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 lack long-term system stability, which caps the maximum possible acquisition volume. Here, we present techniques accelerate while greatly improving reliability, allowing to operate for months generating continuously...

Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7). Only Shs1 is nonessential. The four essential form a complex containing two copies of each, but their arrangement was not known. Single-particle analysis by EM confirmed that the heterooligomer octameric revealed subunits are arrayed in linear rod. Identity each subunit determined examining complexes lacking given septin, antibody decoration, fusion to marker proteins (GFP or maltose binding protein). rod...

Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less known about their assembly and function vivo. We show here carboxysomes, CO(2)-fixing encoded by 10 genes, can be heterologously produced Escherichia coli. Expression carboxysomes E. coli resulted the production icosahedral similar to those from native host. In vivo, were capable both assembling with...

Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic sensors in plants and animals. However, the structural basis ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds bacterial flagellin assembles with NLRC4 to form a multiprotein complex called an inflammasome. Here we report cryo-electron microscopy structure...

The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle–vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence membrane states upon Ca2+-injection at 250–500 μM on 100-ms timescale. Furthermore, detailed morphologies were imaged cryo-electron microscopy before after Ca2+-injection. We discovered heterogeneous network immediate delayed pathways. Remarkably,...

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, scaffolding protein VPS15, tumor suppressor BECN1, and autophagy-specific subunit ATG14. structure ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All ordered domains BECN1 were mapped MBP tagging. dynamics defined using hydrogen–deuterium exchange, novel 20-residue region C-terminal to VPS34 C2 domain....

Understanding the molecular principles of synaptic vesicle fusion is a long-sought goal. It requires development synthetic system that allows manipulations and observations not possible in vivo. Here, we report an vitro with reconstituted proteins meets goal to produce fast content release millisecond time regime upon Ca(2+) triggering. Our simultaneously monitors both lipid exchange, it starts from stable interacting pairs donor acceptor vesicles, mimicking readily releasable pool vesicles...

Clicking into place: Cu-free click chemistry is combined with the aldehyde tag protein modification strategy to produce heterobifunctional fusions. This method relies on linkers that utilize orthogonal triazole and oxime chemistries (see scheme) expand conjugation fGly residue enables site-specific full-length human antibodies.

In prokaryotes, translation can occur on mRNA that is being transcribed in a process called coupling. How the ribosome affects RNA polymerase (RNAP) during coupling not well understood. Here, we reconstituted E. coli system and demonstrated prevent pausing termination of RNAP double overall transcription rate at expense fidelity. Moreover, monitored single RNAPs coupled to ribosomes show increases pause-free velocity mechanical assisting force sufficient explain majority effects Also, by...

The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists multiple protein complexes and is approximately 3.5 MDa in size. At heart this Mediator complex, which helps regulate PIC activity interacts with RNA polymerase II (pol II) enzyme. structure human Mediator–pol interface not well-characterized, whereas attempts structurally define interaction yeast have relied on incomplete assemblies and/or pol yielded...

The ∼230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation affected by deletions within and two tails in asymmetric active EGFR dimer are differently. monitored autophosphorylation cells using flow cytometry found that first ∼80 residues inhibitory, as demonstrated previously. entire ∼80-residue span important for autoinhibition needs to be released from both kinases form dimer. These results...

Microtubule architecture can vary with eukaryotic species, different cell types, and the presence of stabilizing agents. For in vitro assembled microtubules, average number protofilaments is reduced by sarcodictyin A, epothilone B, eleutherobin (similarly to taxol) but increased taxotere. Assembly a slowly hydrolyzable GTP analogue GMPCPP known give 96% 14 protofilament microtubules. We have used electron cryomicroscopy helical reconstruction techniques obtain three-dimensional maps taxotere...

Abstract Stramenopile algae contribute significantly to global primary productivity, and one class, Eustigmatophyceae, is increasingly studied for applications in high-value lipid production. Yet much about their basic biology remains unknown, including the nature of an enigmatic, pigmented globule found vegetative cells. Here, we present in-depth examination this “red body,” focusing on Nannochloropsis oceanica . During cell cycle, red body forms adjacent plastid, but unexpectedly it...

Transcription and its regulation pose a major challenge for genome stability. The helicase RECQL5 has been proposed as an important factor to help safeguard the genome, is only member of human RecQ family that directly binds RNA Polymerase II (Pol II) affects progression. mitigates transcription stress instability in cells, yet molecular mechanism underlying this phenomenon unclear. Here, we employ cryo-electron microscopy (cryo-EM) determine structures stalled Pol elongation complexes (ECs)...

Sequence-specific DNA-binding activators, key regulators of gene expression, stimulate transcription in part by targeting the core promoter recognition TFIID complex and aiding its recruitment to DNA. Although it has been established that activators can interact with multiple components TFIID, is unknown whether common or distinct surfaces within are targeted what changes if any structure may occur upon binding activators. As a first step toward structurally dissecting activator/TFIID...

Significance Carotenoids are essential molecules in oxygenic photoautotrophs, and they fulfill requirements for human animal nutrition. How carotenoid accumulation is regulated the chloroplast, a cyanobacterium-derived organelle, remains poorly understood, despite significant advancements identifying enzymes of biosynthetic pathway. This study identifies role chloroplast Sec14-like 1 (CPSFL1), CRAL-TRIO protein eukaryotic origin, modulation biosynthesis chloroplast. The CPSFL1 represents an...

Significance Embryonic or pluripotent stem cells are unique in their ability to self-renew culture and generate all lineages of an adult organism, making them valuable tools for modeling early developmental processes developing regenerative medicine technologies. An important factor controlling the expression pluripotency genes is Xeroderma pigmentosum complementation group C (XPC) DNA repair complex. This study presents, our knowledge, first complete structures different XPC complexes by...

Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens other biological phenomena. We conducted a structural characterization mAbs against N-terminal domain nucleocapsid protein (NPNTD) from SARS-CoV-2 using small-angle X-ray scattering transmission electron microscopy. Our solution-based results distinguished mAbs' flexibility how this affects assembly multiple on an antigen. By pairing two that bind different epitopes NPNTD, we show flexible form closed...