin-remodeling complex and of human RNA polymerase II. One major effort is to define the structure of the human transcription factor IID (TFIID) and how it is affected by tissue-dependent subunit composition, interaction with activators (in collaboration with Robert Tjian [HHMI, UC Berkeley]), and binding to the core promoter (in collaboration with James Kadonaga [UC San Diego]). We obtained the cryo-EM structure of human TFIID and described its flexibility. We have now started to place TFIID conformational states into a functional context, relating them to the binding effect of regulatory factors (e.g., TFIIA, activators), core promoter recognition, and, ultimately, gene activation. Our long-term goal is to gain a molecular understanding of how the preinitiation complex comes together and how it is regulated synergistically by inputs that need to be integrated at the promoter site of eukaryotic genes.
Translation, RNA interference, and RNA processing. Our studies of gene regulation have extended beyond transcription into studies of translation, RNA processing (exosome studies in collaboration with Ailong Ke [Cornell University]), and RNA interference (RNAi).
We previously described the structure of human eIF3 and its interaction with hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA (in collaboration with Jennifer Doudna [HHMI, UC Berkeley]) and used the existing IRES-40S structure to model the binding of eIF3 to the 40S ribosome. Additionally, our reconstruction of eIF3 bound to eIF4G led us to propose a model of how this factor binds the ribosome. In collaboration with Jamie Cate (UC Berkeley), we are now using recombinant eIF3 subcomplexes to define the subunit architecture of the complex and to dissect the interaction with RNA elements within the HCV-IRES. Our goal is a description of eIF3 interaction with the 40S ribosome.
Our studies of the human RISC (RNA-induced silencing complex) loading complex (RLC) (in collaboration with Jennifer Doudna), composed of the Dicer and AGO2 enzymes and the RNA-binding protein TRBP (TAR RNA binding protein), showed both AGO2 and TRBP to be flexibly attached to the L-shaped Dicer protein, defining an inner cavity where RNA binding and processing occurs. In a process that parallels eukaryotic RNAi, the bacteria and archaea adaptive immune system against viruses and plasmids is a nucleic acid–based system in which short fragments of foreign DNA are integrated into clustered regularly interspaced short palindromic repeats (CRISPRs). CRISPR transcripts are processed into short RNAs that are incorporated into a large ribonucleoprotein surveillance complex. In collaboration with Jennifer Doudna, we used cryo-EM to determine the structure of the Escherichiacoli surveillance complex Cascade. The CRISPR RNA (crRNA) is displayed along a helical arrangement of six CasC subunits. Our studies showed that binding of target nucleic acid triggers a concerted conformational change of the seahorse-shaped complex that may serve as a signal for subsequent destruction of the invading DNA.
Regulated protein degradation by the proteasome. The ubiquitin-proteasome system is the major pathway for selective protein degradation in eukaryotic cells. The proteasome contains at least 32 different subunits that form a barrel-shaped 20S proteolytic core capped on either end by a 19S regulatory particle. The regulatory particle, composed of a lid and base subcomplexes, is required for substrate recognition, deubiquitination, protein unfolding, and translocation. In collaboration with Andreas Martin (UC Berkeley), we have obtained a subnanometer resolution structure of the whole 26S proteosome from budding yeast. By defining the structure of the lid in isolation and labeling each component of both the lid and base, we were able to localize each protein component within the 26S proteasome. This localization led to a model of the coordination of the recognition of ubiquitinated samples, the removal of ubiquitin chains, and the threading of the polypeptide chain into the translocase channel leading to the proteolytic chamber.
Portions of this work are also funded by the National Institute of General Medical Sciences.
