SMA-Lipodisq nanoparticles, with one bacteriorhodopsin (bR) per 12 nm particle on average (protein/lipid molar ratio, 1:172), were prepared without the use of detergents. Using pulsed and continuous wave nitroxide spin label electron paramagnetic resonance, the structural and dynamic integrity of bR was retained when compared with data for bR obtained in the native membrane and in detergents and then with crystal data. This indicates the potential of Lipodisq nanoparticles as a useful membrane mimetic.

Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combined whole-cell cross-linking mass spectrometry, cellular cryo–electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at subnanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome and propose that it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell.

A major challenge for biophysical studies of membrane proteins is obtaining stable, homogenous samples. Traditional detergent solubilization and liposome-based methods of reconstitution may lead to protein inactivation, heterogeneous and polydisperse sized particles, and sample aggregation. [1] While membrane scaffold protein (MSP) stabilized nanodiscs have facilitated the formation of monodisperse protein samples, [2] a drawback is the detergent-based preparation method. Here we present a physicochemical characterization of polymer-stabilized lipid particles termed Lipodisq, a novel nanosized lipid-based platform capable of incorporating membrane proteins. [3] The polymers used in the Lipodisq technology can solubilize commonly used lipids such as dimyristoylphosphatidylcholine (DMPC) without the use of detergents. The small size of Lipodisq (diameter of around 9-10 nm at pH 7.4) renders them potentially suitable for many biophysical methodologies, including electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopies, electron microscopy (EM), and circular dichroism (CD) spectroscopy.DMPC and a polymer formed from a molar styrene to maleic acid ratio of 3:1 (termed 3:1 SMA, see Figure S1 in the Supporting Information) were used as a model system to generate Lipodisq particles. Their formation using 3:1 SMA and DMPC at a weight ratio of 1.25:1.0 was followed by dynamic light scattering (DLS) at 550 nm and 25 8C (Figure 1), resulting in solution clarification within a minute. Higher SMA to DMPC ratios of 3:1 can also be used, though the excess polymer may increase the total solution viscosity, while decreased amounts results in sub-optimal DMPC solubilization. The dynamic light scattering data indicate a monodisperse distribution of Lipodisq particles with an average diameter of 9 nm as previously reported, [3b] which is confirmed by negative stain transmission electron microscopy (TEM, Figure 1 C). Figure 1. Light scattering data and size of Lipodisq particles. Lipodisq particle formation was observed by measuring the light scattering of the resulting polymer-lipid solution at 550 nm (A; Abs = absorption).DLS data (B) suggest that the Lipodisq formed have an average diameter of 9 nm, whereas TEM images (C) reveal that the Lipodisq diameter varies between 5-15 nm.

Structural biology performed inside cells can capture molecular machines in action within their native context. Here we develop an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combine whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpoint NusA at the interface between a NusG-bound elongating RNAP and the ribosome, and propose it could mediate transcription-translation coupling. Translation inhibition dissociates the expressome, whereas transcription inhibition stalls and rearranges it, demonstrating that the active expressome architecture requires both translation and transcription elongation within the cell.

During translation elongation decoding is based on the recognition of codons by corresponding tRNA anticodon triplets. Molecular mechanisms that regulate global protein synthesis via specific base modifications in tRNA anticodons have recently received increasing attention. The conserved eukaryotic Elongator complex specifically modifies uridines located in the wobble base position of tRNAs. Here, we present the crystal structure of Dehalococcoides mccartyi Elp3 (DmcElp3) at 2.15 Å resolution. Our results reveal the unexpected arrangement of Elp3 lysine acetyl transferase (KAT) and radical S-adenosyl-methionine (SAM) domains that share a large interface to form a composite active site and tRNA binding pocket with an iron sulfur cluster located in the dimerization interface of two DmcElp3 molecules. Structure-guided mutagenesis studies of yeast Elp3 confirm the relevance of our findings for eukaryotic Elp3s and for understanding Elongator’s role in the onset of various neurodegenerative diseases and cancer in humans.