@article {65614, title = {Chloroplast Sec14-like 1 (CPSFL1) is essential for normal chloroplast development and affects carotenoid accumulation in Chlamydomonas.}, journal = {Proceedings of the National Academy of Sciences of the U S A}, volume = {117}, year = {2020}, month = {2020 Jun 2}, pages = {1-12}, abstract = {

Plastid isoprenoid-derived carotenoids serve essential roles in chloroplast development and photosynthesis. Although nearly all enzymes that participate in the biosynthesis of carotenoids in plants have been identified, the complement of auxiliary proteins that regulate synthesis, transport, sequestration, and degradation of these molecules and their isoprenoid precursors have not been fully described. To identify such proteins that are necessary for the optimal functioning of oxygenic photosynthesis, we screened a large collection of nonphotosynthetic (acetate-requiring) DNA insertional mutants of and isolated The mutant is extremely light-sensitive and susceptible to photoinhibition and photobleaching. The gene encodes a CRAL-TRIO hydrophobic ligand-binding (Sec14) domain protein. Proteins containing this domain are limited to eukaryotes, but some may have been retargeted to function in organelles of endosymbiotic origin. The mutant showed decreased accumulation of plastidial isoprenoid-derived pigments, especially carotenoids, and whole-cell focused ion-beam scanning-electron microscopy revealed a deficiency of carotenoid-rich chloroplast structures (e.g., eyespot and plastoglobules). The low carotenoid content resulted from impaired biosynthesis at a step prior to phytoene, the committed precursor to carotenoids. The CPSFL1 protein bound phytoene and β-carotene when expressed in and phosphatidic acid in vitro. We suggest that CPSFL1 is involved in the regulation of phytoene synthesis and carotenoid transport and thereby modulates carotenoid accumulation in the chloroplast.

}, issn = {1091-6490}, doi = {10.1073/pnas.1916948117}, author = {Garc{\'\i}a-Cerd{\'a}n, Jos{\'e} G and Schmid, Eva M and Takeuchi, Tomomi and McRae, Ian and McDonald, Kent L and Yordduangjun, Nichakarn and Hassan, Ahmed M and Grob, Patricia and Xu, C Shan and Hess, Harald F and Fletcher, Daniel A and Nogales, Eva and Niyogi, Krishna K} } @article {48490, title = {Enhanced FIB-SEM systems for large-volume 3D imaging.}, journal = {eLife}, volume = {6}, year = {2017}, month = {2017 May 13}, abstract = {

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) can automatically generate 3D images with superior z-axis resolution, yielding data that needs minimal image registration and related post-processing. Obstacles blocking wider adoption of FIB-SEM include slow imaging speed and lack of long-term system stability, which caps the maximum possible acquisition volume. Here we present techniques that accelerate image acquisition while greatly improving FIB-SEM reliability, allowing the system to operate for months and generating continuously imaged volumes \> 10(6) {\textmu}m(3). These volumes are large enough for connectomics, where the excellent z resolution can help in tracing of small neuronal processes and accelerate the tedious and time-consuming human proofreading effort. Even higher resolution can be achieved on smaller volumes. We present example data sets from mammalian neural tissue, Drosophila brain, and Chlamydomonas reinhardtii to illustrate the power of this novel high-resolution technique to address questions in both connectomics and cell biology.

}, issn = {2050-084X}, doi = {10.7554/eLife.25916}, author = {Xu, C Shan and Hayworth, Kenneth J and Lu, Zhiyuan and Grob, Patricia and Hassan, Ahmed M and Garc{\'\i}a-Cerd{\'a}n, Jos{\'e} G and Niyogi, Krishna K and Nogales, Eva and Weinberg, Richard J and Hess, Harald F} } @article {5883, title = {New technologies in imaging.}, journal = {Molecular Biology of the Cell}, volume = {24}, year = {2013}, month = {2013 Mar}, pages = {669}, abstract = {

Visualization of cellular and molecular processes is an indispensable tool for cell biologists, and innovations in microscopy methods unfailingly lead to new biological discoveries. Today, light microscopy (LM) provides ever-higher spatial and temporal resolution and visualization of biological process over enormous ranges. Electron microscopy (EM) is moving into the atomic resolution regime and allowing cellular analyses that are more physiological and sophisticated in scope. Importantly, much is being gained by combining multiple approaches, (e.g., LM and EM) to take advantage of their complementary strengths. The advent of high-throughput microscopies has led to a common need for sophisticated computational methods to quantitatively analyze huge amounts of data and translate images into new biological insights.

}, issn = {1939-4586}, doi = {10.1091/mbc.E12-12-0867}, author = {Galbraith, Catherine G and Keller, Philipp J and Nogales, Eva} } @article {6322, title = {The Ndc80 kinetochore complex forms oligomeric arrays along microtubules.}, journal = {Nature}, volume = {467}, year = {2010}, month = {2010 Oct 14}, pages = {805-10}, abstract = {

The Ndc80 complex is a key site of regulated kinetochore-microtubule attachment (a process required for cell division), but the molecular mechanism underlying its function remains unknown. Here we present a subnanometre-resolution cryo-electron microscopy reconstruction of the human Ndc80 complex bound to microtubules, sufficient for precise docking of crystal structures of the component proteins. We find that the Ndc80 complex binds the microtubule with a tubulin monomer repeat, recognizing α- and β-tubulin at both intra- and inter-tubulin dimer interfaces in a manner that is sensitive to tubulin conformation. Furthermore, Ndc80 complexes self-associate along protofilaments through interactions mediated by the amino-terminal tail of the NDC80 protein, which is the site of phospho-regulation by Aurora B kinase. The complex{\textquoteright}s mode of interaction with the microtubule and its oligomerization suggest a mechanism by which Aurora B could regulate the stability of load-bearing kinetochore-microtubule attachments.

}, keywords = {Binding Sites, Biological, Cryoelectron Microscopy, Humans, Kinetochores, Microtubules, Mitosis, Models, Molecular, Nuclear Proteins, Protein Conformation, Tubulin}, issn = {1476-4687}, doi = {10.1038/nature09423}, author = {Alushin, Gregory M and Ramey, Vincent H and Pasqualato, Sebastiano and Ball, David A and Grigorieff, Nikolaus and Musacchio, Andrea and Nogales, Eva} } @article {2389, title = {Structures of three distinct activator-TFIID complexes.}, journal = {Genes \& Development}, volume = {23}, year = {2009}, month = {2009 Jul 1}, pages = {1510-21}, abstract = {

Sequence-specific DNA-binding activators, key regulators of gene expression, stimulate transcription in part by targeting the core promoter recognition TFIID complex and aiding in its recruitment to promoter DNA. Although it has been established that activators can interact with multiple components of TFIID, it is unknown whether common or distinct surfaces within TFIID are targeted by activators and what changes if any in the structure of TFIID may occur upon binding activators. As a first step toward structurally dissecting activator/TFIID interactions, we determined the three-dimensional structures of TFIID bound to three distinct activators (i.e., the tumor suppressor p53 protein, glutamine-rich Sp1 and the oncoprotein c-Jun) and compared their structures as determined by electron microscopy and single-particle reconstruction. By a combination of EM and biochemical mapping analysis, our results uncover distinct contact regions within TFIID bound by each activator. Unlike the coactivator CRSP/Mediator complex that undergoes drastic and global structural changes upon activator binding, instead, a rather confined set of local conserved structural changes were observed when each activator binds holo-TFIID. These results suggest that activator contact may induce unique structural features of TFIID, thus providing nanoscale information on activator-dependent TFIID assembly and transcription initiation.

}, keywords = {Hela Cells, Humans, Imaging, Immunoglobulins, Models, Molecular, Protein Binding, Protein Multimerization, Protein Structure, Proto-Oncogene Proteins c-jun, Quaternary, TATA-Binding Protein Associated Factors, Three-Dimensional, Transcription Factor TFIID, Tumor Suppressor Protein p53}, issn = {1549-5477}, doi = {10.1073/pnas.1100640108}, author = {Liu, Wei-Li and Coleman, Robert A and Ma, Elizabeth and Grob, Patricia and Yang, Joyce L and Zhang, Yixi and Dailey, Gina and Nogales, Eva and Tjian, Robert} } @article {2399, title = {Structural changes in TAF4b-TFIID correlate with promoter selectivity.}, journal = {Molecular Cell}, volume = {29}, year = {2008}, month = {2008 Jan 18}, pages = {81-91}, abstract = {

Proper ovarian development requires the cell type-specific transcription factor TAF4b, a subunit of the core promoter recognition complex TFIID. We present the 35 A structure of a cell type-specific core promoter recognition complex containing TAF4b and TAF4 (4b/4-IID), which is responsible for directing transcriptional synergy between c-Jun and Sp1 at a TAF4b target promoter. As a first step toward correlating potential structure/function relationships of the prototypic TFIID versus 4b/4-IID, we have compared their 3D structures by electron microscopy and single-particle reconstruction. These studies reveal that TAF4b incorporation into TFIID induces an open conformation at the lobe involved in TFIIA and putative activator interactions. Importantly, this open conformation correlates with differential activator-dependent transcription and promoter recognition by 4b/4-IID. By combining functional and structural analysis, we find that distinct localized structural changes in a megadalton macromolecular assembly can significantly alter its activity and lead to a TAF4b-induced reprogramming of promoter specificity.

}, keywords = {B-Lymphocytes, Cell Line, Computer-Assisted, Electron, Genetic, Hela Cells, Humans, Image Processing, Imaging, Microscopy, Models, Molecular, Organ Specificity, Promoter Regions, Protein Conformation, Protein Interaction Mapping, Proto-Oncogene Proteins c-jun, Sp1 Transcription Factor, Structure-Activity Relationship, TATA-Binding Protein Associated Factors, Three-Dimensional, Transcription Factor TFIIA, Transcription Factor TFIID, Transcriptional Activation, Tumor}, issn = {1097-2765}, doi = {10.1073/pnas.1100640108}, author = {Liu, Wei-Li and Coleman, Robert A and Grob, Patricia and King, David S and Florens, Laurence and Washburn, Michael P and Geles, Kenneth G and Yang, Joyce L and Ramey, Vincent and Nogales, Eva and Tjian, Robert} } @article {2410, title = {Cryo-electron microscopy studies of human TFIID: conformational breathing in the integration of gene regulatory cues.}, journal = {Structure}, volume = {14}, year = {2006}, month = {2006 Mar}, pages = {511-20}, abstract = {

The multisubunit transcription factor TFIID is essential for directing eukaryotic promoter recognition and mediating interactions with activators/cofactors during assembly of the preinitiation complex. Despite its central role in transcription initiation and regulation, structural knowledge of the TFIID complex has so far been largely limited to electron microscopy studies of negatively stained samples. Here, we present a cryo-electron microscopy 3D reconstruction of the large endogenous human TFIID complex. The improved cryopreservation has allowed for a more detailed definition of the structural elements in the complex and for the detection, by an extensive statistical analysis of the data, of a conformational opening and closing of the cavity central to the TFIID architecture. We propose that these density rearrangements in the structure are a likely reflection of the plasticity of the interactions between TFIID and its many partner proteins.

}, keywords = {Cryoelectron Microscopy, Gene Expression Regulation, Genetic, Humans, Protein Binding, Protein Structure, Tertiary, Transcription, Transcription Factor TFIID}, issn = {0969-2126}, doi = {10.1073/pnas.1100640108}, author = {Grob, Patricia and Cruse, Michael J and Inouye, Carla and Peris, Marian and Penczek, Pawel A and Tjian, Robert and Nogales, Eva} } @article {2415, title = {Structural studies of the human PBAF chromatin-remodeling complex.}, journal = {Structure}, volume = {13}, year = {2005}, month = {2005 Feb}, pages = {267-75}, abstract = {

ATP-dependent chromatin remodeling is one of the central processes responsible for imparting fluidity to chromatin and thus regulating DNA transactions. Although knowledge on this process is accumulating rapidly, the basic mechanism (or mechanisms) by which the remodeling complexes alter the structure of a nucleosome is not yet understood. Structural information on these macromolecular machines should aid in interpreting the biochemical and genetic data; to this end, we have determined the structure of the human PBAF ATP-dependent chromatin-remodeling complex preserved in negative stain by electron microscopy and have mapped the nucleosome binding site using two-dimensional (2D) image analysis. PBAF has an overall C-shaped architecture{\textendash}with a larger density to which two smaller knobs are attached{\textendash}surrounding a central cavity; one of these knobs appears to be flexible and occupies different positions in each of the structures determined. The 2D analysis of PBAF:nucleosome complexes indicates that the nucleosome binds in the central cavity.

}, keywords = {Binding Sites, Chromosomal Proteins, Humans, Imaging, Molecular Conformation, Non-Histone, Nucleosomes, Three-Dimensional, Transcription Factors}, issn = {0969-2126}, doi = {10.1073/pnas.1100640108}, author = {Leschziner, Andres E and Lemon, Bryan and Tjian, Robert and Nogales, Eva} } @article {2430, title = {Human CRSP interacts with RNA polymerase II CTD and adopts a specific CTD-bound conformation.}, journal = {Genes \& Development}, volume = {16}, year = {2002}, month = {2002 Jun 1}, pages = {1339-44}, abstract = {

Activation of gene transcription in mammalian cells requires several classes of coactivators that participate in different steps of the activation cascade. Using conventional and affinity chromatography, we have isolated a human coactivator complex that interacts directly with the C-terminal domain (CTD) of RNA polymerase II (Pol II). The CTD-binding complex is structurally and functionally indistinguishable from our previously isolated CRSP coactivator complex. The closely related, but transcriptionally inactive, ARC-L complex failed to interact with the CTD, indicating a significant biochemical difference between CRSP and ARC-L that may, in part, explain their functional divergence. Electron microscopy and three-dimensional single-particle reconstruction reveals a conformation for CTD-CRSP that is structurally distinct from unliganded CRSP or CRSP bound to SREBP-1a, but highly similar to CRSP bound to the VP16 activator. Together, our findings suggest that the human CRSP coactivator functions, at least in part, by mediating activator-dependent recruitment of RNA Pol II via the CTD.

}, keywords = {Binding Sites, Chromatin, Electron, Electrophoresis, Genetic, Glutathione Transferase, Hela Cells, Humans, Immunoblotting, Ligands, Microscopy, Models, Molecular, Polyacrylamide Gel, Protein Binding, Protein Conformation, Protein Structure, Recombinant Fusion Proteins, RNA Polymerase II, Tertiary, Trans-Activators, Transcription}, issn = {0890-9369}, doi = {10.1073/pnas.1100640108}, author = {N{\"a}{\"a}r, Anders M and Taatjes, Dylan J and Zhai, Weiguo and Nogales, Eva and Tjian, Robert} } @article {2431, title = {Structure, function, and activator-induced conformations of the CRSP coactivator.}, journal = {Science}, volume = {295}, year = {2002}, month = {2002 Feb 8}, pages = {1058-62}, abstract = {

The human cofactor complexes ARC (activator-recruited cofactor) and CRSP (cofactor required for Sp1 activation) mediate activator-dependent transcription in vitro. Although these complexes share several common subunits, their structural and functional relationships remain unknown. Here, we report that affinity-purified ARC consists of two distinct multisubunit complexes: a larger complex, denoted ARC-L, and a smaller coactivator, CRSP. Reconstituted in vitro transcription with biochemically separated ARC-L and CRSP reveals differential cofactor functions. The ARC-L complex is transcriptionally inactive, whereas the CRSP complex is highly active. Structural determination by electron microscopy (EM) and three-dimensional reconstruction indicate substantial differences in size and shape between ARC-L and CRSP. Moreover, EM analysis of independently derived CRSP complexes reveals distinct conformations induced by different activators. These results suggest that CRSP may potentiate transcription via specific activator-induced conformational changes.

}, keywords = {CCAAT-Enhancer-Binding Proteins, Chromatin, Computer-Assisted, DNA-Binding Proteins, Electron, Genetic, Hela Cells, Herpes Simplex Virus Protein Vmw65, Humans, Image Processing, Imaging, Macromolecular Substances, Microscopy, Models, Precipitin Tests, Protein Binding, Protein Structure, Protein Subunits, Quaternary, Recombinant Fusion Proteins, Recombinant Proteins, Sterol Regulatory Element Binding Protein 1, Tertiary, Three-Dimensional, Trans-Activators, Transcription, Transcription Factors, Transcriptional Activation}, issn = {1095-9203}, doi = {10.1073/pnas.1100640108}, author = {Taatjes, Dylan J and N{\"a}{\"a}r, Anders M and Andel, Frank and Nogales, Eva and Tjian, Robert} }