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Biondi bodies are filamentous amyloid inclusions of unknown composition in ependymal cells of the choroid plexuses, ependymal cells lining cerebral ventricles and ependymal cells of the central canal of the spinal cord. Their formation is age-dependent and they are commonly associated with a variety of neurodegenerative conditions, including Alzheimer's disease and Lewy body disorders. Here, we show that Biondi bodies are strongly immunoreactive with TMEM239, an antibody specific for inclusions of transmembrane protein 106B (TMEM106B). Biondi bodies were labelled by both this antibody and the amyloid dye pFTAA. Many Biondi bodies were also labelled for TMEM106B and the lysosomal markers Hexosaminidase A and Cathepsin D. By transmission immuno-electron microscopy, Biondi bodies of choroid plexuses were decorated by TMEM239 and were associated with structures that resembled residual bodies or secondary lysosomes. By electron cryo-microscopy, TMEM106B filaments from Biondi bodies of choroid plexuses were similar (Biondi variant), but not identical, to the fold I that was previously identified in filaments from brain parenchyma., (© 2024. The Author(s).)

Neurodegenerative diseases are characterized by the abnormal filamentous assembly of specific proteins in the central nervous system 1 . Human genetic studies have established a causal role for protein assembly in neurodegeneration 2 . However, the underlying molecular mechanisms remain largely unknown, which is limiting progress in developing clinical tools for these diseases. Recent advances in cryo-electron microscopy have enabled the structures of the protein filaments to be determined from the brains of patients 1 . All neurodegenerative diseases studied to date have been characterized by the self-assembly of proteins in homomeric amyloid filaments, including that of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) types A and B 3,4 . Here we used cryo-electron microscopy to determine filament structures from the brains of individuals with FTLD-TDP type C, one of the most common forms of sporadic FTLD-TDP. Unexpectedly, the structures revealed that a second protein, annexin A11 (ANXA11), co-assembles with TDP-43 in heteromeric amyloid filaments. The ordered filament fold is formed by TDP-43 residues G282/G284-N345 and ANXA11 residues L39-Y74 from their respective low-complexity domains. Regions of TDP-43 and ANXA11  that were previously implicated in protein-protein interactions form an extensive hydrophobic interface at the centre of the filament fold. Immunoblots of the filaments revealed that the majority of ANXA11 exists as an approximately 22 kDa N-terminal fragment lacking the annexin core domain. Immunohistochemistry of brain sections showed the colocalization of ANXA11 and TDP-43 in inclusions, redefining the histopathology of FTLD-TDP type C. This work establishes a central role for ANXA11 in FTLD-TDP type C. The unprecedented formation of heteromeric amyloid filaments in the human brain revises our understanding of amyloid assembly and may be of significance for the pathogenesis of neurodegenerative diseases., (© 2024. Crown.)

Mutations in MAPT , the microtubule-associated protein tau gene, give rise to cases of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) with abundant filamentous tau inclusions in brain cells. Individuals with pathological MAPT variants exhibit behavioural changes, cognitive impairment and signs of parkinsonism. Missense mutations of residue P301, which are the most common MAPT mutations associated with FTDP-17, give rise to the assembly of mutant four-repeat tau into filamentous inclusions, in the absence of extracellular deposits. Here we report the cryo-EM structures of tau filaments from five individuals belonging to three unrelated families with mutation P301L and from one individual belonging to a family with mutation P301T. A novel three-lobed tau fold resembling the two-layered tau fold of Pick's disease was present in all cases with the P301L tau mutation. Two different tau folds were found in the case with mutation P301T, the less abundant of which was a variant of the three-lobed fold. The major P301T tau fold was V-shaped, with partial similarity to the four-layered tau folds of corticobasal degeneration and argyrophilic grain disease. These findings suggest that FTDP-17 with mutations in P301 should be considered distinct inherited tauopathies and that model systems with these mutations should be used with caution in the study of sporadic tauopathies., Competing Interests: Competing interests The authors have no competing interests.

Neurodegenerative diseases are characterised by the abnormal filamentous assembly of specific proteins in the central nervous system 1 . Human genetic studies established a causal role for protein assembly in neurodegeneration 2 . However, the underlying molecular mechanisms remain largely unknown, which is limiting progress in developing clinical tools for these diseases. Recent advances in electron cryo-microscopy (cryo-EM) have enabled the structures of the protein filaments to be determined from patient brains 1 . All diseases studied to date have been characterised by the self-assembly of a single intracellular protein in homomeric amyloid filaments, including that of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) Types A and B 3,4 . Here, we used cryo-EM to determine filament structures from the brains of individuals with FTLD-TDP Type C, one of the most common forms of sporadic FTLD-TDP. Unexpectedly, the structures revealed that a second protein, annexin A11 (ANXA11), co-assembles with TDP-43 in heteromeric amyloid filaments. The ordered filament fold is formed by TDP-43 residues G282/284-N345 and ANXA11 residues L39-L74 from their respective low-complexity domains (LCDs). Regions of TDP-43 and ANXA11 previously implicated in protein-protein interactions form an extensive hydrophobic interface at the centre of the filament fold. Immunoblots of the filaments revealed that the majority of ANXA11 exists as a ∼22 kDa N-terminal fragment (NTF) lacking the annexin core domain. Immunohistochemistry of brain sections confirmed the co-localisation of ANXA11 and TDP-43 in inclusions, redefining the histopathology of FTLD-TDP Type C. This work establishes a central role for ANXA11 in FTLD-TDP Type C. The unprecedented formation of heteromeric amyloid filaments in human brain revises our understanding of amyloid assembly and may be of significance for the pathogenesis of neurodegenerative diseases.

Frontotemporal lobar degeneration (FTLD) causes frontotemporal dementia (FTD), the most common form of dementia after Alzheimer's disease, and is often also associated with motor disorders 1 . The pathological hallmarks of FTLD are neuronal inclusions of specific, abnormally assembled proteins 2 . In the majority of cases the inclusions contain amyloid filament assemblies of TAR DNA-binding protein 43 (TDP-43) or tau, with distinct filament structures characterizing different FTLD subtypes 3,4 . The presence of amyloid filaments and their identities and structures in the remaining approximately 10% of FTLD cases are unknown but are widely believed to be composed of the protein fused in sarcoma (FUS, also known as translocated in liposarcoma). As such, these cases are commonly referred to as FTLD-FUS. Here we used cryogenic electron microscopy (cryo-EM) to determine the structures of amyloid filaments extracted from the prefrontal and temporal cortices of four individuals with FTLD-FUS. Surprisingly, we found abundant amyloid filaments of the FUS homologue TATA-binding protein-associated factor 15 (TAF15, also known as TATA-binding protein-associated factor 2N) rather than of FUS itself. The filament fold is formed from residues 7-99 in the low-complexity domain (LCD) of TAF15 and was identical between individuals. Furthermore, we found TAF15 filaments with the same fold in the motor cortex and brainstem of two of the individuals, both showing upper and lower motor neuron pathology. The formation of TAF15 amyloid filaments with a characteristic fold in FTLD establishes TAF15 proteinopathy in neurodegenerative disease. The structure of TAF15 amyloid filaments provides a basis for the development of model systems of neurodegenerative disease, as well as for the design of diagnostic and therapeutic tools targeting TAF15 proteinopathy., (© 2023. The Author(s).)

Intermediate species in the assembly of amyloid filaments are believed to play a central role in neurodegenerative diseases and may constitute important targets for therapeutic intervention 1,2 . However, structural information about intermediate species has been scarce and the molecular mechanisms by which amyloids assemble remain largely unknown. Here we use time-resolved cryogenic electron microscopy to study the in vitro assembly of recombinant truncated tau (amino acid residues 297-391) into paired helical filaments of Alzheimer's disease or into filaments of chronic traumatic encephalopathy 3 . We report the formation of a shared first intermediate amyloid filament, with an ordered core comprising residues 302-316. Nuclear magnetic resonance indicates that the same residues adopt rigid, β-strand-like conformations in monomeric tau. At later time points, the first intermediate amyloid disappears and we observe many different intermediate amyloid filaments, with structures that depend on the reaction conditions. At the end of both assembly reactions, most intermediate amyloids disappear and filaments with the same ordered cores as those from human brains remain. Our results provide structural insights into the processes of primary and secondary nucleation of amyloid assembly, with implications for the design of new therapies., (© 2023. The Author(s).)

The amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of the island of Guam and the Kii peninsula of Japan is a fatal neurodegenerative disease of unknown cause that is characterized by the presence of abundant filamentous tau inclusions in brains and spinal cords. Here, we used electron cryo-microscopy to determine the structures of tau filaments from the cerebral cortex of three cases of ALS/PDC from Guam and eight cases from Kii, as well as from the spinal cord of two of the Guam cases. Tau filaments had the chronic traumatic encephalopathy (CTE) fold, with variable amounts of Type I and Type II filaments. Paired helical tau filaments were also found in three Kii cases and tau filaments with the corticobasal degeneration fold in one Kii case. We identified a new Type III CTE tau filament, where protofilaments pack against each other in an antiparallel fashion. ALS/PDC is the third known tauopathy with CTE-type filaments and abundant tau inclusions in cortical layers II/III, the others being CTE and subacute sclerosing panencephalitis. Because these tauopathies are believed to have environmental causes, our findings support the hypothesis that ALS/PDC is caused by exogenous factors., Competing Interests: Competing interests statement:The authors declare no competing interest.

We used electron cryo-microscopy (cryo-EM) to determine the structures of Aβ40 filaments from the leptomeninges of individuals with Alzheimer's disease and cerebral amyloid angiopathy. In agreement with previously reported structures, which were solved to a resolution of 4.4 Å, we found three types of filaments. However, our new structures, solved to a resolution of 2.4 Å, revealed differences in the sequence assignment that redefine the fold of Aβ40 peptides and their interactions. Filaments are made of pairs of protofilaments, the ordered core of which comprises D1-G38. The different filament types comprise one, two or three protofilament pairs. In each pair, residues H14-G37 of both protofilaments adopt an extended conformation and pack against each other in an anti-parallel fashion, held together by hydrophobic interactions and hydrogen bonds between main chains and side chains. Residues D1-H13 fold back on the adjacent parts of their own chains through both polar and non-polar interactions. There are also several additional densities of unknown identity. Sarkosyl extraction and aqueous extraction gave the same structures. By cryo-EM, parenchymal deposits of Aβ42 and blood vessel deposits of Aβ40 have distinct structures, supporting the view that Alzheimer's disease and cerebral amyloid angiopathy are different Aβ proteinopathies., (© 2023. The Author(s).)

Mice transgenic for human mutant P301S tau are widely used as models for human tauopathies. They develop neurodegeneration and abundant filamentous inclusions made of human mutant four-repeat tau. Here we used electron cryo-microscopy (cryo-EM) to determine the structures of tau filaments from the brains of Tg2541 and PS19 mice. Both lines express human P301S tau (0N4R for Tg2541 and 1N4R for PS19) on mixed genetic backgrounds and downstream of different promoters (murine Thy1 for Tg2541 and murine Prnp for PS19). The structures of tau filaments from Tg2541 and PS19 mice differ from each other and those of wild-type tau filaments from human brains. The structures of tau filaments from the brains of humans with mutations P301L, P301S or P301T in MAPT are not known. Filaments from the brains of Tg2541 and PS19 mice share a substructure at the junction of repeats 2 and 3, which comprises residues I297-V312 of tau and includes the P301S mutation. The filament core from the brainstem of Tg2541 mice consists of residues K274-H329 of tau and two disconnected protein densities. Two non-proteinaceous densities are also in evidence. The filament core from the cerebral cortex of line PS19 extends from residues G271-P364 of tau. One strong non-proteinaceous density is also present. Unlike the tau filaments from human brains, the sequences following repeat 4 are missing from the cores of tau filaments from the brains of Tg2541 and PS19 mice., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The abnormal assembly of TAR DNA-binding protein 43 (TDP-43) in neuronal and glial cells characterizes nearly all cases of amyotrophic lateral sclerosis (ALS) and around half of cases of frontotemporal lobar degeneration (FTLD) 1,2 . A causal role for TDP-43 assembly in neurodegeneration is evidenced by dominantly inherited missense mutations in TARDBP, the gene encoding TDP-43, that promote assembly and give rise to ALS and FTLD 3-7 . At least four types (A-D) of FTLD with TDP-43 pathology (FTLD-TDP) are defined by distinct brain distributions of assembled TDP-43 and are associated with different clinical presentations of frontotemporal dementia 8 . We previously showed, using cryo-electron microscopy, that TDP-43 assembles into amyloid filaments in ALS and type B FTLD-TDP 9 . However, the structures of assembled TDP-43 in FTLD without ALS remained unknown. Here we report the cryo-electron microscopy structures of assembled TDP-43 from the brains of three individuals with the most common type of FTLD-TDP, type A. TDP-43 formed amyloid filaments with a new fold that was the same across individuals, indicating that this fold may characterize type A FTLD-TDP. The fold resembles a chevron badge and is unlike the double-spiral-shaped fold of ALS and type B FTLD-TDP, establishing that distinct filament folds of TDP-43 characterize different neurodegenerative conditions. The structures, in combination with mass spectrometry, led to the identification of two new post-translational modifications of assembled TDP-43, citrullination and monomethylation of R293, and indicate that they may facilitate filament formation and observed structural variation in individual filaments. The structures of TDP-43 filaments from type A FTLD-TDP will guide mechanistic studies of TDP-43 assembly, as well as the development of diagnostic and therapeutic compounds for TDP-43 proteinopathies., (© 2023. The Author(s).)

A 21-nucleotide duplication in one allele of SNCA was identified in a previously described disease with abundant α-synuclein inclusions that we now call juvenile-onset synucleinopathy (JOS). This mutation translates into the insertion of MAAAEKT after residue 22 of α-synuclein, resulting in a protein of 147 amino acids. Both wild-type and mutant proteins were present in sarkosyl-insoluble material that was extracted from frontal cortex of the individual with JOS and examined by electron cryo-microscopy. The structures of JOS filaments, comprising either a single protofilament, or a pair of protofilaments, revealed a new α-synuclein fold that differs from the folds of Lewy body diseases and multiple system atrophy (MSA). The JOS fold consists of a compact core, the sequence of which (residues 36-100 of wild-type α-synuclein) is unaffected by the mutation, and two disconnected density islands (A and B) of mixed sequences. There is a non-proteinaceous cofactor bound between the core and island A. The JOS fold resembles the common substructure of MSA Type I and Type II dimeric filaments, with its core segment approximating the C-terminal body of MSA protofilaments B and its islands mimicking the N-terminal arm of MSA protofilaments A. The partial similarity of JOS and MSA folds extends to the locations of their cofactor-binding sites. In vitro assembly of recombinant wild-type α-synuclein, its insertion mutant and their mixture yielded structures that were distinct from those of JOS filaments. Our findings provide insight into a possible mechanism of JOS fibrillation in which mutant α-synuclein of 147 amino acids forms a nucleus with the JOS fold, around which wild-type and mutant proteins assemble during elongation., (© 2023. The Author(s).)

The Arctic mutation, encoding E693G in the amyloid precursor protein (APP) gene [E22G in amyloid-β (Aβ)], causes dominantly inherited Alzheimer's disease. Here, we report the high-resolution cryo-EM structures of Aβ filaments from the frontal cortex of a previously described case (AβPParc1) with the Arctic mutation. Most filaments consist of two pairs of non-identical protofilaments that comprise residues V12-V40 (human Arctic fold A) and E11-G37 (human Arctic fold B). They have a substructure (residues F20-G37) in common with the folds of type I and type II Aβ42. When compared to the structures of wild-type Aβ42 filaments, there are subtle conformational changes in the human Arctic folds, because of the lack of a side chain at G22, which may strengthen hydrogen bonding between mutant Aβ molecules and promote filament formation. A minority of Aβ42 filaments of type II was also present, as were tau paired helical filaments. In addition, we report the cryo-EM structures of Aβ filaments with the Arctic mutation from mouse knock-in line App NL-G-F . Most filaments are made of two identical mutant protofilaments that extend from D1 to G37 (App NL-G-F murine Arctic fold). In a minority of filaments, two dimeric folds pack against each other in an anti-parallel fashion. The App NL-G-F murine Arctic fold differs from the human Arctic folds, but shares some substructure., (© 2023. The Author(s).)

Parkinson's disease (PD) is the most common movement disorder, with resting tremor, rigidity, bradykinesia and postural instability being major symptoms 1 . Neuropathologically, it is characterized by the presence of abundant filamentous inclusions of α-synuclein in the form of Lewy bodies and Lewy neurites in some brain cells, including dopaminergic nerve cells of the substantia nigra 2 . PD is increasingly recognised as a multisystem disorder, with cognitive decline being one of its most common non-motor symptoms. Many patients with PD develop dementia more than 10 years after diagnosis 3 . PD dementia (PDD) is clinically and neuropathologically similar to dementia with Lewy bodies (DLB), which is diagnosed when cognitive impairment precedes parkinsonian motor signs or begins within one year from their onset 4 . In PDD, cognitive impairment develops in the setting of well-established PD. Besides PD and DLB, multiple system atrophy (MSA) is the third major synucleinopathy 5 . It is characterized by the presence of abundant filamentous α-synuclein inclusions in brain cells, especially oligodendrocytes (Papp-Lantos bodies). We previously reported the electron cryo-microscopy structures of two types of α-synuclein filament extracted from the brains of individuals with MSA 6 . Each filament type is made of two different protofilaments. Here we report that the cryo-electron microscopy structures of α-synuclein filaments from the brains of individuals with PD, PDD and DLB are made of a single protofilament (Lewy fold) that is markedly different from the protofilaments of MSA. These findings establish the existence of distinct molecular conformers of assembled α-synuclein in neurodegenerative disease., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Many age-dependent neurodegenerative diseases, such as Alzheimer's and Parkinson's, are characterized by abundant inclusions of amyloid filaments. Filamentous inclusions of the proteins tau, amyloid-β, α-synuclein and transactive response DNA-binding protein (TARDBP; also known as TDP-43) are the most common 1,2 . Here we used structure determination by cryogenic electron microscopy to show that residues 120-254 of the lysosomal type II transmembrane protein 106B (TMEM106B) also form amyloid filaments in human brains. We determined the structures of TMEM106B filaments from a number of brain regions of 22 individuals with abundant amyloid deposits, including those resulting from sporadic and inherited tauopathies, amyloid-β amyloidoses, synucleinopathies and TDP-43 proteinopathies, as well as from the frontal cortex of 3 individuals with normal neurology and no or only a few amyloid deposits. We observed three TMEM106B folds, with no clear relationships between folds and diseases. TMEM106B filaments correlated with the presence of a 29-kDa sarkosyl-insoluble fragment and globular cytoplasmic inclusions, as detected by an antibody specific to the carboxy-terminal region of TMEM106B. The identification of TMEM106B filaments in the brains of older, but not younger, individuals with normal neurology indicates that they form in an age-dependent manner., (© 2022. The Author(s).)

Abundant filamentous inclusions of tau are characteristic of more than 20 neurodegenerative diseases that are collectively termed tauopathies. Electron cryo-microscopy (cryo-EM) structures of tau amyloid filaments from human brain revealed that distinct tau folds characterise many different diseases. A lack of laboratory-based model systems to generate these structures has hampered efforts to uncover the molecular mechanisms that underlie tauopathies. Here, we report in vitro assembly conditions with recombinant tau that replicate the structures of filaments from both Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE), as determined by cryo-EM. Our results suggest that post-translational modifications of tau modulate filament assembly, and that previously observed additional densities in AD and CTE filaments may arise from the presence of inorganic salts, like phosphates and sodium chloride. In vitro assembly of tau into disease-relevant filaments will facilitate studies to determine their roles in different diseases, as well as the development of compounds that specifically bind to these structures or prevent their formation., Competing Interests: SL, AM, MG No competing interests declared, FK, Bv, AK is affiliated with Thermo Fisher Scientific. The author has no financial interests to declare, SS Reviewing editor, eLife, (© 2022, Lövestam et al.)

Filament assembly of amyloid-β peptides ending at residue 42 (Aβ42) is a central event in Alzheimer’s disease. Here, we report the cryo–electron microscopy (cryo-EM) structures of Aβ42 filaments from human brains. Two structurally related S-shaped protofilament folds give rise to two types of filaments. Type I filaments were found mostly in the brains of individuals with sporadic Alzheimer’s disease, and type II filaments were found in individuals with familial Alzheimer’s disease and other conditions. The structures of Aβ42 filaments from the brain differ from those of filaments assembled in vitro. By contrast, in App NL-F knock-in mice, Aβ42 deposits were made of type II filaments. Knowledge of Aβ42 filament structures from human brains may lead to the development of inhibitors of assembly and improved imaging agents.

The abnormal aggregation of TAR DNA-binding protein 43 kDa (TDP-43) in neurons and glia is the defining pathological hallmark of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and multiple forms of frontotemporal lobar degeneration (FTLD) 1,2 . It is also common in other diseases, including Alzheimer's and Parkinson's. No disease-modifying therapies exist for these conditions and early diagnosis is not possible. The structures of pathological TDP-43 aggregates are unknown. Here we used cryo-electron microscopy to determine the structures of aggregated TDP-43 in the frontal and motor cortices of an individual who had ALS with FTLD and from the frontal cortex of a second individual with the same diagnosis. An identical amyloid-like filament structure comprising a single protofilament was found in both brain regions and individuals. The ordered filament core spans residues 282-360 in the TDP-43 low-complexity domain and adopts a previously undescribed double-spiral-shaped fold, which shows no similarity to those of TDP-43 filaments formed in vitro 3,4 . An abundance of glycine and neutral polar residues facilitates numerous turns and restricts β-strand length, which results in an absence of β-sheet stacking that is associated with cross-β amyloid structure. An uneven distribution of residues gives rise to structurally and chemically distinct surfaces that face external densities and suggest possible ligand-binding sites. This work enhances our understanding of the molecular pathogenesis of ALS and FTLD and informs the development of diagnostic and therapeutic agents that target aggregated TDP-43., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The ordered assembly of tau protein into filaments characterizes several neurodegenerative diseases, which are called tauopathies. It was previously reported that, by cryo-electron microscopy, the structures of tau filaments from Alzheimer's disease 1,2 , Pick's disease 3 , chronic traumatic encephalopathy 4 and corticobasal degeneration 5 are distinct. Here we show that the structures of tau filaments from progressive supranuclear palsy (PSP) define a new three-layered fold. Moreover, the structures of tau filaments from globular glial tauopathy are similar to those from PSP. The tau filament fold of argyrophilic grain disease (AGD) differs, instead resembling the four-layered fold of corticobasal degeneration. The AGD fold is also observed in ageing-related tau astrogliopathy. Tau protofilament structures from inherited cases of mutations at positions +3 or +16 in intron 10 of MAPT (the microtubule-associated protein tau gene) are also identical to those from AGD, suggesting that relative overproduction of four-repeat tau can give rise to the AGD fold. Finally, the structures of tau filaments from cases of familial British dementia and familial Danish dementia are the same as those from cases of Alzheimer's disease and primary age-related tauopathy. These findings suggest a hierarchical classification of tauopathies on the basis of their filament folds, which complements clinical diagnosis and neuropathology and also allows the identification of new entities-as we show for a case diagnosed as PSP, but with filament structures that are intermediate between those of globular glial tauopathy and PSP., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Tau and Aβ assemblies of Alzheimer's disease (AD) can be visualized in living subjects using positron emission tomography (PET). Tau assemblies comprise paired helical and straight filaments (PHFs and SFs). APN-1607 (PM-PBB3) is a recently described PET ligand for AD and other tau proteinopathies. Since it is not known where in the tau folds PET ligands bind, we used electron cryo-microscopy (cryo-EM) to determine the binding sites of APN-1607 in the Alzheimer fold. We identified two major sites in the β-helix of PHFs and SFs and a third major site in the C-shaped cavity of SFs. In addition, we report that tau filaments from posterior cortical atrophy (PCA) and primary age-related tauopathy (PART) are identical to those from AD. In support, fluorescence labelling showed binding of APN-1607 to intraneuronal inclusions in AD, PART and PCA. Knowledge of the binding modes of APN-1607 to tau filaments may lead to the development of new ligands with increased specificity and binding activity. We show that cryo-EM can be used to identify the binding sites of small molecules in amyloid filaments.

Chlamydia trachomatis lacks the canonical genes required for the biosynthesis of p -aminobenzoate (pABA), a component of essential folate cofactors. Previous studies revealed a single gene from C. trachomatis , the CT610 gene, that rescues Escherichia coli Δ pabA , Δ pabB , and Δ pabC mutants, which are otherwise auxotrophic for pABA. CT610 shares low sequence similarity to nonheme diiron oxygenases, and the previously solved crystal structure revealed a diiron active site. Genetic studies ruled out several potential substrates for CT610-dependent pABA biosynthesis, including chorismate and other shikimate pathway intermediates, leaving the actual precursor(s) unknown. Here, we supplied isotopically labeled potential precursors to E. coli Δ pab A cells expressing CT610 and found that the aromatic portion of tyrosine was highly incorporated into pABA, indicating that tyrosine is a precursor for CT610-dependent pABA biosynthesis. Additionally, in vitro enzymatic experiments revealed that purified CT610 exhibits low pABA synthesis activity under aerobic conditions in the absence of tyrosine or other potential substrates, where only the addition of a reducing agent such as dithiothreitol appears to stimulate pABA production. Furthermore, site-directed mutagenesis studies revealed that two conserved active site tyrosine residues are essential for the pABA synthesis reaction in vitro Thus, the current data are most consistent with CT610 being a unique self-sacrificing enzyme that utilizes its own active site tyrosine residue(s) for pABA biosynthesis in a reaction that requires O 2 and a reduced diiron cofactor. IMPORTANCE Chlamydia trachomatis is the most reported sexually transmitted infection in the United States and the leading cause of infectious blindness worldwide. Unlike many other intracellular pathogens that have undergone reductive evolution, C. trachomatis is capable of de novo biosynthesis of the essential cofactor tetrahydrofolate using a noncanonical pathway. Here, we identify the biosynthetic precursor to the p -aminobenzoate (pABA) portion of folate in a process that requires the CT610 enzyme from C. trachomatis We further provide evidence that CT610 is a self-sacrificing or "suicide" enzyme that uses its own amino acid residue(s) as the substrate for pABA synthesis. This work provides the foundation for future investigation of this chlamydial pABA synthase, which could lead to new therapeutic strategies for C. trachomatis infections., (Copyright © 2020 American Society for Microbiology.)

Huntington's disease is a progressive, autosomal dominant, neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin gene. As a result, the translated protein, huntingtin, contains an abnormally long polyglutamine stretch that makes it prone to misfold and aggregating. Aggregation of huntingtin is believed to be the cause of Huntington's disease. However, understanding on how, and why, huntingtin aggregates are deleterious has been hampered by lack of enough relevant structural data. In this review, we discuss our recent findings on a glutamine-based functional amyloid isolated from Drosophila brain and how this information provides plausible structural insight on the structure of huntingtin deposits in the brain.

Synucleinopathies, which include multiple system atrophy (MSA), Parkinson's disease, Parkinson's disease with dementia and dementia with Lewy bodies (DLB), are human neurodegenerative diseases 1 . Existing treatments are at best symptomatic. These diseases are characterized by the presence of, and believed to be caused by the formation of, filamentous inclusions of α-synuclein in brain cells 2,3 . However, the structures of α-synuclein filaments from the human brain are unknown. Here, using cryo-electron microscopy, we show that α-synuclein inclusions from the brains of individuals with MSA are made of two types of filament, each of which consists of two different protofilaments. In each type of filament, non-proteinaceous molecules are present at the interface of the two protofilaments. Using two-dimensional class averaging, we show that α-synuclein filaments from the brains of individuals with MSA differ from those of individuals with DLB, which suggests that distinct conformers or strains characterize specific synucleinopathies. As is the case with tau assemblies 4-9 , the structures of α-synuclein filaments extracted from the brains of individuals with MSA differ from those formed in vitro using recombinant proteins, which has implications for understanding the mechanisms of aggregate propagation and neurodegeneration in the human brain. These findings have diagnostic and potential therapeutic relevance, especially because of the unmet clinical need to be able to image filamentous α-synuclein inclusions in the human brain.

Corticobasal degeneration (CBD) is a neurodegenerative tauopathy-a class of disorders in which the tau protein forms insoluble inclusions in the brain-that is characterized by motor and cognitive disturbances 1-3 . The H1 haplotype of MAPT (the tau gene) is present in cases of CBD at a higher frequency than in controls 4,5 , and genome-wide association studies have identified additional risk factors 6 . By histology, astrocytic plaques are diagnostic of CBD 7,8 ; by SDS-PAGE, so too are detergent-insoluble, 37 kDa fragments of tau 9 . Like progressive supranuclear palsy, globular glial tauopathy and argyrophilic grain disease 10 , CBD is characterized by abundant filamentous tau inclusions that are made of isoforms with four microtubule-binding repeats 11-15 . This distinguishes such '4R' tauopathies from Pick's disease (the filaments of which are made of three-repeat (3R) tau isoforms) and from Alzheimer's disease and chronic traumatic encephalopathy (CTE) (in which both 3R and 4R isoforms are found in the filaments) 16 . Here we use cryo-electron microscopy to analyse the structures of tau filaments extracted from the brains of three individuals with CBD. These filaments were identical between cases, but distinct from those seen in Alzheimer's disease, Pick's disease and CTE 17-19 . The core of a CBD filament comprises residues lysine 274 to glutamate 380 of tau, spanning the last residue of the R1 repeat, the whole of the R2, R3 and R4 repeats, and 12 amino acids after R4. The core adopts a previously unseen four-layered fold, which encloses a large nonproteinaceous density. This density is surrounded by the side chains of lysine residues 290 and 294 from R2 and lysine 370 from the sequence after R4.

How long-lived memories withstand molecular turnover is a fundamental question. Aggregates of a prion-like RNA-binding protein, cytoplasmic polyadenylation element-binding (CPEB) protein, is a putative substrate of long-lasting memories. We isolated aggregated Drosophila CPEB, Orb2, from adult heads and determined its activity and atomic structure, at 2.6-angstrom resolution, using cryo-electron microscopy. Orb2 formed ~75-nanometer-long threefold-symmetric amyloid filaments. Filament formation transformed Orb2 from a translation repressor to an activator and "seed" for further translationally active aggregation. The 31-amino acid protofilament core adopted a cross-β unit with a single hydrophilic hairpin stabilized through interdigitated glutamine packing. Unlike the hydrophobic core of pathogenic amyloids, the hydrophilic core of Orb2 filaments suggests how some neuronal amyloids could be a stable yet regulatable substrate of memory., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The Structural Classification of Proteins (SCOP) database is a classification of protein domains organised according to their evolutionary and structural relationships. We report a major effort to increase the coverage of structural data, aiming to provide classification of almost all domain superfamilies with representatives in the PDB. We have also improved the database schema, provided a new API and modernised the web interface. This is by far the most significant update in coverage since SCOP 1.75 and builds on the advances in schema from the SCOP 2 prototype. The database is accessible from http://scop.mrc-lmb.cam.ac.uk., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Genome3D (https://www.genome3d.eu) is a freely available resource that provides consensus structural annotations for representative protein sequences taken from a selection of model organisms. Since the last NAR update in 2015, the method of data submission has been overhauled, with annotations now being 'pushed' to the database via an API. As a result, contributing groups are now able to manage their own structural annotations, making the resource more flexible and maintainable. The new submission protocol brings a number of additional benefits including: providing instant validation of data and avoiding the requirement to synchronise releases between resources. It also makes it possible to implement the submission of these structural annotations as an automated part of existing internal workflows. In turn, these improvements facilitate Genome3D being opened up to new prediction algorithms and groups. For the latest release of Genome3D (v2.1), the underlying dataset of sequences used as prediction targets has been updated using the latest reference proteomes available in UniProtKB. A number of new reference proteomes have also been added of particular interest to the wider scientific community: cow, pig, wheat and mycobacterium tuberculosis. These additions, along with improvements to the underlying predictions from contributing resources, has ensured that the number of annotations in Genome3D has nearly doubled since the last NAR update article. The new API has also been used to facilitate the dissemination of Genome3D data into InterPro, thereby widening the visibility of both the annotation data and annotation algorithms., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Chronic traumatic encephalopathy (CTE) is a neurodegenerative tauopathy that is associated with repetitive head impacts or exposure to blast waves. First described as punch-drunk syndrome and dementia pugilistica in retired boxers 1-3 , CTE has since been identified in former participants of other contact sports, ex-military personnel and after physical abuse 4-7 . No disease-modifying therapies currently exist, and diagnosis requires an autopsy. CTE is defined by an abundance of hyperphosphorylated tau protein in neurons, astrocytes and cell processes around blood vessels 8,9 . This, together with the accumulation of tau inclusions in cortical layers II and III, distinguishes CTE from Alzheimer's disease and other tauopathies 10,11 . However, the morphologies of tau filaments in CTE and the mechanisms by which brain trauma can lead to their formation are unknown. Here we determine the structures of tau filaments from the brains of three individuals with CTE at resolutions down to 2.3 Å, using cryo-electron microscopy. We show that filament structures are identical in the three cases but are distinct from those of Alzheimer's and Pick's diseases, and from those formed in vitro 12-15 . Similar to Alzheimer's disease 12,14,16-18 , all six brain tau isoforms assemble into filaments in CTE, and residues K274-R379 of three-repeat tau and S305-R379 of four-repeat tau form the ordered core of two identical C-shaped protofilaments. However, a different conformation of the β-helix region creates a hydrophobic cavity that is absent in tau filaments from the brains of patients with Alzheimer's disease. This cavity encloses an additional density that is not connected to tau, which suggests that the incorporation of cofactors may have a role in tau aggregation in CTE. Moreover, filaments in CTE have distinct protofilament interfaces to those of Alzheimer's disease. Our structures provide a unifying neuropathological criterion for CTE, and support the hypothesis that the formation and propagation of distinct conformers of assembled tau underlie different neurodegenerative diseases.

Assembly of microtubule-associated protein tau into filamentous inclusions underlies a range of neurodegenerative diseases. Tau filaments adopt different conformations in Alzheimer's and Pick's diseases. Here, we used cryo- and immuno- electron microscopy to characterise filaments that were assembled from recombinant full-length human tau with four (2N4R) or three (2N3R) microtubule-binding repeats in the presence of heparin. 2N4R tau assembles into multiple types of filaments, and the structures of three types reveal similar 'kinked hairpin' folds, in which the second and third repeats pack against each other. 2N3R tau filaments are structurally homogeneous, and adopt a dimeric core, where the third repeats of two tau molecules pack in a parallel manner. The heparin-induced tau filaments differ from those of Alzheimer's or Pick's disease, which have larger cores with different repeat compositions. Our results illustrate the structural versatility of amyloid filaments, and raise questions about the relevance of in vitro assembly., Competing Interests: WZ, BF, AM, JF, RC No competing interests declared, MG, SS Reviewing editor, eLife, (© 2019, Zhang et al.)

The ordered assembly of tau protein into abnormal filaments is a defining characteristic of Alzheimer's disease (AD) and other neurodegenerative disorders. It is not known if the structures of tau filaments vary within, or between, the brains of individuals with AD. We used a combination of electron cryo-microscopy (cryo-EM) and immuno-gold negative-stain electron microscopy (immuno-EM) to determine the structures of paired helical filaments (PHFs) and straight filaments (SFs) from the frontal cortex of 17 cases of AD (15 sporadic and 2 inherited) and 2 cases of atypical AD (posterior cortical atrophy). The high-resolution structures of PHFs and SFs from the frontal cortex of 3 cases of AD, 2 sporadic and 1 inherited, were determined by cryo-EM. We also used immuno-EM to study the PHFs and SFs from a number of cortical and subcortical brain regions. PHFs outnumbered SFs in all AD cases. By cryo-EM, PHFs and SFs were made of two C-shaped protofilaments with a combined cross-β/β-helix structure, as described previously for one case of AD. The higher resolution structures obtained here showed two additional amino acids at each end of the protofilament. The immuno-EM findings, which indicated the presence of repeats 3 and 4, but not of the N-terminal regions of repeats 1 and 2, of tau in the filament cores of all AD cases, were consistent with the cryo-EM results. These findings show that there is no significant variation in tau filament structures between individuals with AD. This knowledge will be crucial for understanding the mechanisms that underlie tau filament formation and for developing novel diagnostics and therapies.

The ordered assembly of tau protein into abnormal filamentous inclusions underlies many human neurodegenerative diseases 1 . Tau assemblies seem to spread through specific neural networks in each disease 2 , with short filaments having the greatest seeding activity 3 . The abundance of tau inclusions strongly correlates with disease symptoms 4 . Six tau isoforms are expressed in the normal adult human brain-three isoforms with four microtubule-binding repeats each (4R tau) and three isoforms that lack the second repeat (3R tau) 1 . In various diseases, tau filaments can be composed of either 3R or 4R tau, or of both. Tau filaments have distinct cellular and neuroanatomical distributions 5 , with morphological and biochemical differences suggesting that they may be able to adopt disease-specific molecular conformations 6,7 . Such conformers may give rise to different neuropathological phenotypes 8,9 , reminiscent of prion strains 10 . However, the underlying structures are not known. Using electron cryo-microscopy, we recently reported the structures of tau filaments from patients with Alzheimer's disease, which contain both 3R and 4R tau 11 . Here we determine the structures of tau filaments from patients with Pick's disease, a neurodegenerative disorder characterized by frontotemporal dementia. The filaments consist of residues Lys254-Phe378 of 3R tau, which are folded differently from the tau filaments in Alzheimer's disease, establishing the existence of conformers of assembled tau. The observed tau fold in the filaments of patients with Pick's disease explains the selective incorporation of 3R tau in Pick bodies, and the differences in phosphorylation relative to the tau filaments of Alzheimer's disease. Our findings show how tau can adopt distinct folds in the human brain in different diseases, an essential step for understanding the formation and propagation of molecular conformers.

Alzheimer's disease is the most common neurodegenerative disease, and there are no mechanism-based therapies. The disease is defined by the presence of abundant neurofibrillary lesions and neuritic plaques in the cerebral cortex. Neurofibrillary lesions comprise paired helical and straight tau filaments, whereas tau filaments with different morphologies characterize other neurodegenerative diseases. No high-resolution structures of tau filaments are available. Here we present cryo-electron microscopy (cryo-EM) maps at 3.4-3.5 Å resolution and corresponding atomic models of paired helical and straight filaments from the brain of an individual with Alzheimer's disease. Filament cores are made of two identical protofilaments comprising residues 306-378 of tau protein, which adopt a combined cross-β/β-helix structure and define the seed for tau aggregation. Paired helical and straight filaments differ in their inter-protofilament packing, showing that they are ultrastructural polymorphs. These findings demonstrate that cryo-EM allows atomic characterization of amyloid filaments from patient-derived material, and pave the way for investigation of a range of neurodegenerative diseases.

SCOP2 is a successor to the Structural Classification of Proteins (SCOP) database that organizes proteins of known structure according to their structural and evolutionary relationships. It was designed to provide a more advanced framework for the classification of proteins. The SCOP2 classification is described in terms of a directed acyclic graph in which each node defines a relationship of particular type that is represented by a region of protein structure and sequence. The SCOP2 data are accessible via SCOP2-Browser and SCOP2-Graph. This protocol unit describes different ways to explore and investigate the SCOP2 evolutionary and structural groupings., (Copyright © 2015 John Wiley & Sons, Inc.)

Genome3D (http://www.genome3d.eu) is a collaborative resource that provides predicted domain annotations and structural models for key sequences. Since introducing Genome3D in a previous NAR paper, we have substantially extended and improved the resource. We have annotated representatives from Pfam families to improve coverage of diverse sequences and added a fast sequence search to the website to allow users to find Genome3D-annotated sequences similar to their own. We have improved and extended the Genome3D data, enlarging the source data set from three model organisms to 10, and adding VIVACE, a resource new to Genome3D. We have analysed and updated Genome3D's SCOP/CATH mapping. Finally, we have improved the superposition tools, which now give users a more powerful interface for investigating similarities and differences between structural models., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-D-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins.

Background: CA_C2195 from Clostridium acetobutylicum is a protein of unknown function. Sequence analysis predicted that part of the protein contained a metallopeptidase-related domain. There are over 200 homologs of similar size in large sequence databases such as UniProt, with pairwise sequence identities in the range of ~40-60%. CA_C2195 was chosen for crystal structure determination for structure-based function annotation of novel protein sequence space., Results: The structure confirmed that CA_C2195 contained an N-terminal metallopeptidase-like domain. The structure revealed two extra domains: an α+β domain inserted in the metallopeptidase-like domain and a C-terminal circularly permuted winged-helix-turn-helix domain., Conclusions: Based on our sequence and structural analyses using the crystal structure of CA_C2195 we provide a view into the possible functions of the protein. From contextual information from gene-neighborhood analysis, we propose that rather than being a peptidase, CA_C2195 and its homologs might play a role in biosynthesis of a modified cell-surface carbohydrate in conjunction with several sugar-modification enzymes. These results provide the groundwork for the experimental verification of the function.

We present a prototype of a new structural classification of proteins, SCOP2 (http://scop2.mrc-lmb.cam.ac.uk/), that we have developed recently. SCOP2 is a successor to the Structural Classification of Proteins (SCOP, http://scop.mrc-lmb.cam.ac.uk/scop/) database. Similarly to SCOP, the main focus of SCOP2 is to organize structurally characterized proteins according to their structural and evolutionary relationships. SCOP2 was designed to provide a more advanced framework for protein structure annotation and classification. It defines a new approach to the classification of proteins that is essentially different from SCOP, but retains its best features. The SCOP2 classification is described in terms of a directed acyclic graph in which nodes form a complex network of many-to-many relationships and are represented by a region of protein structure and sequence. The new classification project is expected to ensure new advances in the field and open new areas of research.

Maf (for multicopy associated filamentation) proteins represent a large family of conserved proteins implicated in cell division arrest but whose biochemical activity remains unknown. Here, we show that the prokaryotic and eukaryotic Maf proteins exhibit nucleotide pyrophosphatase activity against 5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP, which represent the most abundant modified bases in all organisms, as well as against canonical nucleotides dTTP, UTP, and CTP. Overexpression of the Maf protein YhdE in E. coli cells increased intracellular levels of dTMP and UMP, confirming that dTTP and UTP are the in vivo substrates of this protein. Crystal structures and site-directed mutagenesis of Maf proteins revealed the determinants of their activity and substrate specificity. Thus, pyrophosphatase activity of Maf proteins toward canonical and modified nucleotides might provide the molecular mechanism for a dual role of these proteins in cell division arrest and house cleaning., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The NTF2-like superfamily is a versatile group of protein domains sharing a common fold. The sequences of these domains are very diverse and they share no common sequence motif. These domains serve a range of different functions within the proteins in which they are found, including both catalytic and non-catalytic versions. Clues to the function of protein domains belonging to such a diverse superfamily can be gleaned from analysis of the proteins and organisms in which they are found., Results: Here we describe three protein domains of unknown function found mainly in bacteria: DUF3828, DUF3887 and DUF4878. Structures of representatives of each of these domains: BT_3511 from Bacteroides thetaiotaomicron (strain VPI-5482) [PDB:3KZT], Cj0202c from Campylobacter jejuni subsp. jejuni serotype O:2 (strain NCTC 11168) [PDB:3K7C], rumgna_01855) and RUMGNA_01855 from Ruminococcus gnavus (strain ATCC 29149) [PDB:4HYZ] have been solved by X-ray crystallography. All three domains are similar in structure and all belong to the NTF2-like superfamily. Although the function of these domains remains unknown at present, our analysis enables us to present a hypothesis concerning their role., Conclusions: Our analysis of these three protein domains suggests a potential non-catalytic ligand-binding role. This may regulate the activities of domains with which they are combined in the same polypeptide or via operonic linkages, such as signaling domains (e.g. serine/threonine protein kinase), peptidoglycan-processing hydrolases (e.g. NlpC/P60 peptidases) or nucleic acid binding domains (e.g. Zn-ribbons).

Background: Every genome contains a large number of uncharacterized proteins that may encode entirely novel biological systems. Many of these uncharacterized proteins fall into related sequence families. By applying sequence and structural analysis we hope to provide insight into novel biology., Results: We analyze a previously uncharacterized Pfam protein family called DUF4424 [Pfam:PF14415]. The recently solved three-dimensional structure of the protein lpg2210 from Legionella pneumophila provides the first structural information pertaining to this family. This protein additionally includes the first representative structure of another Pfam family called the YARHG domain [Pfam:PF13308]. The Pfam family DUF4424 adopts a 19-stranded beta-sandwich fold that shows similarity to the N-terminal domain of leukotriene A-4 hydrolase. The YARHG domain forms an all-helical domain at the C-terminus. Structure analysis allows us to recognize distant similarities between the DUF4424 domain and individual domains of M1 aminopeptidases and tricorn proteases, which form massive proteasome-like capsids in both archaea and bacteria., Conclusions: Based on our analyses we hypothesize that the DUF4424 domain may have a role in forming large, multi-component enzyme complexes. We suggest that the YARGH domain may play a role in binding a moiety in proximity with peptidoglycan, such as a hydrophobic outer membrane lipid or lipopolysaccharide.

Genome3D, available at http://www.genome3d.eu, is a new collaborative project that integrates UK-based structural resources to provide a unique perspective on sequence-structure-function relationships. Leading structure prediction resources (DomSerf, FUGUE, Gene3D, pDomTHREADER, Phyre and SUPERFAMILY) provide annotations for UniProt sequences to indicate the locations of structural domains (structural annotations) and their 3D structures (structural models). Structural annotations and 3D model predictions are currently available for three model genomes (Homo sapiens, E. coli and baker's yeast), and the project will extend to other genomes in the near future. As these resources exploit different strategies for predicting structures, the main aim of Genome3D is to enable comparisons between all the resources so that biologists can see where predictions agree and are therefore more trusted. Furthermore, as these methods differ in whether they build their predictions using CATH or SCOP, Genome3D also contains the first official mapping between these two databases. This has identified pairs of similar superfamilies from the two resources at various degrees of consensus (532 bronze pairs, 527 silver pairs and 370 gold pairs).

C-1 carriers are essential cofactors in all domains of life, and in Archaea, these can be derivatives of tetrahydromethanopterin (H(4)-MPT) or tetrahydrofolate (H(4)-folate). Their synthesis requires 6-hydroxymethyl-7,8-dihydropterin diphosphate (6-HMDP) as the precursor, but the nature of pathways that lead to its formation were unknown until the recent discovery of the GTP cyclohydrolase IB/MptA family that catalyzes the first step, the conversion of GTP to dihydroneopterin 2',3'-cyclic phosphate or 7,8-dihydroneopterin triphosphate [El Yacoubi, B.; et al. (2006) J. Biol. Chem., 281, 37586-37593 and Grochowski, L. L.; et al. (2007) Biochemistry46, 6658-6667]. Using a combination of comparative genomics analyses, heterologous complementation tests, and in vitro assays, we show that the archaeal protein families COG2098 and COG1634 specify two of the missing 6-HMDP synthesis enzymes. Members of the COG2098 family catalyze the formation of 6-hydroxymethyl-7,8-dihydropterin from 7,8-dihydroneopterin, while members of the COG1634 family catalyze the formation of 6-HMDP from 6-hydroxymethyl-7,8-dihydropterin. The discovery of these missing genes solves a long-standing mystery and provides novel examples of convergent evolutions where proteins of dissimilar architectures perform the same biochemical function.

The YgjD/Kae1 family (COG0533) has been on the top-10 list of universally conserved proteins of unknown function for over 5 years. It has been linked to DNA maintenance in bacteria and mitochondria and transcription regulation and telomere homeostasis in eukaryotes, but its actual function has never been found. Based on a comparative genomic and structural analysis, we predicted this family was involved in the biosynthesis of N(6)-threonylcarbamoyl adenosine, a universal modification found at position 37 of tRNAs decoding ANN codons. This was confirmed as a yeast mutant lacking Kae1 is devoid of t(6)A. t(6)A(-) strains were also used to reveal that t(6)A has a critical role in initiation codon restriction to AUG and in restricting frameshifting at tandem ANN codons. We also showed that YaeZ, a YgjD paralog, is required for YgjD function in vivo in bacteria. This work lays the foundation for understanding the pleiotropic role of this universal protein family.

The crystal structure of a putative NTPase, YP_001813558.1 from Exiguobacterium sibiricum 255-15 (PF09934, DUF2166) was determined to 1.78 Å resolution. YP_001813558.1 and its homologs (dimeric dUTPases, MazG proteins and HisE-encoded phosphoribosyl ATP pyrophosphohydrolases) form a superfamily of all-α-helical NTP pyrophosphatases. In dimeric dUTPase-like proteins, a central four-helix bundle forms the active site. However, in YP_001813558.1, an unexpected intertwined swapping of two of the helices that compose the conserved helix bundle results in a `linked dimer' that has not previously been observed for this family. Interestingly, despite this novel mode of dimerization, the metal-binding site for divalent cations, such as magnesium, that are essential for NTPase activity is still conserved. Furthermore, the active-site residues that are involved in sugar binding of the NTPs are also conserved when compared with other α-helical NTPases, but those that recognize the nucleotide bases are not conserved, suggesting a different substrate specificity.

During the past decade, the Protein Structure Initiative (PSI) centres have become major contributors of new families, superfamilies and folds to the Structural Classification of Proteins (SCOP) database. The PSI results have increased the diversity of protein structural space and accelerated our understanding of it. This review article surveys a selection of protein structures determined by the Joint Center for Structural Genomics (JCSG). It presents previously undescribed β-sheet architectures such as the double barrel and spiral β-roll and discusses new examples of unusual topologies and peculiar structural features observed in proteins characterized by the JCSG and other Structural Genomics centres.

We have identified a novel family of proteins, in which the N-terminal cystathionine beta-synthase (CBS) domain is fused to the C-terminal Zn ribbon domain. Four proteins were overexpressed in Escherichia coli and purified: TA0289 from Thermoplasma acidophilum, TV1335 from Thermoplasma volcanium, PF1953 from Pyrococcus furiosus, and PH0267 from Pyrococcus horikoshii. The purified proteins had a red/purple color in solution and an absorption spectrum typical of rubredoxins (Rds). Metal analysis of purified proteins revealed the presence of several metals, with iron and zinc being the most abundant metals (2-67% of iron and 12-74% of zinc). Crystal structures of both mercury- and iron-bound TA0289 (1.5-2.0 A resolution) revealed a dimeric protein whose intersubunit contacts are formed exclusively by the alpha-helices of two cystathionine beta-synthase subdomains, whereas the C-terminal domain has a classical Zn ribbon planar architecture. All proteins were reversibly reduced by chemical reductants (ascorbate or dithionite) or by the general Rd reductase NorW from E. coli in the presence of NADH. Reduced TA0289 was found to be capable of transferring electrons to cytochrome C from horse heart. Likewise, the purified Zn ribbon protein KTI11 from Saccharomyces cerevisiae had a purple color in solution and an Rd-like absorption spectrum, contained both iron and zinc, and was reduced by the Rd reductase NorW from E. coli. Thus, recombinant Zn ribbon domains from archaea and yeast demonstrate an Rd-like electron carrier activity in vitro. We suggest that, in vivo, some Zn ribbon domains might also bind iron and therefore possess an electron carrier activity, adding another physiological role to this large family of important proteins.