Krios I-Titan Krios I at Diamond
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Abstract: The first automated data set of a test specimen, GroEL, has been successfully collected on Diamond's cryo-electron microscope, Titan Krios I.
The data set was collected with the world's first EPU embedded bioquantum-K2 detector, and was produced in the form of films so that researchers could see a moving image of the specimen. The films were collected in super-resolution counting mode with a 30eV energy selecting slit.
The films were then aligned using IMOD followed by particle selection, alignment and 3D reconstriuction in relion. From a total of 30,000 particles, 17,500 were used to calculate a 3.2Å 3D reconstruction of GroEL. This is currently the highest resolution 3D reconstruction of GroEL determined by cryo-electron microscopy - a significant achievement for Diamond's Titan Krios I.
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Nov 2015
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[13256]
Open Access
Abstract: Type 1 and P pili are prototypical bacterial cell surface appendages playing essential roles in mediating recognition and adhesion of bacteria to the urinary tract. These pili, assembled by the chaperone-usher pathway, are polymers of pilus subunits assembling into two parts: a thin, short, tip fibrillum at the top, mounted on a long pilus rod. The rod adopts a helical quaternary structure and is thought to play essential roles: its formation may drive pilus extrusion by preventing backsliding of the nascent growing pilus within the secretion pore; the rod also has striking spring-like properties being able to uncoil and recoil depending on the intensity of shear forces generated by urine flow. Here we present an atomic model of the P pilus generated from a 3.8 Å resolution cryo-electron microscopy reconstruction. This structure provides the molecular basis for the rod’s remarkable mechanical properties and illuminates its role in pilus secretion.
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Dec 2015
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[13256]
Abstract: One of the first users of Diamond Light Source’s new cryo-electron microscope (cryo-EM) has elucidated the structure of the hair-like appendages found on Escherichia coli (E.coli). The remarkable atomic model generated from a 3.8 Å resolution cryo-EM reconstruction has recently been published in the high-profile journal, Cell (Fig. 1).
Bacteria, such as E.coli, naturally possess hair-like structures exposed at the cell surface, which are known as pili. The pili play an essential role in infection as they are responsible for seeking out and latching onto specific host tissues. By fully understanding the structure and function of these unassuming bacterial appendages, it is thought that new medicines could be developed to prevent infection.
A team of scientists from University College London (UCL) and Birkbeck, University of London, the University of Washington, and the University of Virginia, took on the challenge to delve into the inner workings of the pili. By using the cutting edge cryo-EM facility at Diamond, the group discovered that the pili were shaped like springs, which allowed the bacteria to cling on to host tissues even in adverse environments.
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Jan 2016
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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Transmission electron microscopy (EM) is a versatile technique that can be used to image biological specimens ranging from intact eukaryotic cells to individual proteins greater than 150 kDa. There are several strategies for preparing samples for imaging by EM, including negative staining and cryogenic freezing. In the last few years, cryo-EM has undergone a ’resolution revolution’, owing to both advances in imaging hardware, image processing software, and improvements in sample preparation, leading to growing number of researchers using cryo-EM as a research tool. However, cryo-EM is still a rapidly growing field, with unique challenges. Here, we summarise considerations for imaging of a range of specimens from macromolecular complexes to cells using EM.
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Feb 2016
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[12388]
Open Access
Abstract: In response to complement activation, the membrane attack complex (MAC) assembles from fluid-phase proteins to form pores in lipid bilayers. MAC directly lyses pathogens by a ‘multi-hit’ mechanism; however, sublytic MAC pores on host cells activate signalling pathways. Previous studies have described the structures of individual MAC components and subcomplexes; however, the molecular details of its assembly and mechanism of action remain unresolved. Here we report the electron cryo-microscopy structure of human MAC at subnanometre resolution. Structural analyses define the stoichiometry of the complete pore and identify a network of interaction interfaces that determine its assembly mechanism. MAC adopts a ‘split-washer’ configuration, in contrast to the predicted closed ring observed for perforin and cholesterol-dependent cytolysins. Assembly precursors partially penetrate the lipid bilayer, resulting in an irregular β-barrel pore. Our results demonstrate how differences in symmetric and asymmetric components of the MAC underpin a molecular basis for pore formation and suggest a mechanism of action that extends beyond membrane penetration.
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Mar 2016
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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high resolutions and robust approaches for model assessment. Flex-EM/MODELLER has been used for flexible fitting of atomic models in intermediate-to-low resolution density maps of different biological systems. Here, we demonstrate the suitability of the method to successfully refine structures at higher resolutions (2.5-4.5 Å) using both simulated and experimental data, including a newly processed map of Apo-GroEL. A hierarchical refinement protocol was adopted where the rigid body definitions are relaxed and atom displacement steps are reduced progressively at successive stages of refinement. For the assessment of local fit, we used the SMOC (segment-based Manders’ overlap coefficient) score, while the model quality was checked using the Qmean score. Comparison of SMOC profiles at different stages of refinement helped in detecting regions that are poorly fitted. We also show how initial model errors can have significant impact on the goodness-of-fit. Finally, we discuss the implementation of Flex-EM in the CCP-EM software suite.
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Mar 2016
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[12388]
Abstract: For the first time ever, scientists have defined the structure of a fundamental part of our immune system, the membrane attack complex (MAC), with the aid of the state-of-the-art cryo-electron microscope (cryo-EM) facility at Diamond Light Source. The structure was recently unveiled in an article published by Nature Communications.
The MAC is a critical player in our response to infection. When pathogens are detected, a collection of signalling proteins known as ‘complement’ are activated and this process triggers the assembly of the MAC. The MAC is a huge pore made from a set of proteins that literally punches holes in the wall of bacteria to kill them. Although the lesions made by this fascinating protein complex were first noted in 1964, the actual structure of the MAC was unknown.
The mystery was solved at the electron Bio-Imaging Centre (eBIC) at Diamond over half a century later with the collaborative efforts of scientists from Imperial College and Cardiff University who determined that the MAC adopted a ‘split washer’ configuration. Their structural analyses also gave them an insight into how this unusual complex breaks through microbial membranes.
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Mar 2016
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[13275]
Open Access
Abstract: In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly processive, duplex unwinding and degrading machines that require tight regulation. Here, we report the structure of E.coli RecBCD, determined by cryoEM at 3.8 Å resolution, with a DNA substrate that reveals how the nuclease activity of the complex is activated once unwinding progresses. Extension of the 5'-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit. The process involves a SH3 domain that binds to a region of the RecB subunit in a binding mode that is distinct from others observed previously in SH3 domains and, to our knowledge, this is the first example of peptide-binding of an SH3 domain in a bacterial system.
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Sep 2016
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[12877, 13590]
Open Access
Abstract: The β-barrel assembly machinery (BAM) is a ∼203 kDa complex of five proteins (BamA–E), which is essential for viability in E. coli. BAM promotes the folding and insertion of β-barrel proteins into the outer membrane via a poorly understood mechanism. Several current models suggest that BAM functions through a ‘lateral gating’ motion of the β-barrel of BamA. Here we present a cryo-EM structure of the BamABCDE complex, at 4.9 Å resolution. The structure is in a laterally open conformation showing that gating is independent of BamB binding. We describe conformational changes throughout the complex and interactions between BamA, B, D and E, and the detergent micelle that suggest communication between BAM and the lipid bilayer. Finally, using an enhanced reconstitution protocol and functional assays, we show that for the outer membrane protein OmpT, efficient folding in vitro requires lateral gating in BAM.
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Sep 2016
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[13278]
Open Access
Abstract: Retinoschisin, an octameric retinal-specific protein, is essential for retinal architecture with mutations causing X-linked retinoschisis (XLRS), a monogenic form of macular degeneration. Most XLRS-associated mutations cause intracellular retention, however a subset are secreted as octamers and the cause of their pathology is ill-defined. Therefore, here we investigated the solution structure of the retinoschisin monomer and the impact of two XLRS-causing mutants using a combinatorial approach of biophysics and cryo-EM. The retinoschisin monomer has an elongated structure which persists in the octameric assembly. Retinoschisin forms a dimer of octamers with each octameric ring adopting a planar propeller structure. Comparison of the octamer with the hexadecamer structure indicated little conformational change in the retinoschisin octamer upon dimerization, suggesting that the octamer provides a stable interface for construction of the hexadecamer. The H207Q XLRS-associated mutation was found in the interface between octamers and destabilized both monomeric and octameric retinoschisin. Octamer dimerization is consistent with the adhesive function of retinoschisin supporting interactions between retinal cell layers, so disassembly would prevent structural coupling between opposing membranes. In contrast, cryo-EM structural analysis of the R141H mutation at ~4.2Å resolution was found to only cause a subtle conformational change in the propeller tips, potentially perturbing an interaction site. Together, these findings support distinct mechanisms of pathology for two classes of XLRS-associated mutations in the retinoschisin assembly.
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Oct 2016
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