Publications by year
In Press
Neuhaus A, Selvaraj M, Salzer R, Langer JD, Kruse K, Sanders K, Daum B, Averhoff B, Gold VAM (In Press). A new twist on bacterial motility – two distinct type IV pili revealed by cryoEM.
Abstract:
A new twist on bacterial motility – two distinct type IV pili revealed by cryoEM
SummaryMany bacteria express flexible protein filaments on their surface that enable a variety of important cellular functions. Type IV pili are examples of such filaments and are comprised of a helical assembly of repeating pilin subunits. Type IV pili are involved in motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). They are therefore powerful structures that enable bacterial proliferation and genetic adaptation, potentially leading to the development of pathogenicity and antibiotic resistance. They are also targets for drug development.By a complement of experimental approaches, we show that the bacteriumThermus thermophilusproduces two different forms of type IV pilus. We have determined the structures of both and built atomic models. The structures answer key unresolved questions regarding the molecular architecture of type IV pili and identify a new type of pilin. We also delineate the roles of the two filaments in promoting twitching and natural transformation.
Abstract.
Gambelli L, Meyer B, McLaren M, Sanders K, Quax TEF, Gold V, Albers S-V, Daum B (In Press). Architecture and modular assembly of<i>Sulfolobus</i>S-layers revealed by electron cryo-tomography.
Abstract:
Architecture and modular assembly ofSulfolobusS-layers revealed by electron cryo-tomography
AbstractSurface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic and mechanical stability, the formation of a semi-permeable protective barrier around the cell, cell-cell interaction, as well as surface adhesion. Despite the central importance of the S-layer for archaeal life, their three-dimensional architecture is still poorly understood. Here we present the first detailed 3D electron cryo-microscopy maps of archaeal S-layers from three differentSulfolobusstrains. We were able to pinpoint the positions and determine the structure of the two subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer.
Abstract.
Conners R, León-Quezada RI, McLaren M, Bennett NJ, Daum B, Rakonjac J, Gold VAM (In Press). Cryo-electron microscopy of the f1 filamentous phage reveals a new paradigm in viral infection and assembly.
Abstract:
Cryo-electron microscopy of the f1 filamentous phage reveals a new paradigm in viral infection and assembly
AbstractPhages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ff has seen an extraordinary range of applications, including in phage display and nanotechnology. However, the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. Using cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods, we have determined the first structure of a filamentous virus, including the filament tips. Structure combined with mutagenesis was employed to identify domains of the phage that are important in bacterial attack and for release of new phage progeny. These data allow new models to be proposed for the phage lifecycle and will undoubtedly enable the development of novel biotechnological applications.
Abstract.
Gaines MC, Sivabalasarma S, Isupov MN, Haque RU, McLaren M, Hanus C, Gold VAM, Albers S-V, Daum B (In Press). CryoEM reveals the structure of an archaeal pilus involved in twitching motility.
Abstract:
CryoEM reveals the structure of an archaeal pilus involved in twitching motility
AbstractAmongst the major archaeal filament types, several have been shown to closely resemble bacterial homologues of the Type IV pili (T4P). WithinSulfolobales,member species encode for three types of T4P, namely the archaellum, the UV-inducible pilus (Uvp) and the archaeal adhesive pilus (Aap). Whereas the archaellum functions primarily in swimming motility, and the Uvp in UV-induced cell aggregation and DNA-exchange, the Aap plays an important role in adhesion and twitching motility. All previously solved Aap appear to have almost identical helical structures. Here, we present a cryoEM structure of the Aap of the archaeal model organismSulfolobus acidocaldarius.We identify the component subunit as AapB and find that while its structure follows the canonical T4P blueprint, it adopts three distinct conformations within the pilus. The tri-conformer Aap structure that we describe challenges our current understanding of pilus structure and sheds new light on the principles of twitching motility.
Abstract.
Daum B, Gaines M, Sivabalasarma S, Isupov M, Haque R, McLaren M, Hanus C, Gold V, Albers S-V (In Press). CryoEM reveals the structure of an archaeal pilus involved in twitching motility.
Abstract:
CryoEM reveals the structure of an archaeal pilus involved in twitching motility.
Abstract
. Amongst the major archaeal filament types, several have been shown to closely resemble bacterial homologues of the Type IV pili (T4P). Within Sulfolobales, member species encode for three types of T4P, namely the archaellum, the UV-inducible pilus (Uvp) and the archaeal adhesive pilus (Aap). Whereas the archaellum functions primarily in swimming motility, and the Uvp in UV-induced cell aggregation and DNA-exchange, the Aap plays an important role in adhesion and twitching motility. All previously solved Aap appear to have almost identical helical structures. Here, we present a cryoEM structure of the Aap of the archaeal model organism Sulfolobus acidocaldarius. We identify the component subunit as AapB and find that while its structure follows the canonical T4P blueprint, it adopts three distinct conformations within the pilus. The tri-conformer Aap structure that we describe challenges our current understanding of pilus structure and sheds new light on the principles of twitching motility.
Abstract.
Conners R, McLaren M, Łapińska U, Sanders K, Stone MRL, Blaskovich MAT, Pagliara S, Daum B, Rakonjac J, Gold VAM, et al (In Press). CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage.
Abstract:
CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage
AbstractThe Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.
Abstract.
Troman L, Alvira S, Daum B, Gold VAM, Collinson I (In Press). INTERACTION OF THE PERIPLASMIC CHAPERONE SURA WITH THE INNER MEMBRANE PROTEIN SECRETION (SEC) MACHINERY.
Abstract:
INTERACTION OF THE PERIPLASMIC CHAPERONE SURA WITH THE INNER MEMBRANE PROTEIN SECRETION (SEC) MACHINERY
ABSTRACTGram-negative bacteria are surrounded by two protein-rich membranes with a peptidoglycan layer sandwiched between them. Together they form the envelope (or cell wall), crucial for energy production, lipid biosynthesis, structural integrity, and for protection against the physical and chemical environmental challenges. To achieve envelope biogenesis, periplasmic and outer-membrane proteins (OMPs) must be transported from the cytosol and through the inner-membrane, via the ubiquitous SecYEG protein-channel. Emergent proteins either fold in the periplasm or cross the peptidoglycan (PG) layer towards the outer-membrane for insertion through the β-barrel assembly machinery (BAM). Trafficking of hydrophobic proteins through the periplasm is particularly treacherous given the high protein density and the absence of energy (ATP or chemiosmotic potential). Numerous molecular chaperones assist in the prevention and recovery from aggregation, and of these SurA is known to interact with BAM, facilitating delivery to the outer-membrane. However, it is unclear how proteins emerging from the Sec-machinery are received and protected from aggregation and proteolysis prior to an interaction with SurA. Through biochemical analysis and electron microscopy we demonstrate the binding capabilities of the unoccupied and substrate-engaged SurA to the inner-membrane translocation machinery complex of SecYEG-SecDF-YidC – aka the holo-translocon (HTL). Supported by AlphaFold predictions, we suggest a role for periplasmic domains of SecDF in chaperone recruitment to the protein translocation exit site in SecYEG. We propose that this immediate interaction with a recruited chaperone helps to prevent aggregation and degradation of nascent envelope proteins, facilitating their safe passage to the periplasm and outer-membrane.
Abstract.
Alvira S, Watkins DW, Troman L, Allen WJ, Lorriman J, Degliesposti G, Cohen EJ, Beeby M, Daum B, Gold VAM, et al (In Press). Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis.
Abstract:
Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis
SUMMARYThe outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent – hydrophobic β-barrelOuter-MembraneProteins (OMPs) – are secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperonese.g.SurA, which prevent aggregation. OMPs are then offloaded to the β-BarrelAssemblyMachinery (BAM) in the outer-membrane for insertion and folding. We show theHolo-TransLocon (HTL: an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane ‘insertase’ YidC) contacts SurA and BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Our results show the trans-membrane proton-motive-force (PMF) acts at distinct stages of protein secretion: for SecA-driven translocation across the inner-membrane through SecYEG; and to communicate conformational changesviaSecDF to the BAM machinery. The latter presumably ensures efficient passage of OMPs. These interactions provide insights of inter-membrane organisation, the importance of which is becoming increasingly apparent.
Abstract.
Gambelli L, Isupov MN, Conners R, McLaren M, Bellack A, Gold V, Rachel R, Daum B (In Press). New insights into the architecture and dynamics of archaella.
Abstract:
New insights into the architecture and dynamics of archaella
AbstractArchaea swim by means of a unique molecular machine called the archaellum. The archaellum consists of an ATP-powered intracellular motor that drives the rotation of an extracellular filament, allowing the cell to rapidly propel itself through liquid media.The archaellum filament comprises multiple copies of helically organised subunits named archaellins. While in many species several archaellin homologs are encoded in the same operon, structural studies conducted to date have suggested that archaella consist of only one protein species. Thus, the role of the remaining archaellin genes remains elusive.Here we present the structure of the Methanocaldococcus villosus archaellum filament at 3.08 Å resolution. We find that the filament is composed of two alternating archaellins - ArlB1 and ArlB2, suggesting that the architecture and assembly of archaella is more complex than previously thought. Moreover, we identify two major structural elements that enable the archaellum filament to move.Our findings provide new insights into archaeal motility and challenge the current view on the archaellum architecture and assembly.
Abstract.
Gambelli L, McLaren M, Conners R, Sanders K, Gaines MC, Clark L, Gold V, Kattnig D, Sikora M, Hanus C, et al (In Press). Structure of the two-component S-layer of the archaeon <i>Sulfolobus acidocaldarius</i>.
Abstract:
Structure of the two-component S-layer of the archaeon Sulfolobus acidocaldarius
AbstractSurface layers (S-layers) are resilient two-dimensional protein lattices that encapsulate many bacteria and most archaea. In archaea, S-layers usually form the only structural component of the cell wall and thus act as the final frontier between the cell and its environment. Therefore, S-layers are crucial for supporting microbial life. Notwithstanding their importance, little is known about archaeal S-layers at the atomic level. Here, we combined single particle cryo electron microscopy (cryoEM), cryo electron tomography (cryoET) and Alphafold2 predictions to generate an atomic model of the two-component S-layer of Sulfolobus acidocaldarius. The outer component of this S-layer (SlaA) is a flexible, highly glycosylated, and stable protein. Together with the inner and membrane-bound component (SlaB), they assemble into a porous and interwoven lattice. We hypothesize that jackknife-like conformational changes, as well as pH-induced alterations in the surface charge of SlaA, play important roles in S-layer assembly.
Abstract.
2023
Conners R, León-Quezada RI, McLaren M, Bennett NJ, Daum B, Rakonjac J, Gold VAM (2023). Cryo-electron microscopy of the f1 filamentous phage reveals insights into viral infection and assembly.
Nature Communications,
14(1).
Abstract:
Cryo-electron microscopy of the f1 filamentous phage reveals insights into viral infection and assembly
AbstractPhages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ffs have seen an extraordinary range of applications, yet the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. In this work, we use cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods to determine a structure of a filamentous virus including the tips. We show that structure combined with mutagenesis can identify phage domains that are important in bacterial attack and for release of new progeny, allowing new models to be proposed for the phage lifecycle.
Abstract.
Buzzard E, McLaren M, Bragoszewski P, Brancaccio A, Ford H, Daum B, Kuwabara P, Collinson I, Gold VAM (2023). Cryo-electron tomography of<i>C. elegans</i>mitochondria reveals how the ATP synthase dimer interface shapes crista membranes.
Abstract:
Cryo-electron tomography ofC. elegansmitochondria reveals how the ATP synthase dimer interface shapes crista membranes
Mitochondrial ATP synthases form rows of dimers, which induce membrane curvature to give cristae their characteristic lamellar or tubular morphology. The angle formed between the central stalks of ATP synthase dimers varies between species. Using cryo-electron tomography and sub-tomogram averaging, we determined the structure of the ATP synthase dimer from the nematode worm C. elegans. We showed that the angle formed by the ATP synthase dimer is different to previously determined structures. The consequences of species-specific differences at the dimer interface were investigated by comparing mitochondrial morphology between C. elegans and S. cerevisiae. We reveal that a larger ATP synthase dimer angle in C. elegans is consequent with more lamellar (flatter) cristae compared to yeast. The underlying cause of this difference was investigated by generating an atomic model of the C. elegans ATP synthase dimer by homology modelling and comparing it to an existing S. cerevisiae structure. We reveal extensions and rearrangements of C. elegans subunits that maintain the dimer interface. We propose that increased dimer angles resulting in flatter cristae could provide an energetic advantage for species that inhabit variable-oxygen environments. Significance Statement ATP synthase, the world’s smallest rotary motor, generates the energy required for all living processes. The universal formation of ATP synthase dimers in mitochondria induces membrane curvature and cristae formation, generating the famously convoluted inner mitochondrial membrane that maximises ATP production. Intriguingly, the angle between ATP synthase dimers varies between species. Here, we report the discovery of a novel ATP synthase dimer angle in mitochondria from the nematode worm C. elegans, identify a relationship between dimer angle and crista membrane morphology, and exploit AlphaFold homology modelling to investigate associated structural changes. In summary, we have taken steps towards understanding the importance of subunit composition at the ATP synthase dimer interface, providing insights into the origins of this evolutionary divergence.
Abstract.
McLaren M, Conners R, Isupov MN, Gil-Díez P, Gambelli L, Gold VAM, Walter A, Connell SR, Williams B, Daum B, et al (2023). CryoEM reveals that ribosomes in microsporidian spores are locked in a dimeric hibernating state.
Nature MicrobiologyAbstract:
CryoEM reveals that ribosomes in microsporidian spores are locked in a dimeric hibernating state
AbstractTranslational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of electron cryo-tomography and single-particle electron cryo-microscopy. We show that microsporidian spores contain hibernating ribosomes that are locked in a dimeric (100S) state, which is formed by a unique dimerization mechanism involving the beak region. The ribosomes within the dimer are fully assembled, suggesting that they are ready to be activated once the host cell is invaded. This study provides structural evidence for dimerization acting as a mechanism for ribosomal hibernation in microsporidia, and therefore demonstrates that eukaryotes utilize this mechanism in translational control.
Abstract.
Troman L, Alvira S, Daum B, Gold VAM, Collinson I (2023). Interaction of the periplasmic chaperone SurA with the inner membrane protein secretion (SEC) machinery.
Biochemical Journal,
480(4), 283-296.
Abstract:
Interaction of the periplasmic chaperone SurA with the inner membrane protein secretion (SEC) machinery
Gram-negative bacteria are surrounded by two protein-rich membranes with a peptidoglycan layer sandwiched between them. Together they form the envelope (or cell wall), crucial for energy production, lipid biosynthesis, structural integrity, and for protection against physical and chemical environmental challenges. To achieve envelope biogenesis, periplasmic and outer-membrane proteins (OMPs) must be transported from the cytosol and through the inner-membrane, via the ubiquitous SecYEG protein–channel. Emergent proteins either fold in the periplasm or cross the peptidoglycan (PG) layer towards the outer-membrane for insertion through the β-barrel assembly machinery (BAM). Trafficking of hydrophobic proteins through the periplasm is particularly treacherous given the high protein density and the absence of energy (ATP or chemiosmotic potential). Numerous molecular chaperones assist in the prevention and recovery from aggregation, and of these SurA is known to interact with BAM, facilitating delivery to the outer-membrane. However, it is unclear how proteins emerging from the Sec-machinery are received and protected from aggregation and proteolysis prior to an interaction with SurA. Through biochemical analysis and electron microscopy we demonstrate the binding capabilities of the unoccupied and substrate-engaged SurA to the inner-membrane translocation machinery complex of SecYEG–SecDF–YidC — aka the holo-translocon (HTL). Supported by AlphaFold predictions, we suggest a role for periplasmic domains of SecDF in chaperone recruitment to the protein translocation exit site in SecYEG. We propose that this immediate interaction with the enlisted chaperone helps to prevent aggregation and degradation of nascent envelope proteins, facilitating their safe passage to the periplasm and outer-membrane.
Abstract.
2022
McLaren M, Gil-Diez P, Isupov M, Conners R, Gambelli L, Gold V, Walter A, Connell S, Williams B, Daum B, et al (2022). <i>In situ</i> structure of a dimeric hibernating ribosome from a eukaryotic intracellular pathogen.
Abstract:
In situ structure of a dimeric hibernating ribosome from a eukaryotic intracellular pathogen
Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of cryo-electron tomography (cryoET) and single particle cryoEM. We show that microsporidian spores contain ribosomes primed for host cell invasion and thus shed new light on the infection mechanism of this important pathogen. Prior to host infection, virtually all ribosomes are locked in the 100 S dimeric state, which appears to be formed by a unique dimerization mechanism that is distinct from its bacterial counterparts. Within the dimer, the hibernation factor MDF1 is bound within the E site, locking the L1 stalk in a closed conformation, and thus preventing the translation of mRNAs to polypeptides.
Abstract.
Gambelli L, Isupov MN, Conners R, McLaren M, Bellack A, Gold V, Rachel R, Daum B (2022). An archaellum filament composed of two alternating subunits.
Nature Communications,
13(1).
Abstract:
An archaellum filament composed of two alternating subunits
AbstractArchaea use a molecular machine, called the archaellum, to swim. The archaellum consists of an ATP-powered intracellular motor that drives the rotation of an extracellular filament composed of multiple copies of proteins named archaellins. In many species, several archaellin homologs are encoded in the same operon; however, previous structural studies indicated that archaellum filaments mainly consist of only one protein species. Here, we use electron cryo-microscopy to elucidate the structure of the archaellum from Methanocaldococcus villosus at 3.08 Å resolution. The filament is composed of two alternating archaellins, suggesting that the architecture and assembly of archaella is more complex than previously thought. Moreover, we identify structural elements that may contribute to the filament’s flexibility.
Abstract.
Gaines MC, Isupov MN, Sivabalasarma S, Haque RU, McLaren M, Tripp P, Neuhaus A, Gold V, Albers S-V, Daum B, et al (2022). Donor strand complementation, isopeptide bonds and glycosylation reinforce highly resilient archaeal thread filaments.
Gaines MC, Isupov MN, Sivabalasarma S, Haque RU, McLaren M, Mollat CL, Tripp P, Neuhaus A, Gold VAM, Albers S-V, et al (2022). Electron cryo-microscopy reveals the structure of the archaeal thread filament.
Nature Communications,
13(1).
Abstract:
Electron cryo-microscopy reveals the structure of the archaeal thread filament
AbstractPili are filamentous surface extensions that play roles in bacterial and archaeal cellular processes such as adhesion, biofilm formation, motility, cell-cell communication, DNA uptake and horizontal gene transfer. The model archaeaon Sulfolobus acidocaldarius assembles three filaments of the type-IV pilus superfamily (archaella, archaeal adhesion pili and UV-inducible pili), as well as a so-far uncharacterised fourth filament, named “thread”. Here, we report on the cryo-EM structure of the archaeal thread. The filament is highly glycosylated and consists of subunits of the protein Saci_0406, arranged in a head-to-tail manner. Saci_0406 displays structural similarity, but low sequence homology, to bacterial type-I pilins. Thread subunits are interconnected via donor strand complementation, a feature reminiscent of bacterial chaperone-usher pili. However, despite these similarities in overall architecture, archaeal threads appear to have evolved independently and are likely assembled by a distinct mechanism.
Abstract.
Gambelli L, Isupov MN, Daum B (2022). Escaping the symmetry trap in helical reconstruction.
Faraday Discussions,
240, 303-311.
Abstract:
Escaping the symmetry trap in helical reconstruction
In this study, we reflect on widely-used helical processing routines in cryoEM and suggest a new workflow capable of uncovering heterogeneities and complex symmetries that could otherwise be overlooked.
Abstract.
Bakker SE, Bhella D, Brescia R, Bullough P, Clare DK, Daum B, Frank RAW, Gold VAM, Jackson Hirst I, Kühlbrandt W, et al (2022). Pushing the limits in single particle cryo-EM: general discussion. Faraday Discussions, 240, 312-322.
Al-Otaibi N, Baatsen P, Bhella D, Brescia R, Bullough P, Daum B, de Bruin R, Frank R, Kühlbrandt W, López-Iglesias C, et al (2022). Tomographic analysis, CLEM: general discussion. Faraday Discussions, 240(0), 142-151.
Nuno de Sousa Machado J, Albers S-V, Daum B (2022). Towards Elucidating the Rotary Mechanism of the Archaellum Machinery.
Front Microbiol,
13Abstract:
Towards Elucidating the Rotary Mechanism of the Archaellum Machinery.
Motile archaea swim by means of a molecular machine called the archaellum. This structure consists of a filament attached to a membrane-embedded motor. The archaellum is found exclusively in members of the archaeal domain, but the core of its motor shares homology with the motor of type IV pili (T4P). Here, we provide an overview of the different components of the archaellum machinery and hypothetical models to explain how rotary motion of the filament is powered by the archaellum motor.
Abstract.
Author URL.
2021
Conners R, McLaren M, Łapińska U, Sanders K, Stone MRL, Blaskovich MAT, Pagliara S, Daum B, Rakonjac J, Gold VAM, et al (2021). CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage.
Nature Communications,
12(1).
Abstract:
CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage
AbstractThe Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.
Abstract.
Gambelli L, Mesman R, Versantvoort W, Diebolder CA, Engel A, Evers W, Jetten MSM, Pabst M, Daum B, van Niftrik L, et al (2021). The Polygonal Cell Shape and Surface Protein Layer of Anaerobic Methane-Oxidizing Methylomirabilislanthanidiphila Bacteria.
Frontiers in Microbiology,
12Abstract:
The Polygonal Cell Shape and Surface Protein Layer of Anaerobic Methane-Oxidizing Methylomirabilislanthanidiphila Bacteria
Methylomirabilis bacteria perform anaerobic methane oxidation coupled to nitrite reduction via an intra-aerobic pathway, producing carbon dioxide and dinitrogen gas. These diderm bacteria possess an unusual polygonal cell shape with sharp ridges that run along the cell body. Previously, a putative surface protein layer (S-layer) was observed as the outermost cell layer of these bacteria. We hypothesized that this S-layer is the determining factor for their polygonal cell shape. Therefore, we enriched the S-layer from M. lanthanidiphila cells and through LC-MS/MS identified a 31 kDa candidate S-layer protein, mela_00855, which had no homology to any other known protein. Antibodies were generated against a synthesized peptide derived from the mela_00855 protein sequence and used in immunogold localization to verify its identity and location. Both on thin sections of M. lanthanidiphila cells and in negative-stained enriched S-layer patches, the immunogold localization identified mela_00855 as the S-layer protein. Using electron cryo-tomography and sub-tomogram averaging of S-layer patches, we observed that the S-layer has a hexagonal symmetry. Cryo-tomography of whole cells showed that the S-layer and the outer membrane, but not the peptidoglycan layer and the cytoplasmic membrane, exhibited the polygonal shape. Moreover, the S-layer consisted of multiple rigid sheets that partially overlapped, most likely giving rise to the unique polygonal cell shape. These characteristics make the S-layer of M. lanthanidiphila a distinctive and intriguing case to study.
Abstract.
2020
Gambelli L, Meyer BH, McLaren M, Sanders K, Quax TEF, Gold VAM, Albers SV, Daum B (2020). Architecture and modular assembly of Sulfolobus S-layers revealed by electron cryotomography.
Proceedings of the National Academy of Sciences of the United States of America,
116(50), 25278-25286.
Abstract:
Architecture and modular assembly of Sulfolobus S-layers revealed by electron cryotomography
© 2019 National Academy of Sciences. All rights reserved. Surface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic, and mechanical stability, the formation of a semipermeable protective barrier around the cell, and cell-cell interaction, as well as surface adhesion. Despite the central importance of S-layers for archaeal life, their 3-dimensional (3D) architecture is still poorly understood. Here we present detailed 3D electron cryomicroscopy maps of archaeal S-layers from 3 different Sulfolobus strains. We were able to pinpoint the positions and determine the structure of the 2 subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer.
Abstract.
Neuhaus A, Selvaraj M, Salzer R, Langer JD, Kruse K, Kirchner L, Sanders K, Daum B, Averhoff B, Gold VAM, et al (2020). Cryo-electron microscopy reveals two distinct type IV pili assembled by the same bacterium.
Nature Communications,
11(1).
Abstract:
Cryo-electron microscopy reveals two distinct type IV pili assembled by the same bacterium
Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus (‘wide’ and ‘narrow’), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.
Abstract.
Alvira S, Watkins DW, Troman L, Allen WJ, Lorriman JS, Degliesposti G, Cohen EJ, Beeby M, Daum B, Gold VAM, et al (2020). Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis.
eLife,
9Abstract:
Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis
The outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent – hydrophobic β-barrelOuter-MembraneProteins (OMPs) – are first secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperones, for example SurA, which prevent aggregation. OMPs are then offloaded to the β-BarrelAssemblyMachinery (BAM) in the outer-membrane for insertion and folding. We show theHolo-TransLocon (HTL) – an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane ‘insertase’ YidC – contacts BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Furthermore, the proton-motive force (PMF) across the inner-membrane acts at distinct stages of protein secretion: (1) SecA-driven translocation through SecYEG and (2) communication of conformational changes via SecDF across the periplasm to BAM. The latter presumably drives efficient passage of OMPs. These interactions provide insights of inter-membrane organisation and communication, the importance of which is becoming increasingly apparent.
Abstract.
2018
Quax TEF, Daum B (2018). Structure and assembly mechanism of virus-associated pyramids.
Biophys Rev,
10(2), 551-557.
Abstract:
Structure and assembly mechanism of virus-associated pyramids.
Viruses have developed intricate molecular machines to infect, replicate within and escape from their host cells. Perhaps one of the most intriguing of these mechanisms is the pyramidal egress structure that has evolved in archaeal viruses, such as SIRV2 or STIV1. The structure and mechanism of these virus-associated pyramids (VAPs) has been studied by cryo-electron tomography and complementary biochemical techniques, revealing that VAPs are formed by multiple copies of a virus-encoded 10-kDa protein (PVAP) that integrate into the cell membrane and assemble into hollow, sevenfold symmetric pyramids. In this process, growing VAPs puncture the protective surface layer and ultimately open to release newly replicated viral particles into the surrounding medium. PVAP has the striking capability to spontaneously integrate and self-assemble into VAPs in biological membranes of the archaea, bacteria and eukaryotes. This renders the VAP a universal membrane remodelling system. In this review, we provide an overview of the VAP structure and assembly mechanism and discuss the possible use of VAPs in nano-biotechnology.
Abstract.
Author URL.
Daum B, Gold V (2018). Twitch or swim: Towards the understanding of prokaryotic motion based on the type IV pilus blueprint.
Biological Chemistry,
399(7), 799-808.
Abstract:
Twitch or swim: Towards the understanding of prokaryotic motion based on the type IV pilus blueprint
Bacteria and archaea are evolutionarily distinct prokaryotes that diverged from a common ancestor billions of years ago. However, both bacteria and archaea assemble long, helical protein filaments on their surface through a machinery that is conserved at its core. In both domains of life, the filaments are required for a diverse array of important cellular processes including cell motility, adhesion, communication and biofilm formation. In this review, we highlight the recent structures of both the type IV pilus machinery and the archaellum determined in situ. We describe the current level of functional understanding and discuss how this relates to the pressures facing bacteria and archaea throughout evolution.
Abstract.
2017
Davies K, Muehleip A, Blum T, Daum B, Anselmi C, Faraldo-Gómez J, Kühlbrandt W (2017). Structure and in Situ Organization of ATP Sythase and Respiratory Chain Complexes. Biophysical Journal, 112(3), 179a-180a.
Daum B, Vonck J, Bellack A, Chaudhury P, Reichelt R, Albers SV, Rachel R, Kühlbrandt W (2017). Structure and in situ organisation of the pyrococcus furiosus archaellum machinery.
eLife,
6Abstract:
Structure and in situ organisation of the pyrococcus furiosus archaellum machinery
The archaellum is the macromolecular machinery that Archaea use for propulsion or surface adhesion, enabling them to proliferate and invade new territories. The molecular composition of the archaellum and of the motor that drives it appears to be entirely distinct from that of the functionally equivalent bacterial flagellum and flagellar motor. Yet, the structure of the archaellum machinery is scarcely known. Using combined modes of electron cryo-microscopy (cryoEM), we have solved the structure of the Pyrococcus furiosus archaellum filament at 4.2 Å resolution and visualise the architecture and organisation of its motor complex in situ. This allows us to build a structural model combining the archaellum and its motor complex, paving the way to a molecular understanding of archaeal swimming motion.
Abstract.
2016
Daum B, Auerswald A, Gruber T, Hause G, Balbach J, Kühlbrandt W, Meister A (2016). Supramolecular organization of the human N-BAR domain in shaping the sarcolemma membrane. Journal of Structural Biology, 194(3), 375-382.
2015
Perras AK, Daum B, Ziegler C, Takahashi LK, Ahmed M, Wanner G, Klingl A, Leitinger G, Kolb-Lenz D, Gribaldo S, et al (2015). S-layers at second glance? Altiarchaeal grappling hooks (hami) resemble archaeal S-layer proteins in structure and sequence. Frontiers in Microbiology, 6
2014
Daum B, Quax TEF, Sachse M, Mills DJ, Reimann J, Yildiz Ö, Häder S, Saveanu C, Forterre P, Albers S-V, et al (2014). Self-assembly of the general membrane-remodeling protein PVAP into sevenfold virus-associated pyramids.
Proceedings of the National Academy of Sciences,
111(10), 3829-3834.
Abstract:
Self-assembly of the general membrane-remodeling protein PVAP into sevenfold virus-associated pyramids
Significance
.
. The
. Sulfolobus islandicus
. rod-shaped virus 2 (SIRV2) has developed unique mechanisms to penetrate the plasma membrane and S-layer of its host
. Sulfolobus islandicus
. in order to leave the cell after replication. SIRV2 encodes the 10-kDa protein PVAP, which assembles into sevenfold symmetric virus-associated pyramids (VAPs) in the host cell plasma membrane. Toward the end of the viral replication cycle, these VAPs open to form pores through the plasma membrane and S-layer, allowing viral egress. Here we show that PVAP inserts spontaneously and forms VAPs in any kind of biological membrane. By electron cryotomography we have obtained a 3D map of the VAP and present a model describing the assembly of PVAP into VAPs. Our findings open new avenues for a large variety of biotechnological applications.
.
Abstract.
Davies KM, Daum B, Gold VAM, Mühleip AW, Brandt T, Blum TB, Mills DJ, Kühlbrandt W (2014). Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography. Journal of Visualized Experiments(91).
Davies KM, Daum B, Gold VAM, Mühleip AW, Brandt T, Blum TB, Mills DJ, Kühlbrandt W (2014). Visualization of ATP synthase dimers in mitochondria by electron cryo-tomography.
J Vis Exp(91).
Abstract:
Visualization of ATP synthase dimers in mitochondria by electron cryo-tomography.
Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.
Abstract.
Author URL.
2013
Daum B, Walter A, Horst A, Osiewacz HD, Kühlbrandt W (2013). Age-dependent dissociation of ATP synthase dimers and loss of inner-membrane cristae in mitochondria.
Proceedings of the National Academy of Sciences,
110(38), 15301-15306.
Abstract:
Age-dependent dissociation of ATP synthase dimers and loss of inner-membrane cristae in mitochondria
. Aging is one of the most fundamental, yet least understood biological processes that affect all forms of eukaryotic life. Mitochondria are intimately involved in aging, but the underlying molecular mechanisms are largely unknown. Electron cryotomography of whole mitochondria from the aging model organism
. Podospora anserina
. revealed profound age-dependent changes in membrane architecture. With increasing age, the typical cristae disappear and the inner membrane vesiculates. The ATP synthase dimers that form rows at the cristae tips dissociate into monomers in inner-membrane vesicles, and the membrane curvature at the ATP synthase inverts. Dissociation of the ATP synthase dimer may involve the peptidyl prolyl isomerase cyclophilin D. Finally, the outer membrane ruptures near large contact-site complexes, releasing apoptogens into the cytoplasm. Inner-membrane vesiculation and dissociation of ATP synthase dimers would impair the ability of mitochondria to supply the cell with sufficient ATP to maintain essential cellular functions.
.
Abstract.
Quemin ERJ, Lucas S, Daum B, Quax TEF, Kühlbrandt W, Forterre P, Albers S-V, Prangishvili D, Krupovic M (2013). First Insights into the Entry Process of Hyperthermophilic Archaeal Viruses.
Journal of Virology,
87(24), 13379-13385.
Abstract:
First Insights into the Entry Process of Hyperthermophilic Archaeal Viruses
ABSTRACTA decisive step in a virus infection cycle is the recognition of a specific receptor present on the host cell surface, subsequently leading to the delivery of the viral genome into the cell interior. Until now, the early stages of infection have not been thoroughly investigated for any virus infecting hyperthermophilic archaea. Here, we present the first study focusing on the primary interactions between the archaeal rod-shaped virusSulfolobus islandicus rod-shaped virus 2(SIRV2) (familyRudiviridae) and its hyperthermoacidophilic host,S. islandicus. We show that SIRV2 adsorption is very rapid, with the majority of virions being irreversibly bound to the host cell within 1 min. We utilized transmission electron microscopy and whole-cell electron cryotomography to demonstrate that SIRV2 virions specifically recognize the tips of pilus-like filaments, which are highly abundant on the host cell surface. Following the initial binding, the viral particles are found attached to the sides of the filaments, suggesting a movement along these appendages toward the cell surface. Finally, we also show that SIRV2 establishes superinfection exclusion, a phenomenon not previously described for archaeal viruses.
Abstract.
Davies KM, Daum B (2013). Role of cryo-ET in membrane bioenergetics research.
Biochemical Society Transactions,
41(5), 1227-1234.
Abstract:
Role of cryo-ET in membrane bioenergetics research
To truly understand bioenergetic processes such as ATP synthesis, membrane-bound substrate transport or flagellar rotation, systems need to be analysed in a cellular context. Cryo-ET (cryo-electron tomography) is an essential part of this process, as it is currently the only technique which can directly determine the spatial organization of proteins at the level of both the cell and the individual protein complexes. The need to assess bioenergetic processes at a cellular level is becoming more and more apparent with the increasing interest in mitochondrial diseases. In recent years, cryo-ET has contributed significantly to our understanding of the molecular organization of mitochondria and chloroplasts. The present mini-review first describes the technique of cryo-ET and then discusses its role in membrane bioenergetics specifically in chloroplasts and mitochondrial research.
Abstract.
2012
Davies KM, Anselmi C, Daum B, Faraldo-Gómez JD, Kühlbrandt W (2012). In situ structure of the mitochondrial F1Fo ATP synthase dimer and its role in shaping membrane morphology. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1817, s11-s12.
Daum B, Walter A, Horst A, Heide H, Werner A, Brandt U, Osiewacz HD, Kühlbrandt W (2012). Macromolecular reorganization of mitochondria during aging. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1817
Davies KM, Daum B, Kühlbrandt W, Anselmi C, Faraldo-Gómez J (2012). Structure of the Mitochondrial ATP Synthase and its Role in Shaping Mitochondria Cristae. Microscopy and Microanalysis, 18(S2), 56-57.
2011
Sommer MS, Daum B, Gross LE, Weis BLM, Mirus O, Abram L, Maier U-G, Kühlbrandt W, Schleiff E (2011). Chloroplast Omp85 proteins change orientation during evolution.
Proceedings of the National Academy of Sciences,
108(33), 13841-13846.
Abstract:
Chloroplast Omp85 proteins change orientation during evolution
The majority of outer membrane proteins (OMPs) from Gram-negative bacteria and many of mitochondria and chloroplasts are β-barrels. Insertion and assembly of these proteins are catalyzed by the Omp85 protein family in a seemingly conserved process. All members of this family exhibit a characteristic N-terminal polypeptide-transport–associated (POTRA) and a C-terminal 16-stranded β-barrel domain. In plants, two phylogenetically distinct and essential Omp85's exist in the chloroplast outer membrane, namely Toc75-III and Toc75-V. Whereas Toc75-V, similar to the mitochondrial Sam50, is thought to possess the original bacterial function, its homolog, Toc75-III, evolved to the pore-forming unit of the TOC translocon for preprotein import. In all current models of OMP biogenesis and preprotein translocation, a topology of Omp85 with the POTRA domain in the periplasm or intermembrane space is assumed. Using self-assembly GFP-based in vivo experiments and in situ topology studies by electron cryotomography, we show that the POTRA domains of both Toc75-III and Toc75-V are exposed to the cytoplasm. This unexpected finding explains many experimental observations and requires a reevaluation of current models of OMP biogenesis and TOC complex function.
Abstract.
Daum B, Kuhlbrandt W (2011). Electron tomography of plant thylakoid membranes. Journal of Experimental Botany, 62(7), 2393-2402.
Davies KM, Strauss M, Daum B, Kief JH, Osiewacz HD, Rycovska A, Zickermann V, Kühlbrandt W (2011). Macromolecular organization of ATP synthase and complex I in whole mitochondria.
Proceedings of the National Academy of Sciences,
108(34), 14121-14126.
Abstract:
Macromolecular organization of ATP synthase and complex I in whole mitochondria
We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance betweenF1heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species exceptSaccharomyces cerevisiaeand identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.
Abstract.
2010
Daum B, Nicastro D, Austin J, McIntosh JR, Kühlbrandt W (2010). Arrangement of Photosystem II and ATP Synthase in Chloroplast Membranes of Spinach and Pea
.
The Plant Cell,
22(4), 1299-1312.
Abstract:
Arrangement of Photosystem II and ATP Synthase in Chloroplast Membranes of Spinach and Pea
Abstract
. We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.
Abstract.
Brust D, Daum B, Breunig C, Hamann A, Kühlbrandt W, Osiewacz HD (2010). Cyclophilin D links programmed cell death and organismal aging in Podospora anserina. Aging Cell, 9(5), 761-775.
