@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 {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} }