Project description:The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral replication. Here, we report the cryogenic EM (cryo-EM) structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells - providing both an architectural overview of the entire complex and an understanding of how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the 'W-loop' of Smc4 or 'F-loop' of Smc1, mediates an important interaction with Nse1. Notably, mutations that alter the surface-charge profile of the region of Nse1 which accepts the Smc5-loop, lead to a slow-growth phenotype and a global reduction in the chromatin-associated fraction of the Smc5/6 complex, as judged by single molecule localisation microscopy experiments in live yeast. Moreover, when taken together, our data indicates functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.
Project description:CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism1-3. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.
Project description:Current single-cell RNA-seq approaches are hindered by preamplification bias, loss of strand of origin information, and the inability to observe small-RNA and mRNA dual transcriptomes. Here, we introduce a single-cell holo-transcriptome sequencing (Holo-Seq) that overcomes all three hurdles. Holo-Seq has the same quantitative accuracy and uniform coverage with a complete strand of origin information as bulk RNA-seq. Most importantly, Holo-Seq can simultaneously observe small RNAs and mRNAs in a single cell. Furthermore, we acquire small RNA and mRNA dual transcriptomes of 32 human hepatocellular carcinoma single cells, which display the genome-wide super-enhancer activity and hepatic neoplasm kinetics of these cells.
Project description:The TOM complex is the main entry point for precursor proteins (preproteins) into mitochondria. Preproteins containing targeting sequences are recognized by the TOM complex and imported into mitochondria. We have determined the structure of the TOM core complex from Neurospora crassa by single-particle electron cryomicroscopy at 3.3 Å resolution, showing its interaction with a bound preprotein at 4 Å resolution, and of the TOM holo complex including the Tom20 receptor at 6 to 7 Å resolution. TOM is a transmembrane complex consisting of two β-barrels, three receptor subunits, and three short transmembrane subunits. Tom20 has a transmembrane helix and a receptor domain on the cytoplasmic side. We propose that Tom20 acts as a dynamic gatekeeper, guiding preproteins into the pores of the TOM complex. We analyze the interactions of Tom20 with other TOM subunits, present insights into the structure of the TOM holo complex, and suggest a translocation mechanism.
Project description:Statistical models play a significant role in designing competent breeding programs related to complex traits. Recently; the holo-omics framework has been productively utilized in trait prediction; but it contains many complexities. Therefore; it is desirable to establish prediction accuracy while combining the host's genome and microbiome data. Several methods can be used to combine the two data in the model and study their effectiveness by estimating the prediction accuracy. We validate our holo-omics interaction models with analysis from two publicly available datasets and compare them with genomic and microbiome prediction models. We illustrate that the holo-omics interactive models achieved the highest prediction accuracy in ten out of eleven traits. In particular; the holo-omics interaction matrix estimated using the Hadamard product displayed the highest accuracy in nine out of eleven traits, with the direct holo-omics model and microbiome model showing the highest prediction accuracy in the remaining two traits. We conclude that comparing prediction accuracy in different traits using real data showed important intuitions into the holo-omics architecture of complex traits.
Project description:Two crystal forms of Escherichia coli tryptophanase (tryptophan indole-lyase, Trpase) were obtained under the same crystallization conditions. Both forms belonged to the same space group P43212 but had slightly different unit-cell parameters. The holo crystal form, with pyridoxal phosphate (PLP) bound to Lys270 of both polypeptide chains in the asymmetric unit, diffracted to 2.9 Å resolution. The second crystal form diffracted to 3.2 Å resolution. Of the two subunits in the asymmetric unit, one was found in the holo form, while the other appeared to be in the apo form in a wide-open conformation with two sulfate ions bound in the vicinity of the active site. The conformation of all holo subunits is the same in both crystal forms. The structures suggest that Trpase is flexible in the apo form. Its conformation partially closes upon binding of PLP. The closed conformation might correspond to the enzyme in its active state with both cofactor and substrate bound in a similar way as in tyrosine phenol-lyase.
Project description:The bacterial Sec translocon, SecYEG, associates with accessory proteins YidC and the SecDF-YajC subcomplex to form the bacterial holo-translocon (HTL). The HTL is a dynamic and flexible protein transport machine capable of coordinating protein secretion across the membrane and efficient lateral insertion of nascent membrane proteins. It has been hypothesized that a central lipid core facilitates the controlled passage of membrane proteins into the bilayer, ensuring the efficient formation of their native state. By performing small-angle neutron scattering on protein solubilized in "match-out" deuterated detergent, we have been able to interrogate a "naked" HTL complex, with the scattering contribution of the surrounding detergent micelle rendered invisible. Such an approach has allowed the confirmation of a lipid core within the HTL, which accommodates between 8 and 29 lipids. Coarse-grained molecular dynamics simulations of the HTL also demonstrate a dynamic, central pool of lipids. An opening at this lipid-rich region between YidC and the SecY lateral gate may provide an exit gateway for newly synthesized, correctly oriented, membrane protein helices, or even small bundles of helices, to emerge from the HTL.
Project description:General transcription factor TFIID is a cornerstone of RNA polymerase II transcription initiation in eukaryotic cells. How human TFIID-a megadalton-sized multiprotein complex composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs)-assembles into a functional transcription factor is poorly understood. Here we describe a heterotrimeric TFIID subcomplex consisting of the TAF2, TAF8 and TAF10 proteins, which assembles in the cytoplasm. Using native mass spectrometry, we define the interactions between the TAFs and uncover a central role for TAF8 in nucleating the complex. X-ray crystallography reveals a non-canonical arrangement of the TAF8-TAF10 histone fold domains. TAF2 binds to multiple motifs within the TAF8 C-terminal region, and these interactions dictate TAF2 incorporation into a core-TFIID complex that exists in the nucleus. Our results provide evidence for a stepwise assembly pathway of nuclear holo-TFIID, regulated by nuclear import of preformed cytoplasmic submodules.