Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain (RLC) promotes an inactive, 'shutdown' state with the filament-forming tail folded onto the two heads 1 , preventing filament formation and inactivating the motors 2 . The mechanism by which this happens is obscure.Here we report a cryo-electron microscopy structure of shutdown smooth muscle myosin, with a resolution of 6 Å in the head region. A pseudo-atomic model, obtained by flexible fitting of crystal structures into the density and molecular dynamics simulations, describes interaction interfaces at the atomic level. The N-terminal extension of one RLC interacts with the tail and the other with the partner head, revealing how the RLCs stabilise the shutdown state in different ways and how their phosphorylation would allow myosin activation. Additional interactions between the three segments of the coiled coil, the motor domains and LCs stabilise the shutdown molecule. The structure of the lever in each head is competent to generate force upon activation. This shutdown structure is relevant to all myosin-2 isoforms and provides a framework for understanding their disease-causing mutations.Myosin-2 is a molecular motor that interacts with actin using the energy from ATP hydrolysis to generate force and movement. It comprises two heavy chains, two essential light chains (ELC) and two regulatory light chains (RLC). The first ~800 residues of the heavy chain form the globular motor, which contains binding sites for actin and nucleotide. The remaining heavy chain forms an α-helix, in which the proximal region complexes with one ELC and one RLC to form a stiff light chain domain (LCD) that transmits motor force via a lever mechanism. The motor and the LCD comprise the myosin head. C-terminal to the LCD, the two heavy chains dimerise to form a 160-nm coiled-coil tail. The coiled coil contains the typical seven residue heptad repeat 3 , interrupted by three 'skip' residues in smooth muscle and non-muscle myosin-2 isoforms.In the active state of smooth muscle myosin (SmM), the two RLCs are phosphorylated 4 and the tails are polymerised into thick filaments. Dephosphorylation of S20 and/or T19 within the N-terminal extension of the RLCs 4,5 triggers an extraordinary transformation into a monomeric, shutdown state, in which both motors are trapped in a primed state, phosphate release is inhibited and ATPase activity is very low 6 . The two motor domains are organised into an asymmetric, interacting heads motif (IHM) (Fig. 1a), in which part of the actin-binding interface of the motor in the 'blocked' head is positioned onto the converter domain of the motor in the 'free' head, preventing interaction with actin 7 . The coiled-coil tail folds into three segments and wraps around the heads 8,9 .

Antibodies have long been the main approach used for localizing proteins of interest by light microscopy. In the past 5 yr or so, and with the advent of superresolution microscopy, the diversity of tools for imaging has rapidly expanded. One main area of expansion has been in the area of nanobodies, small single-chain antibodies from camelids or sharks. The other has been the use of artificial scaffold proteins, including Affimers. The small size of nanobodies and Affimers compared with the traditional antibody provides several advantages for superresolution imaging.

Dilated Cardiomyopathy is a common form of heart failure. Determining how this disease affects the structure and organization of cardiomyocytes in the human heart is important in understanding how the heart becomes less effective at contraction. Here we isolated and characterised Affimers (small non-antibody binding proteins) to Z-disc proteins ACTN2 (α-actinin-2), ZASP (also known as LIM domain binding protein 3 or LDB3) and the N-terminal region of the giant protein titin (TTN Z1-Z2). These proteins are known to localise in both the sarcomere Z-discs and the transitional junctions, found close to the intercalated discs that connect adjacent cardiomyocytes. We use cryosections of left ventricles from two patients diagnosed with end-stage Dilated Cardiomyopathy who underwent Orthotopic Heart Transplantation and were whole genome sequenced. We describe how Affimers substantially improve the resolution achieved by confocal and STED microscopy compared to conventional antibodies. We quantified the expression of ACTN2, ZASP and TTN proteins in two patients with dilated cardiomyopathy and compared them with a sex- and age-matched healthy donor. The small size of the Affimer reagents, combined with a small linkage error (the distance from the epitope to the dye label covalently bound to the Affimer) revealed new structural details in Z-discs and intercalated discs in the failing samples. Affimers are thus useful for analysis of changes to cardiomyocyte structure and organisation in diseased hearts.

Antibodies are the most widely used, traditional tool for labelling molecules in cells. In the past five to ten years, many new labelling tools have been developed with significant advantages over the traditional antibody. Here, we focus on nanobodies and the non-antibody binding scaffold proteins called Affimers. We explain how they are generated, selected and produced, and we describe how their small size, high binding affinity and specificity provides them with many advantages compared to antibodies. Of particular importance, their small size enables them to better penetrate dense cytoskeletal regions within cells, as well as tissues, providing them with specific advantage for super-resolution imaging, as they place the fluorophore with a few nanometres of the target protein being imaged. We expect these novel tools to be of broad interest to many cell biologists and anticipate them becoming the tools of choice for super-resolution imaging.

The crystal structure of PD1 from the Haemophilus surface fibril was determined at a resolution of 3.3 Å, revealing a novel domain arrangement.