Structural basis for broad anti-phage immunity by DISARM

In the evolutionary arms race against phage, bacteria have assembled a diverse arsenal of antiviral immune strategies. While the recently discovered DISARM (Defense Island System Associated with Restriction-Modification) systems can provide protection against a wide range of phage, the molecular mechanisms that underpin broad antiviral targeting but avoiding autoimmunity remain enigmatic. Here, we report cryo-EM structures of the core DISARM complex, DrmAB, both alone and in complex with an unmethylated phage DNA mimetic. These structures reveal that DrmAB core complex is autoinhibited by a trigger loop (TL) within DrmA and binding to DNA substrates containing a 5' overhang dislodges the TL, initiating a long-range structural rearrangement for DrmAB activation. Together with structure-guided in vivo studies, our work provides insights into the mechanism of phage DNA recognition and specific activation of this widespread antiviral defense system.

Structure and mechanism of NALCN-FAM155A-UNC79-UNC80 channel complex

NALCN channel mediates sodium leak currents and is important for maintaining proper resting membrane potential. NALCN and FAM155A form the core complex of the channel, the activity of which essentially depends on the presence of both UNC79 and UNC80, two auxiliary proteins. NALCN, FAM155A, UNC79, and UNC80 co-assemble into a large hetero-tetrameric channel complex. Genetic mutations of NALCN channel components lead to neurodevelopmental diseases. However, the structure and mechanism of the intact channel complex remain elusive. Here, we present the cryo-EM structure of the mammalian NALCN-FAM155A-UNC79-UNC80 quaternary complex. The structure showed that UNC79-UNC80 form a large piler-shaped heterodimer which was tethered to the intracellular side of the NALCN channel through tripartite interactions with the cytoplasmic loops of NALCN. Two interactions are essential for proper cell surface localization of NALCN. The other interaction relieves the self-inhibition of NALCN by pulling the auto-inhibitory CTD Interacting Helix (CIH) out of its binding site.

Asymmetric Structure of the Native Rhodobacter sphaeroides Dimeric LH1-RC Complex

The light-harvesting-reaction center (LH1-RC) core complex of purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides is characterized by the presence of both a dimeric form and a monomeric form. Following structure determination of the monomeric LH1-RC including its previously unrecognized component designated protein-U (Nat. Common.12, 6300, 2021), here we present cryo-EM structures of the dimeric LH1-RC from native Rba. sphaeroides IL106 at 2.75 Å resolution and from an LH1-RC monomer lacking protein-U (ΔU) at 2.64 Å resolution. The native dimeric core complex reveals many asymmetric features in the arrangement of its two monomeric components including the structural integrity of protein-U, the overall LH1 organization, and the rigidities of the proteins and pigments that form the complex. PufX polypeptides play a critical role in connecting two monomers, with one PufX interacting at its N-terminus with another PufX and an LH1 β-polypeptide in another monomer, in good agreement with biochemical analyses. One of the proteins-U was only partially identified in the dimeric structure, signaling significantly different degrees of disorder in the two monomers. The ΔU LH1-RC monomer revealed a half-moon-shaped structure containing 11 α- and 10 β-polypeptides (compared with 14 of each in the wild type), indicating a critical role for protein-U in controlling the number of αβ-subunits required for correct assembly and stabilization of the LH1-RC dimer. The structural features are discussed in relation to the unusual topology of intracytoplasmic photosynthetic membranes and an assembly model proposed for the native Rba. sphaeroides dimeric LH1-RC complex in membranes of wild-type cells.

Novel Bi-Allelic Variants of FANCM Cause Sertoli Cell-Only Syndrome and Non-Obstructive Azoospermia

Non-obstructive azoospermia (NOA) is the most severe disease in male infertility, but the genetic causes for the majority of NOA remain unknown. FANCM is a member of Fanconi Anemia (FA) core complex, whose defects are associated with cell hypersensitivity to DNA interstrand crosslink (ICL)-inducing agents. It was reported that variants in FANCM (MIM: 609644) might cause azoospermia or oligospermia. However, there is still a lack of evidence to explain the association between different FANCM variants and male infertility phenotypes. Herein, we identified compound heterozygous variants in FANCM in two NOA-affected brothers (c. 1778delG:p. R593Qfs*76 and c. 1663G > T:p. V555F), and a homozygous variant in FANCM (c. 1972C > T:p. R658X) in a sporadic case with NOA, respectively. H&E staining and immunohistochemistry showed Sertoli cell-only Syndrome (SCOS) in the three patients with NOA. Collectively, our study expands the knowledge of variants in FANCM, and provides a new insight to understand the genetic etiology of NOA.

Absence of PEXEL-Dependent Protein Export in Plasmodium Liver Stages Cannot Be Restored by Gain of the HSP101 Protein Translocon ATPase

Host cell remodeling is critical for successful Plasmodium replication inside erythrocytes and achieved by targeted export of parasite-encoded proteins. In contrast, during liver infection the malarial parasite appears to avoid protein export, perhaps to limit exposure of parasite antigens by infected liver cells. HSP101, the force-generating ATPase of the protein translocon of exported proteins (PTEX) is the only component that is switched off during early liver infection. Here, we generated transgenic Plasmodium berghei parasite lines that restore liver stage expression of HSP101. HSP101 expression in infected hepatocytes was achieved by swapping the endogenous promoter with the ptex150 promoter and by inserting an additional copy under the control of the elongation one alpha (ef1α) promoter. Both promoters drive constitutive and, hence, also pre-erythrocytic expression. Transgenic parasites were able to complete the life cycle, but failed to export PEXEL-proteins in early liver stages. Our results suggest that PTEX-dependent early liver stage export cannot be restored by addition of HSP101, indicative of alternative export complexes or other functions of the PTEX core complex during liver infection.

The thermo-tectonic evolution of the Suckling-Dayman metamorphic core complex, southeastern Papua New Guinea

<p>The Suckling-Dayman metamorphic core complex (SDMCC) in the Woodlark Rift of southeastern Papua New Guinea is being exhumed along the Mai’iu Fault, an active low-angle normal fault dipping ~20-22° northwards at the surface. The spectacularly smooth topography of the Mai’iu Fault footwall clearly is expressive of geologically recent uplift. The precise timing and rates of the exhumation of this continental metamorphic core complex (MCC) have, however, never been studied in detail. This thesis provides the first systematic set of U-Pb, fission track (FT), (U-Th[-Sm])/He and ²⁶Al/¹⁰Be ages from metaigneous and metasedimentary rocks of the footwall of the SDMCC, clasts and a tephra deposit contained within syn-tectonic conglomerates (the Gwoira Conglomerate) in a rider block, and modern stream sediments in the footwall and hanging wall of the Mai’iu Fault. The ages are complemented by whole-rock compositional and thermobarometric data (Al-in-amphibole, Al-in-biotite, Raman spectroscopy of carbonaceous material). Based on these data, the timing of the onset of extension along the Mai’iu Fault, its long-term dip-slip rate and its initial dip were constrained. These data are presented in the context of the evolution of the SDMCC from the Cretaceous to the present. The dominant lithology of the SDMCC, the Goropu Metabasalt, formed in a marginal basin to the northeast of the Australian continent. Two zircon U-Pb ages of 103.0 ± 5.7 and 71.6 ± 3.3 Ma, indicative of maximum depositional ages, from metasedimentary intercalations (the Bonenau Schist) in the Goropu Metabasalt, suggest formation of the oceanic protolith in the Late Cretaceous. Between 60.4 ± 2.5 and 56.6 ± 2.3 Ma (zircon U-Pb), tholeiitic to mildly calc-alkaline gabbroic to tonalitic rocks of the Yau Igneous Complex intruded the Goropu Metabasalt. The age of the Yau Igneous Complex overlaps with the known timing of north-directed subduction of the oceanic lithosphere along the Owen Stanley Fault (OSF) beneath the Cape Vogel Arc and provides a minimum age for the oceanic protolith. A second phase of magmatism, consisting of peraluminous-metaluminous calc-alkaline (Suckling Granite) and high-K (Mai’iu Monzonite, Bonua Porphyry) granitoids and basaltic andesite dikes that were cut by the Mai’iu Fault, was associated with the tectonic inversion of the OSF. Zircons from these syn-extensional intrusions suggest crystallization between 3.8 ± 0.2 and 2.0 ± 0.1 Ma. The oldest age of this range is inferred to mark the time by which the OSF had been re-activated as an extensional structure, the Mai’iu Fault. Al-in-amphibole and -biotite thermobarometry suggests crystallization of the Suckling Granite and Mai’iu Monzonite in a relatively shallow crust (~2-8 km depth) at pressures of ~0.4-2.3 kbar. Inherited zircons in the Plio-Pleistocene granitoids indicate that the Goropu Metabasalt carapace of the SDMCC is underlain by Australian-derived Cretaceous crustal material that is inferred to be the continuation of the Kagi Metamorphics in the central Papuan Peninsula. Further constraints of the timing of unroofing of the SDMCC were determined from three quartz clasts in the Gwoira Conglomerate. ²⁶Al/¹⁰Be burial ages of these samples indicate deposition in the Pliocene between 4.6 ± 2.9 and 3.4 ± 2.1 Ma. A tephra in the upper section of the exposed conglomerates was dated employing U-Pb methods on zircon, combined with apatite, zircon and magnetite (U-Th[-Sm])/He chronometers, yielding a complex age spectrum. An eruption age of 0.6 ± 0.4 Ma was extrapolated for this tephra. FT and (U-Th[-Sm])/He low-temperature thermochronometry details a young (≤3 Ma) and rapid exhumation history. Based on the crystallization ages of the syn-extensional granitoids, the depositional age of the Gwoira Conglomerate, the extensional cooling recorded by low-temperature thermochronometry, and the backwards projection of the published Holocene dip-slip rate of the Mai’iu Fault, the timing of the onset of extension is estimated at ~4 Ma. A minimum dip-slip rate of 8.1 ± 1.3 km/myr has been calculated from the inverse slope of zircon (U-Th)/He (ZHe) ages with slip-parallel distance from Mai’iu Fault trace. This is slightly lower than the >12 km/myr required to restore the intrusion depths (2-8 km) of the syn-extensional granitoids, now exposed 20-25 km south of the Mai’iu Fault trace at elevations up to 3.4 km. Collectively, these constraints suggest that the Mai’iu Fault has moved at cm-per-year rates since ~3 Ma. Evidence for both a fossil zircon FT (ZFT) partial annealing zone (PAZ) and a ZHe partial retention zone (PRZ) on the footwall of the SDMCC is presented. Combining paleo-temperature estimates from the inferred bases of the zircon PAZ and PRZ, peak-metamorphic temperatures inferred from Raman spectroscopy of carbonaceous material (RSCM), and published peak-metamorphic temperature constraints on the extensional shear zone mylonites near the Mai’iu Fault trace, a minimum slip-parallel, down-dip paleo-temperature gradient of 9.7 ± 2.2°C/km has been estimated for the exhumed Mai’iu Fault plane. Assuming that the modern regional geothermal gradient in the Woodlark Rift is a maximum estimate of that which existed prior to extensional exhumation of the SDMCC, the paleo-temperature gradient was used to estimate an average initial dip of the Mai’iu Fault of ~44° for pre-extensional geothermal gradients ranging between 10 to 20°C/km. Presently dipping 20-22° at the surface, the constraints on the initial dip suggest that the Mai’iu Fault may have been back-rotated by >20° since the onset of extension, consistent with a rolling hinge-style evolution of this continental MCC.</p>

The thermo-tectonic evolution of the Suckling-Dayman metamorphic core complex, southeastern Papua New Guinea

<p>The Suckling-Dayman metamorphic core complex (SDMCC) in the Woodlark Rift of southeastern Papua New Guinea is being exhumed along the Mai’iu Fault, an active low-angle normal fault dipping ~20-22° northwards at the surface. The spectacularly smooth topography of the Mai’iu Fault footwall clearly is expressive of geologically recent uplift. The precise timing and rates of the exhumation of this continental metamorphic core complex (MCC) have, however, never been studied in detail. This thesis provides the first systematic set of U-Pb, fission track (FT), (U-Th[-Sm])/He and ²⁶Al/¹⁰Be ages from metaigneous and metasedimentary rocks of the footwall of the SDMCC, clasts and a tephra deposit contained within syn-tectonic conglomerates (the Gwoira Conglomerate) in a rider block, and modern stream sediments in the footwall and hanging wall of the Mai’iu Fault. The ages are complemented by whole-rock compositional and thermobarometric data (Al-in-amphibole, Al-in-biotite, Raman spectroscopy of carbonaceous material). Based on these data, the timing of the onset of extension along the Mai’iu Fault, its long-term dip-slip rate and its initial dip were constrained. These data are presented in the context of the evolution of the SDMCC from the Cretaceous to the present. The dominant lithology of the SDMCC, the Goropu Metabasalt, formed in a marginal basin to the northeast of the Australian continent. Two zircon U-Pb ages of 103.0 ± 5.7 and 71.6 ± 3.3 Ma, indicative of maximum depositional ages, from metasedimentary intercalations (the Bonenau Schist) in the Goropu Metabasalt, suggest formation of the oceanic protolith in the Late Cretaceous. Between 60.4 ± 2.5 and 56.6 ± 2.3 Ma (zircon U-Pb), tholeiitic to mildly calc-alkaline gabbroic to tonalitic rocks of the Yau Igneous Complex intruded the Goropu Metabasalt. The age of the Yau Igneous Complex overlaps with the known timing of north-directed subduction of the oceanic lithosphere along the Owen Stanley Fault (OSF) beneath the Cape Vogel Arc and provides a minimum age for the oceanic protolith. A second phase of magmatism, consisting of peraluminous-metaluminous calc-alkaline (Suckling Granite) and high-K (Mai’iu Monzonite, Bonua Porphyry) granitoids and basaltic andesite dikes that were cut by the Mai’iu Fault, was associated with the tectonic inversion of the OSF. Zircons from these syn-extensional intrusions suggest crystallization between 3.8 ± 0.2 and 2.0 ± 0.1 Ma. The oldest age of this range is inferred to mark the time by which the OSF had been re-activated as an extensional structure, the Mai’iu Fault. Al-in-amphibole and -biotite thermobarometry suggests crystallization of the Suckling Granite and Mai’iu Monzonite in a relatively shallow crust (~2-8 km depth) at pressures of ~0.4-2.3 kbar. Inherited zircons in the Plio-Pleistocene granitoids indicate that the Goropu Metabasalt carapace of the SDMCC is underlain by Australian-derived Cretaceous crustal material that is inferred to be the continuation of the Kagi Metamorphics in the central Papuan Peninsula. Further constraints of the timing of unroofing of the SDMCC were determined from three quartz clasts in the Gwoira Conglomerate. ²⁶Al/¹⁰Be burial ages of these samples indicate deposition in the Pliocene between 4.6 ± 2.9 and 3.4 ± 2.1 Ma. A tephra in the upper section of the exposed conglomerates was dated employing U-Pb methods on zircon, combined with apatite, zircon and magnetite (U-Th[-Sm])/He chronometers, yielding a complex age spectrum. An eruption age of 0.6 ± 0.4 Ma was extrapolated for this tephra. FT and (U-Th[-Sm])/He low-temperature thermochronometry details a young (≤3 Ma) and rapid exhumation history. Based on the crystallization ages of the syn-extensional granitoids, the depositional age of the Gwoira Conglomerate, the extensional cooling recorded by low-temperature thermochronometry, and the backwards projection of the published Holocene dip-slip rate of the Mai’iu Fault, the timing of the onset of extension is estimated at ~4 Ma. A minimum dip-slip rate of 8.1 ± 1.3 km/myr has been calculated from the inverse slope of zircon (U-Th)/He (ZHe) ages with slip-parallel distance from Mai’iu Fault trace. This is slightly lower than the >12 km/myr required to restore the intrusion depths (2-8 km) of the syn-extensional granitoids, now exposed 20-25 km south of the Mai’iu Fault trace at elevations up to 3.4 km. Collectively, these constraints suggest that the Mai’iu Fault has moved at cm-per-year rates since ~3 Ma. Evidence for both a fossil zircon FT (ZFT) partial annealing zone (PAZ) and a ZHe partial retention zone (PRZ) on the footwall of the SDMCC is presented. Combining paleo-temperature estimates from the inferred bases of the zircon PAZ and PRZ, peak-metamorphic temperatures inferred from Raman spectroscopy of carbonaceous material (RSCM), and published peak-metamorphic temperature constraints on the extensional shear zone mylonites near the Mai’iu Fault trace, a minimum slip-parallel, down-dip paleo-temperature gradient of 9.7 ± 2.2°C/km has been estimated for the exhumed Mai’iu Fault plane. Assuming that the modern regional geothermal gradient in the Woodlark Rift is a maximum estimate of that which existed prior to extensional exhumation of the SDMCC, the paleo-temperature gradient was used to estimate an average initial dip of the Mai’iu Fault of ~44° for pre-extensional geothermal gradients ranging between 10 to 20°C/km. Presently dipping 20-22° at the surface, the constraints on the initial dip suggest that the Mai’iu Fault may have been back-rotated by >20° since the onset of extension, consistent with a rolling hinge-style evolution of this continental MCC.</p>

Architecture of the outer-membrane core complex from a conjugative type IV secretion system

Conjugation is one of the most important processes that bacteria utilize to spread antibiotic resistance genes among bacterial populations. Interbacterial DNA transfer requires a large double membrane-spanning nanomachine called the type 4 secretion system (T4SS) made up of the inner-membrane complex (IMC), the outer-membrane core complex (OMCC) and the conjugative pilus. The iconic F plasmid-encoded T4SS has been central in understanding conjugation for several decades, however atomic details of its structure are not known. Here, we report the structure of a complete conjugative OMCC encoded by the pED208 plasmid from E. coli, solved by cryo-electron microscopy at 3.3 Å resolution. This 2.1 MDa complex has a unique arrangement with two radial concentric rings, each having a different symmetry eventually contributing to remarkable differences in protein stoichiometry and flexibility in comparison to other OMCCs. Our structure suggests that F-OMCC is a highly dynamic complex, with implications for pilus extension and retraction during conjugation.