22 23 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 2 / 2 3 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 2 / 2 3 Understanding the Power of Movement in Plants Plant Science – Biochemistry – Agriculture & Fisheries – Chemistry – Structural Biology – Biophysics – Life Sciences & Biotech Auxins are hormones playing a central role and controlling nearly all aspects of plant growthanddevelopment. CharlesDarwinobserved that plants could grow directionally in response to environmental stimuli such as light or gravity. In his book, The Power of Movement in Plants , published in 1880, Darwin showed that the part of the plant responding to such a stimulus differs from the part that perceives it. He proposed that some kind of ‘influence’ must travel from the perception site to the response area. However, Darwin was unable to identify the influence. Darwin’s ‘growth accelerating substance’ was identified in 1926 as the hormone auxin. Later research identified that auxin is the growth factor that determines almost all plant responses to environmental changes. Directional transport of the auxin molecule between cells is required to ensure that the auxin response occurs in the correct part of the plant. It wasn’t until the 1990s that scientists identified the proteins involved in the process. PIN-FORMED (PIN) proteins are auxin transporters, and they are essential for the development of auxin gradients within plant tissues that guide plant growth. They’re named from the distinct needle-like ‘pin’ form, without shoots or flowers, into which plants with dysfunctional PIN proteins grow. Even then, how PIN proteins fold, how they recognise substrates and inhibitors and the molecular mechanism behind transport have remained unknown. Now researchers from Aarhus University and the Technical University of Munich have used single particle cryo-EM at eBIC to provide the first structural basis of auxin transport by PIN proteins. By combining three structures of Arabidopsis thaliana PIN8 with a comprehensive biochemical characterisation, their results finally provide the molecular mechanism behind auxin transport. They reveal an elevator-type transport mechanism similar to sodium/proton antiporters, bile acid/sodium symporters and bicarbonate/ sodium symporters. Their work provides a comprehensive molecular model for auxin recognition and transport by PINs and links and expands on a well-known conceptual framework for transport. In addition, it explains a central mechanism of polar auxin transport, a core feature of plant physiology, growth and development. Furthermore, their results offer insights into how a broad range of widely used herbicides, collectively known as synthetic auxins and anti-auxins, can be recognised by PIN proteins. It could therefore help to create more specific herbicides and minimise their environmental impact. Related publication: Ung, KL. et al. Structures and mechanism of the plant PIN-FORMED auxin transporter. Nature 609 , 605–610 (2022). DOI: 10.1038/s41586-022-04883-y Funding acknowledgement: ERC: grant agreement No 101000936 Deutsche Forschungsgemeinschaft (HA3468/6-1 and HA3468/6-3) and SFB924 National Institutes of Health (R35 GM144109) Corresponding authors: Ulrich Z. Hammes, Technical University of Munich,
[email protected] Bjørn Panyella Pedersen, Aarhus University,
[email protected] Biological Cryo-Imaging Group eBIC PIN8 is a 40 kDa membrane protein that transports the plant hormone Auxin. It forms a homodimer with each monomer containing two domains: transporter (green) and scaffold (blue). In the transporter domain a distinct crossover (red) is localized at the middle of the membrane plane that defines the auxin binding site. Below the structure show 8 representative 2D classes from the data collected at eBIC that resulted in 3 distinct conformations solved. To the left a schematic of the transport of Auxin (IAA) with two key conformations coloured that summarizes the transport mechanism as described by the data obtained at eBIC is shown. Deciphering amethane-oxidising enzyme using cryo-Electron Tomography Earth Science & Environment – Biotechnology – Climate Change – Biochemistry – Chemistry – Structural Biology – Engineering & Technology – Life Sciences & Biotech Methane is a greenhouse gas whose atmospheric concentration is currently around two-and-a-half times greater than pre-industrial levels and is increasing steadily. This rise has important implications for climate change. Methane-oxidising bacteria (methanotrophs) play a central role in greenhouse gas mitigation and have potential applications in biomanufacturing. Their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is housed in copper- induced intracytoplasmic membranes (ICMs). pMMO’s methane oxidation activity critically depends on its lipid environment, as detergent-solubilised enzymes used for crystallization or single particle cryo-EM structural analysis show no enzymatic activity. Therefore, to fully understand the molecular mechanism of pMMO, it is essential to study it in its native membrane environment. A team of scientists used serial cryo-Focused Ion Beam (cryoFIB) milling/ Scanning Electron Microscope (SEM) volume imaging and lamellae-based cellular cryo-Electron Tomography (cryo-ET) to study pMMO in native cells. They showed that these pMMO-embedded ICMs are derived from the inner cell membrane. Furthermore, pMMO forms ordered hexagonal arrays of trimers in intact cells. The structure of pMMO trimer in the ICM, composed of PmoA, PmoB and PmoC (88 KD together), was resolved by cryo-ET and subtomogram averaging to 4.8 Å resolution. Data were collected at Diamond’s electron Bio- Imaging Centre (eBIC) using the 300 kv Titan Krios microscope with technical support from staff scientists at eBIC. The structure reveals that the critical helices, which were missing or disordered from previous structural studies, are stabilised by the lipid membrane and form an active Cu centre for enzyme function. pMMO array formation correlates with increased enzymatic activity, highlighting the importance of studying the enzyme in its native environment. The remarkable hexagonal arrays of pMMO trimers in the native ICM provide new insights into the mechanistic understanding of the stimulated enzymatic activity of pMMO in intact cells. Understanding how pMMO arrays assemble and promote methane oxidation will be integral to future efforts to deploy methanotrophs in biotechnology. The findings also demonstrate, for the first time, the power of cryo-ET to structurally characterise small (< 100 KD) native transmembrane enzymes in the cellular context. Related publication: Zhu, Y. et al. Structure and activity of particulate methane monooxygenase arrays in methanotrophs. Nature Communications 13 , 5221 (2022). DOI: 10.1038/s41467-022-32752-9 Funding acknowledgement: UKWellcome Trust Investigator Award 206422/Z/17/Z (P.Z.) National Institutes of Health Grants R35GM118035 (A.C.R.) and T32GM008382 (C.W.K.) UK Biotechnology and Biological Sciences Research Council grant BB/ S003339/1 (P.Z.) ERC AdG grant (101021133) (P.Z.) Corresponding authors: Prof Peijun Zhang, Diamond Light Source and University of Oxford, peijun.
[email protected] Dr Yanan Zhu, University of Oxford,
[email protected] Biological Cryo-Imaging Group eBIC Native pMMO structure from methanotrophic bacteria. (A) A slice from serial cryoFIB/SEM volume of M. capsulatus (Bath). (B) A projection image of cryoFIB lamella of Bath. Inset, enlarged view of boxed area, displaying comb-like edge-on view of membrane-bound protein arrays. (C) A tomographic slice of Bath lamella displaying hexagonal arrays of particles. Inset, a subtomogram average of the 7-particle volume (yellow circle). (D) The structure pMMO trimer in ICM at 4.8 Å resolution, with each pMMO monomer consisting of PmoA (pink), PmoB (blue), and PmoC (cyan).