22 results found
Ramlaul K, Fu W, Li H, et al., 2021, Architecture of the Tuberous Sclerosis protein complex, Journal of Molecular Biology, Vol: 433, ISSN: 0022-2836
The Tuberous Sclerosis Complex (TSC) protein complex (TSCC), comprising TSC1, TSC2, and TBC1D7, is widely recognised as a key integration hub for cell growth and intracellular stress signals upstream of the mammalian target of rapamycin complex 1 (mTORC1). The TSCC negatively regulates mTORC1 by acting as a GTPase-activating protein (GAP) towards the small GTPase Rheb. Both human TSC1 and TSC2 are important tumour suppressors, and mutations in them underlie the disease tuberous sclerosis.We used single-particle cryo-EM to reveal the organisation and architecture of the complete human TSCC. We show that TSCC forms an elongated scorpion-like structure, consisting of a central “body”, with a “pincer” and a “tail” at the respective ends. The “body” is composed of a flexible TSC2 HEAT repeat dimer, along the surface of which runs the TSC1 coiled-coil backbone, breaking the symmetry of the dimer. Each end of the body is structurally distinct, representing the N- and C-termini of TSC1; a “pincer” is formed by the highly flexible N-terminal TSC1 core domains and a barbed “tail” makes up the TSC1 coiled-coil-TBC1D7 junction. The TSC2 GAP domain is found abutting the centre of the body on each side of the dimerisation interface, poised to bind a pair of Rheb molecules at a similar separation to the pair in activated mTORC1.Our architectural dissection reveals the mode of association and topology of the complex, casts light on the recruitment of Rheb to the TSCC, and also hints at functional higher order oligomerisation, which has previously been predicted to be important for Rheb-signalling suppression.
Aylett C, 2020, Direct transfer of electron microscopy samples to wetted carbon and graphene films via a support floatation block, Journal of Structural Biology, Vol: 213, ISSN: 1047-8477
Support films are commonly used during cryo-EM specimen preparation to both immobilise the sample and minimise the exposure of particles at the air-water interface. Here we report preparation protocols for carbon and graphene supported single particle electron microscopy samples using a novel 3D-printed sample transfer block to facilitate the direct, wetted, movement of both carbon and graphene supports from the substrate on which they were generated to small volumes (10 μL) of sample. These approaches are simple and inexpensive to implement, minimise hydrophobic contamination of the support films, and are widely applicable to single particle studies. Our approach also allows the direct exchange of the sample buffer on the support film in cases in which it is unsuitable for vitrification, e.g. for samples from centrifugal density gradients that help to preserve sample integrity.
Ramlaul K, Palmer C, Nakane T, et al., 2020, Mitigating local over-fitting during single particle reconstruction with SIDESPLITTER, Journal of Structural Biology, Vol: 211, Pages: 1-9, ISSN: 1047-8477
Single particle analysis has become a key structural biology technique. Experimental images are extremely noisy, and during iterative refinement it is possible to stably incorporate noise into the reconstruction. Such “over-fitting” can lead to misinterpretation of the structure and flawed biological results. Several strategies are routinely used to prevent over-fitting, the most common being independent refinement of two sides of a split dataset. In this study, we show that over-fitting remains an issue within regions of low local signal-to-noise, despite independent refinement of half datasets. We propose a modification of the refinement process through the application of a local signal-to-noise filter: SIDESPLITTER. We show that our approach can reduce over-fitting for both idealised and experimental data while maintaining independence between the two sides of a split refinement. SIDESPLITTER refinement leads to improved density, and can also lead to improvement of the final resolution in extreme cases where datasets are prone to severe over-fitting, such as small membrane proteins.
de Martin Garrido N, Aylett C, 2020, Nutrient signalling and lysosome positioning crosstalk through a multifunctional protein, Folliculin, Frontiers in Cell and Developmental Biology, Vol: 8, ISSN: 2296-634X
FLCN was identified as the gene responsible for Birt-Hogg-Dubé (BHD) syndrome, a hereditary syndrome associated with the appearance of familiar renal oncocytomas. Most mutations affecting FLCN result in the truncation of the protein, and therefore loss of its associated functions, as typical for a tumor suppressor. FLCN encodes the protein folliculin (FLCN), which is involved in numerous biological processes; mutations affecting this protein thus lead to different phenotypes depending on the cellular context. FLCN forms complexes with two large interacting proteins, FNIP1 and FNIP2. Structural studies have shown that both FLCN and FNIPs contain longin and differentially expressed in normal versus neoplastic cells (DENN) domains, typically involved in the regulation of small GTPases. Accordingly, functional studies show that FLCN regulates both the Rag and the Rab GTPases depending on nutrient availability, which are respectively involved in the mTORC1 pathway and lysosomal positioning. Although recent structural studies shed light on the precise mechanism by which FLCN regulates the Rag GTPases, which in turn regulate mTORC1, how FLCN regulates membrane trafficking through the Rab GTPases or the significance of the intriguing FLCN-FNIP-AMPK complex formation are questions that still remain unanswered. We discuss the recent progress in our understanding of FLCN regulation of both growth signaling and lysosomal positioning, as well as future approaches to establish detailed mechanisms to explain the disparate phenotypes caused by the loss of FLCN function and the development of BHD-associated and other tumors.
de Martín Garrido N, Crone MA, Ramlaul K, et al., 2020, Bacteriophage MS2 displays unreported capsid variability assembling T = 4 and mixed capsids, Molecular Microbiology, Vol: 113, Pages: 143-152, ISSN: 0950-382X
Bacteriophage MS2 is a positive-sense, single-stranded RNA virus encapsulated in an asymmetric T = 3 pseudo-icosahedral capsid. It infects Escherichia coli through the F-pilus, which it binds through a maturation protein incorporated into its capsid. Cryogenic electron microscopy has previously shown that its genome is highly ordered within virions, and that it regulates the assembly process of the capsid. In this study we have assembled recombinant MS2 capsids with non-genomic RNA containing the capsid incorporation sequence, and investigated the structures formed, revealing that T = 3, T = 4 and mixed capsids between these two triangulation numbers are generated, and resolving structures of T = 3 and T = 4 capsids to 4 Å and 6 Å respectively. We conclude that the basic MS2 capsid can form a mix of T = 3 and T = 4 structures, supporting a role for the ordered genome in favouring the formation of functional T = 3 virions.
Ramlaul K, Palmer CM, Aylett CHS, 2019, A local agreement filtering algorithm for transmission EM reconstructions, Journal of Structural Biology, Vol: 205, Pages: 30-40, ISSN: 1047-8477
We present LAFTER, an algorithm for de-noising single particle reconstructions from cryo-EM.Single particle analysis entails the reconstruction of high-resolution volumes from tens of thousands of particle images with low individual signal-to-noise. Imperfections in this process result in substantial variations in the local signal-to-noise ratio within the resulting reconstruction, complicating the interpretation of molecular structure. An effective local de-noising filter could therefore improve interpretability and maximise the amount of useful information obtained from cryo-EM maps.LAFTER is a local de-noising algorithm based on a pair of serial real-space filters. It compares independent half-set reconstructions to identify and retain shared features that have power greater than the noise. It is capable of recovering features across a wide range of signal-to-noise ratios, and we demonstrate recovery of the strongest features at Fourier shell correlation (FSC) values as low as 0.144 over a 2563-voxel cube. A fast and computationally efficient implementation of LAFTER is freely available.We also propose a new way to evaluate the effectiveness of real-space filters for noise suppression, based on the correspondence between two FSC curves: 1) the FSC between the filtered and unfiltered volumes, and 2) Cref, the FSC between the unfiltered volume and a hypothetical noiseless volume, which can readily be estimated from the FSC between two half-set reconstructions.
Ramlaul K, Aylett CHS, 2018, Signal integration in the (m)TORC1 growth pathway, Frontiers in Biology, Vol: 13, Pages: 237-262, ISSN: 1674-7984
BackgroundThe protein kinase Target Of Rapamycin (TOR) is a nexus for the regulation of eukaryotic cell growth. TOR assembles into one of two distinct signalling complexes, TOR complex 1 (TORC1) and TORC2 (mTORC1/2 in mammals), with a set of largely non-overlapping protein partners. (m)TORC1 activation occurs in response to a series of stimuli relevant to cell growth, including nutrient availability, growth factor signals and stress, and regulates much of the cell’s biosynthetic activity, from proteins to lipids, and recycling through autophagy. mTORC1 regulation is of great therapeutic significance, since in humans many of these signalling complexes, alongside subunits of mTORC1 itself, are implicated in a wide variety of pathophysiologies, including multiple types of cancer, neurological disorders, neurodegenerative diseases and metabolic disorders including diabetes.MethodologyRecent years have seen numerous structures determined of (m)TOR, which have provided mechanistic insight into (m)TORC1 activation in particular, however the integration of cellular signals occurs upstream of the kinase and remains incompletely understood. Here we have collected and analysed in detail as many as possible of the molecular and structural studies which have shed light on (m)TORC1 repression, activation and signal integration.ConclusionsA molecular understanding of this signal integration pathway is required to understand how (m)TORC1 activation is reconciled with the many diverse and contradictory stimuli affecting cell growth. We discuss the current level of molecular understanding of the upstream components of the (m)TORC1 signalling pathway, recent progress on this key biochemical frontier, and the future studies necessary to establish a mechanistic understanding of this master-switch for eukaryotic cell growth.
Imseng S, Aylett CHS, Maier T, 2018, Architecture and activation of phosphatidylinositol 3-kinase related kinases, Current Opinion in Structural Biology, Vol: 49, Pages: 177-189, ISSN: 0959-440X
The phosphatidylinositol 3-kinase related protein kinases (PIKKs) are key to the regulation of a variety of eukaryotic cellular processes including DNA repair and growth regulation. While these massive proteins had long resisted structural analysis, recent advances in electron cryo-microscopy have now facilitated structural analysis of the major examples of PIKKs, including mTOR, DNA-PK, ATM, ATR and TRAPP/Tra1. In these PIKKs, the carboxy-terminal kinase domains and their proximal regions are structurally conserved. The structural organization of their extensive amino-terminal repeat regions, however, as well as their oligomeric organization and their interactions with accessory proteins, differ markedly amongst PIKKs. This architectural divergence provides the structural basis for the complex regulatory roles and functional diversity of PIKKs.
The mammalian target of rapamycin (mTOR) is a key protein kinase controlling cellular metabolism and growth. It is part of the two structurally and functionally distinct multiprotein complexes mTORC1 and mTORC2. Dysregulation of mTOR occurs in diabetes, cancer and neurological disease. We report the architecture of human mTORC2 at intermediate resolution, revealing a conserved binding site for accessory proteins on mTOR and explaining the structural basis for the rapamycin insensitivity of the complex.
Weisser M, Schäfer T, Leibundgut M, et al., 2017, Structural and functional insights into human re-initiation complexes., Molecular Cell, Vol: 67, Pages: 447-456.e7, ISSN: 1097-2765
After having translated short upstream open reading frames, ribosomes can re-initiate translation on the same mRNA. This process, referred to as re-initiation, controls the translation of a large fraction of mammalian cellular mRNAs, many of which are important in cancer. Key ribosomal binding proteins involved in re-initiation are the eukaryotic translation initiation factor 2D (eIF2D) or the homologous complex of MCT-1/DENR. We determined the structures of these factors bound to the human 40S ribosomal subunit in complex with initiator tRNA positioned on an mRNA start codon in the P-site using a combination of cryoelectron microscopy and X-ray crystallography. The structures, supported by biochemical experiments, reveal how eIF2D emulates the function of several canonical translation initiation factors by using three independent, flexibly connected RNA binding domains to simultaneously monitor codon-anticodon interactions in the ribosomal P-site and position the initiator tRNA.
Aylett CHS, Duggin IG, 2017, The Tubulin Superfamily in Archaea., Subcell Biochem, Vol: 84, Pages: 393-417, ISSN: 0306-0225
In comparison with bacteria and eukaryotes, the large and diverse group of microorganisms known as archaea possess a great diversity of cytoskeletal proteins, including members of the tubulin superfamily. Many species contain FtsZ, CetZ and even possible tubulins; however, some major taxonomic groups do not contain any member of the tubulin superfamily. Studies using the model archaeon, Halferax volcanii have recently been instrumental in defining the fundamental roles of FtsZ and CetZ in archaeal cell division and cell shape regulation. Structural studies of archaeal tubulin superfamily proteins provide a definitive contribution to the cytoskeletal field, showing which protein-types must have developed prior to the divergence of archaea and eukaryotes. Several regions of the globular core domain - the "signature" motifs - combine in the 3D structure of the common molecular fold to form the GTP-binding site. They are the most conserved sequence elements and provide the primary basis for identification of new superfamily members through homology searches. The currently well-characterised proteins also all share a common mechanism of GTP-dependent polymerisation, in which GTP molecules are sandwiched between successive subunits that are arranged in a head-to-tail manner. However, some poorly-characterised archaeal protein families retain only some of the signature motifs and are unlikely to be capable of dynamic polymerisation, since the promotion of depolymerisation by hydrolysis to GDP depends on contributions from both subunits that sandwich the nucleotide in the polymer.
Aylett CHS, Ban N, 2017, Eukaryotic aspects of translation initiation brought into focus., Philos Trans R Soc Lond B Biol Sci, Vol: 372
In all organisms, mRNA-directed protein synthesis is catalysed by ribosomes. Although the basic aspects of translation are preserved in all kingdoms of life, important differences are found in the process of translation initiation, which is rate-limiting and the most important step for translation regulation. While great strides had been taken towards a complete structural understanding of the initiation of translation in eubacteria, our understanding of the eukaryotic process, which includes numerous eukaryotic-specific initiation factors, was until recently limited owing to a lack of structural information. In this review, we discuss recent results in the field that provide an increasingly complete molecular description of the eukaryotic initiation process. The structural snapshots obtained using a range of methods now provide insights into the architecture of the initiation complex, start-codon recognition by the initiator tRNA and the process of subunit joining. Future advances will require both higher-resolution insights into previously characterized complexes and mapping of initiation factors that control translation on an additional level by interacting only peripherally or transiently with ribosomal subunits.This article is part of the themed issue 'Perspectives on the ribosome'.
Fink G, Aylett CHS, 2017, Tubulin-Like Proteins in Prokaryotic DNA Positioning., Subcell Biochem, Vol: 84, Pages: 323-356, ISSN: 0306-0225
A family of tubulin-related proteins (TubZs) has been identified in prokaryotes as being important for the inheritance of virulence plasmids of several pathogenic Bacilli and also being implicated in the lysogenic life cycle of several bacteriophages. Cell biological studies and reconstitution experiments revealed that TubZs function as prokaryotic cytomotive filaments, providing one-dimensional motive forces. Plasmid-borne TubZ filaments most likely transport plasmid centromeric complexes by depolymerisation, pulling on the plasmid DNA, in vitro. In contrast, phage-borne TubZ (PhuZ) pushes bacteriophage particles (virions) to mid cell by filament growth. Structural studies by both crystallography and electron cryo-microscopy of multiple proteins, both from the plasmid partitioning sub-group and the bacteriophage virion centring group of TubZ homologues, allow a detailed consideration of the structural phylogeny of the group as a whole, while complete structures of both crystallographic protofilaments at high resolution and fully polymerised filaments at intermediate resolution by cryo-EM have revealed details of the polymerisation behaviour of both TubZ sub-groups.
Target of rapamycin (TOR), a conserved protein kinase and central controller of cell growth, functions in two structurally and functionally distinct complexes: TORC1 and TORC2. Dysregulation of mammalian TOR (mTOR) signaling is implicated in pathologies that include diabetes, cancer, and neurodegeneration. We resolved the architecture of human mTORC1 (mTOR with subunits Raptor and mLST8) bound to FK506 binding protein (FKBP)-rapamycin, by combining cryo-electron microscopy at 5.9 angstrom resolution with crystallographic studies of Chaetomium thermophilum Raptor at 4.3 angstrom resolution. The structure explains how FKBP-rapamycin and architectural elements of mTORC1 limit access to the recessed active site. Consistent with a role in substrate recognition and delivery, the conserved amino-terminal domain of Raptor is juxtaposed to the kinase active site.
Duggin IG, Aylett CHS, Walsh JC, et al., 2015, CetZ tubulin-like proteins control archaeal cell shape, Nature, Vol: 519, Pages: 362-365, ISSN: 0028-0836
Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homologue FtsZ establishes the cytokinetic ring that constricts during cell division1,2. How such different roles of tubulin and FtsZ evolved is unknown. Studying Archaea may provide clues as these organisms share characteristics with Eukarya and Bacteria3. Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ X-ray crystal structures showed the FtsZ/tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of CetZ proteins in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of the microbial rod shape is to facilitate swimming.
Aylett CHS, Boehringer D, Erzberger JP, et al., 2015, Structure of a yeast 40S-eIF1-eIF1A-eIF3-eIF3j initiation complex., Nat Struct Mol Biol, Vol: 22, Pages: 269-271
Eukaryotic translation initiation requires cooperative assembly of a large protein complex at the 40S ribosomal subunit. We have resolved a budding yeast initiation complex by cryo-EM, allowing placement of prior structures of eIF1, eIF1A, eIF3a, eIF3b and eIF3c. Our structure highlights differences in initiation-complex binding to the ribosome compared to that of mammalian eIF3, demonstrates a direct contact between eIF3j and eIF1A and reveals the network of interactions between eIF3 subunits.
Erzberger JP, Stengel F, Pellarin R, et al., 2014, Molecular architecture of the 40S⋅eIF1⋅eIF3 translation initiation complex., Cell, Vol: 158, Pages: 1123-1135, ISSN: 0092-8674
Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA.
Aylett CHS, Izore T, Amos LA, et al., 2013, Structure of the Tubulin/FtsZ-Like Protein TubZ from Pseudomonas Bacteriophage Phi KZ, JOURNAL OF MOLECULAR BIOLOGY, Vol: 425, Pages: 2164-2173, ISSN: 0022-2836
Aylett CHS, Loewe J, 2012, Superstructure of the centromeric complex of TubZRC plasmid partitioning systems, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 109, Pages: 16522-16527, ISSN: 0027-8424
Gorrec F, Perisic O, Aylett C, et al., 2011, Macromolecular crystallization: robotics, procedures and Innovations, Publisher: INT UNION CRYSTALLOGRAPHY, Pages: C765-C765, ISSN: 2053-2733
Aylett CHS, Loewe J, Amos LA, 2011, NEW INSIGHTS INTO THE MECHANISMS OF CYTOMOTIVE ACTIN AND TUBULIN FILAMENTS, INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY, VOL 292, Editors: Jeon, Publisher: ELSEVIER ACADEMIC PRESS INC, Pages: 1-71, ISBN: 978-0-12-386033-0
Aylett CHS, Wang Q, Michie KA, et al., 2010, Filament structure of bacterial tubulin homologue TubZ, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 107, Pages: 19766-19771, ISSN: 0027-8424
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