Current data processing strategies for cryo-electron tomography and subtomogram averaging

Cryo-electron tomography (cryo-ET) can be used to reconstruct three-dimensional (3D) volumes, or tomograms, from a series of tilted two-dimensional images of biological objects in their near-native states in situ or in vitro. 3D subvolumes, or subtomograms, containing particles of interest can be extracted from tomograms, aligned, and averaged in a process called subtomogram averaging (STA). STA overcomes the low signal to noise ratio within the individual subtomograms to generate structures of the particle(s) of interest. In recent years, cryo-ET with STA has increasingly been capable of reaching subnanometer resolution due to improvements in microscope hardware and data processing strategies. There has also been an increase in the number and quality of software packages available to process cryo-ET data with STA. In this review, we describe and assess the data processing strategies available for cryo-ET data and highlight the recent software developments which have enabled the extraction of high-resolution information from cryo-ET datasets

The impact of meteorology on the interannual growth rate of atmospheric methane

The impact of interannual changes in meteorology on the local and global growth rates of atmospheric methane is assessed in a nineteen year simulation using a tropospheric chemical transport model forced by ECMWF meteorological analyses from 1980 to 1998. A very simple CH4 chemistry scheme has been implemented, using prescribed OH fields. There are no interannual variations in modeled methane emissions or in the OH fields, so any changes in the modeled growth rate arise from changes in meteorology. The methane simulation shows significant interannual variability at both local and global scales. The local scale variability is comparable in magnitude to the interannual variability found in surface observations and shows some clear correlation with observed changes in growth rates. This suggests that, even over interannual timescales, meteorology could be important in driving the interannual fluctuations of atmospheric methane at the surface

A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0

We present a new approach for macromolecular structure determination from multiple particles in electron cryo-tomography (cryo-ET) data sets. Whereas existing subtomogram averaging approaches are based on 3D data models, we propose to optimise a regularised likelihood target that approximates a function of the 2D experimental images. In addition, analogous to Bayesian polishing and contrast transfer function (CTF) refinement in single-particle analysis, we describe the approaches that exploit the increased signal-to-noise ratio in the averaged structure to optimise tilt-series alignments, beam-induced motions of the particles throughout the tilt-series acquisition, defoci of the individual particles, as well as higher-order optical aberrations of the microscope. Implementation of our approaches in the open-source software package RELION aims to facilitate their general use, particularly for those researchers who are already familiar with its single-particle analysis tools. We illustrate for three applications that our approaches allow structure determination from cryo-ET data to resolutions sufficient for de novo atomic modelling.This work was funded by the UK Research and Innovation (UKRI) Medical Research Council (MC_UP_A025_1013 to SHWS; and MC_UP_1201/16 to JAGB), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC-CoG-2014, grant 648432, MEMBRANEFUSION to JAGB and ERC StG-2019, grant 852915 CRYTOCOP to GZ); the Swiss National Science Foundation (grant 205321_179041/1 to DC-D), the Max Planck Society (to JAGB) and the UKRI Biotechnology and Biological Sciences Research Council (grant BB/T002670/1 to GZ). TAMB is a recipient of a Sir Henry Dale Fellowship, jointly funded by the Wellcome Trust and the Royal Society (202231/Z/16/Z). JZ was partially funded by the European Union’s Horizon 2020 research and innovation program (ERC-ADG-2015, grant 692726, GlobalBioIm to Michael Unser)

ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinery

Funding: Royal Society for a University Research Fellowship; Wellcome Multi-User Equipment Grant (099149/Z/12/Z) (JEL).Transport of proteins across membranes is a fundamental process, achieved in every cell by the 'Sec' translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide [Allen et al. eLife 2016]. Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogen-deuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.Publisher PDFPeer reviewe

Insights into protein-lipid interactions by structural mass spectrometry

Interactions with membrane lipids have long been known to affect a wide range of membrane protein properties including folding, stability, and function. Our understanding of the precise molecular basis of these interactions is currently limited. Here, we combine mass spectrometry (MS)-based techniques with molecular dynamics (MD) simulations, in vivo biochemical assays, and other biophysical techniques to characterise how protein-lipid interactions affect the oligomerisation, conformation, and function of the structurally related xanthine/uric acid transporter UapA from Aspergillus nidulans and the boron transporter BOR1 from Saccharomyces cerevisiae (ScBOR1p). Using native MS and lipidomics, we found that UapA requires phosphatidylethanolamine or phosphatidylinositol binding to form the physiological dimer. A putative lipid binding site at the dimer interface was identified using MD simulations. Lipid binding at this site is essential for formation of functional UapA dimers. Similar analysis of ScBOR1p revealed that this protein is primarily monomeric in detergent-based solution and that phosphatidylethanolamine or phosphatidylserine binding is also essential for its dimerization. Mutation of a putative lipid binding site in ScBOR1p results in loss of lipid-induced dimer formation but has no effect on transport function. Thus, the lipids play slightly different roles in UapA and ScBOR1p. Additional research probed the conformational dynamics of UapA in different lipid compositions using hydrogen-deuterium exchange MS (HDX-MS). The results revealed that protein-lipid interactions stabilise the mobile domain of UapA responsible for substrate transport. HDX-MS also revealed how substrate binding stabilises the inward facing conformation of UapA and that specific mutations stabilise the outward facing conformation of UapA. Together, this work represents the first detailed analyses of the interactions between eukaryotic membrane transporters and their associated lipids.Open Acces