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Journal articleWebb A, Allan F, Kelwick R, et al., 2022,
Specific Nucleic AcId Ligation for the detection of Schistosomes: SNAILS
, PLOS Neglected Tropical Diseases, Vol: 16, Pages: 1-19, ISSN: 1935-2727Schistosomiasis, also known as bilharzia or snail fever, is a debilitating neglected tropical disease (NTD), caused by parasitic trematode flatworms of the genus Schistosoma, that has an annual mortality rate of 280,000 people in sub-Saharan Africa alone. Schistosomiasis is transmitted via contact with water bodies that are home to the intermediate host snail which shed the infective cercariae into the water. Schistosome lifecycles are complex, and while not all schistosome species cause human disease, endemic regions also typically feature animal infecting schistosomes that can have broader economic and/or food security implications. Therefore, the development of species-specific Schistosoma detection technologies may help to inform evidence-based local environmental, food security and health systems policy making. Crucially, schistosomiasis disproportionally affects low- and middle-income (LMIC) countries and for that reason, environmental screening of water bodies for schistosomes may aid with the targeting of water, sanitation, and hygiene (WASH) interventions and preventive chemotherapy to regions at highest risk of schistosomiasis transmission, and to monitor the effectiveness of such interventions at reducing the risk over time. To this end, we developed a DNA-based biosensor termed Specific Nucleic AcId Ligation for the detection of Schistosomes or ‘SNAILS’. Here we show that ‘SNAILS’ enables species-specific detection from genomic DNA (gDNA) samples that were collected from the field in endemic areas.
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Journal articleHao M, Ye F, Jovanovic M, et al., 2022,
Structures of Class I and Class II transcription complexes reveal the molecular basis of RamA-dependent transcription activation
, Advanced Science, Vol: 9, Pages: 1-10, ISSN: 2198-3844Transcription activator RamA is linked to multidrug resistance of Klebsiella pneumoniae through controlling genes that encode efflux pumps (acrA) and porin-regulating antisense RNA (micF). In bacteria, σ70, together with activators, controls the majority of genes by recruiting RNA polymerase (RNAP) to the promoter regions. RNAP and σ70 form a holoenzyme that recognizes -35 and -10 promoter DNA consensus sites. Many activators bind upstream from the holoenzyme and can be broadly divided into two classes. RamA acts as a class I activator on acrA and class II activator on micF, respectively. The authors present biochemical and structural data on RamA in complex with RNAP-σ70 at the two promoters and the data reveal the molecular basis for how RamA assembles and interacts with core RNAP and activates transcription that contributes to antibiotic resistance. Further, comparing with CAP/TAP complexes reveals common and activator-specific features in activator binding and uncovers distinct roles of the two C-terminal domains of RNAP α subunit.
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Journal articleGarrido NDM, Orekhova M, Loong YTELW, et al., 2021,
Structure of the bacteriophage PhiKZ non-virion RNA polymerase (vol 49, pg 7732, 2021)
, Nucleic Acids Research, Vol: 49, Pages: 10806-10806, ISSN: 0305-1048 -
Journal articleGarrido NDM, Fu W, Ramlaul K, et al., 2021,
Direct transfer of electron microscopy samples to wetted carbon and graphene films via a support floatation block (vol 213, 107677, 2021)
, Journal of Structural Biology, Vol: 213, Pages: 1-2, ISSN: 1047-8477 -
Journal articledeYMartín Garrido N, Orekhova M, Lai Wan Loong YTE, et al., 2021,
Structure of the bacteriophage PhiKZ non-virion RNA polymerase
, Nucleic Acids Research, Vol: 49, Pages: 7732-7739, ISSN: 0305-1048Bacteriophage ΦKZ (PhiKZ) is the archetype of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa, is an opportunistic, intrinsically antibiotic resistant, pathogen that kills tens of thousands worldwide each year. ΦKZ is an incredibly interesting virus, expressing many systems that the host already possesses. On infection, it forms a ‘nucleus’, erecting a barrier around its genome to exclude host endonucleases and CRISPR-Cas systems. ΦKZ infection is independent of the host transcriptional apparatus. It expresses two different multi-subunit RNA polymerases (RNAPs): the virion RNAP (vRNAP) is injected with the viral DNA during infection to transcribe early genes, including those encoding the non-virion RNAP (nvRNAP), which transcribes all further genes. ΦKZ nvRNAP is formed by four polypeptides thought to represent homologues of the eubacterial β/β′ subunits, and a fifth with unclear homology, but essential for transcription. We have resolved the structure of ΦKZ nvRNAP to better than 3.0 Å, shedding light on its assembly, homology, and the biological role of the fifth subunit: it is an embedded, integral member of the complex, the position, structural homology and biochemical role of which imply that it has evolved from an ancestral homologue to σ-factor.
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Journal articleZhang X, Carver A, 2021,
Rad51 filament dynamics and its antagonistic modulators
, Seminars in Cell and Developmental Biology, Vol: 113, Pages: 3-13, ISSN: 1084-9521Rad51 recombinase is the central player in homologous recombination, the faithful repair pathway for double-strand breaks and key event during meiosis. Rad51 forms nucleoprotein filaments on single-stranded DNA, exposed by a double-strand break. These filaments are responsible for homology search and strand invasion, which lead to homology-directed repair. Due to its central roles in DNA repair and genome stability, Rad51 is modulated by multiple factors and post-translational modifications. In this review, we summarize our current understanding of the dynamics of Rad51 filaments, the roles of other factors and their modes of action in modulating key stages of Rad51 filaments: formation, stability and disassembly.
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Journal articleAylett C, de Martin Garrido N, Ramlaul K, 2021,
Preparation of sample support films in transmission electron microscopy using a support floatation block
, Jove-Journal of Visualized Experiments, Vol: 170, Pages: 1-11, ISSN: 1940-087XStructure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.
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Journal articleRiglar DT, Richmond DL, Potvin-Trottier L, et al., 2021,
Bacterial variability in the mammalian gut captured by a single-cell synthetic oscillator (vol 10, 4665, 2019)
, NATURE COMMUNICATIONS, Vol: 12, ISSN: 2041-1723 -
Journal articleRamlaul K, Fu W, Li H, et al., 2021,
Architecture of the Tuberous Sclerosis protein complex
, Journal of Molecular Biology, Vol: 433, ISSN: 0022-2836The 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.
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Journal articleAylett C, 2021,
Direct transfer of electron microscopy samples to wetted carbon and graphene films via a support floatation block
, Journal of Structural Biology, Vol: 213, ISSN: 1047-8477Support 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.
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