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• W4225585148 abstract "Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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. Editor's evaluation In this paper 76 cryo-EM structures of recombinant tau filaments assembled in vitro are described. This is a scientific tour-de-force, and will provide an immense database that can be used by everyone working in the amyloid field. When this knowledge is combined with the structure of tau filaments in vivo, it will help shape the design of laboratory experiments in the future to generate the conditions to replicate the in vivo forms in vitro. https://doi.org/10.7554/eLife.76494.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Many neurodegenerative diseases, including Alzheimer’s disease, the most common form of dementia, are characterised by knotted clumps of a protein called tau. In these diseases, tau misfolds, stacks together and forms abnormal filaments, which have a structured core and fuzzy coat. These sticky, misfolded proteins are thought to be toxic to brain cells, the loss of which ultimately causes problems with how people move, think, feel or behave. Reconstructing the shape of tau filaments using an atomic-level imaging technique called electron cryo-microscopy, or cryo-EM, researchers have found distinct types of tau filaments present in certain diseases. In Alzheimer’s disease, for example, a mixture of paired helical and straight filaments is found. Different tau filaments are seen again in chronic traumatic encephalopathy (CTE), a condition associated with repetitive brain trauma. It remains unclear, however, how tau folds into these distinct shapes and under what conditions it forms certain types of filaments. The role that distinct tau folds play in different diseases is also poorly understood. This is largely because researchers making tau proteins in the lab have yet to replicate the exact structure of tau filaments found in diseased brain tissue. Lövestam et al. describe the conditions for making tau filaments in the lab identical to those isolated from the brains of people who died from Alzheimer’s disease and CTE. Lövestam et al. instructed bacteria to make tau protein, optimised filament assembly conditions, including shaking time and speed, and found that bona fide filaments formed from shortened versions of tau. On cryo-EM imaging, the lab-produced filaments had the same left-handed twist and helical symmetry as filaments characteristic of Alzheimer’s disease. Adding salts, however, changed the shape of tau filaments. In the presence of sodium chloride, otherwise known as kitchen salt, tau formed filaments with a filled cavity at the core, identical to tau filaments observed in CTE. Again, this structure was confirmed on cryo-EM imaging. Being able to make tau filaments identical to those found in human tauopathies will allow scientists to study how these filaments form and elucidate what role they play in disease. Ultimately, a better understanding of tau filament formation could lead to improved diagnostics and treatments for neurodegenerative diseases involving tau. Introduction Six tau isoforms that range from 352 to 441 amino acids in length are expressed in adult human brain from a single gene by alternative mRNA splicing (Goedert et al., 1989). The tau sequence can be separated into an N-terminal projection domain (residues 1–150), a proline-rich region (residues 151–243), the microtubule-binding repeat region (residues 244–368), and a C-terminal domain (residues 369–441). Isoforms differ by the presence or absence of 1 or 2 inserts of 29 amino acids each, in the N-terminal region (0N, 1N or 2N), and the presence or absence of the second of four microtubule-binding repeats of 31 or 32 amino acids each (resulting in three-repeat, 3R, and four-repeat, 4R, isoforms). The second N-terminal insert is only expressed together with the first. Tau filaments can differ in isoform composition in disease (Goedert et al., 2017). Thus, a mixture of all six isoforms is present in the tau filaments of Alzheimer’s disease (AD), chronic traumatic encephalopathy (CTE), and other diseases; in Pick’s disease (PiD), filaments are composed of only 3R tau isoforms; and in progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), globular glial tauopathy (GGT), argyrophilic grain disease (AGD), and other tauopathies, filaments are composed of only 4R tau isoforms. Tau filaments consist of a structured core made mostly of the repeat domain, with less structured N- and C-terminal regions forming the fuzzy coat (Goedert et al., 1988; Wischik et al., 1988a). Previously, we and others used electron cryo-microscopy (cryo-EM) to determine the atomic structures of the cores of tau filaments extracted from the brains of individuals with a number of neurodegenerative diseases (Fitzpatrick et al., 2017; Falcon et al., 2018a; Falcon et al., 2019; Falcon et al., 2019; Arakhamia et al., 2021; Zhang et al., 2020; Shi et al., 2021). Whereas, tau filaments from different individuals with a given disease share the same structures, different diseases tend to be characterised by distinct tau folds. Identical protofilaments can arrange in different ways to give rise to distinct filaments (ultrastructural polymorphs). In AD, two protofilaments can combine in a symmetrical way to form paired helical filaments (PHFs), or in an asymmetrical manner to form straight filaments (SFs). The extent of the ordered cores of the protofilament folds explains the isoform composition of tau filaments. Alzheimer and CTE folds consist almost entirely of R3 and R4; the Pick fold comprises most of R1, as well as all of R3 and R4; and the CBD, PSP, GGT, and AGD folds comprise the whole of R2, R3, and R4. All known tau folds also contain residues 369–379/380 from the C-terminal domain. Based on these findings, we proposed a hierarchical classification of tauopathies, which complements clinical and neuropathological characterisation, and allows identification of new disease entities (Shi et al., 2021). Nevertheless, it remains unclear what roles distinct tau folds play in different diseases and what factors determine structural specificity. In human diseases, full-length tau assembles into filaments intracellularly (Goedert et al., 2017). Proteolytic cleavage of the fuzzy coat is characteristic of extracellular ghost tangles (Endoh et al., 1993). Yet, full-length recombinant tau is highly soluble and in vitro assembly into filaments requires the addition of anionic co-factors, such as sulphated glycosaminoglycans, RNA, fatty acids, and polyglutamate (Goedert et al., 1996; Kampers et al., 1996; Pérez et al., 1996; Wilson and Binder, 1997; Friedhoff et al., 1998) or the use of harsh shaking conditions with small beads and sodium azide (Chakraborty et al., 2021). Whether the structures of filaments assembled in vitro resemble those observed in disease requires verification by structure determination using solid-state NMR or cryo-EM. We previously showed that the addition of heparin to full-length 3R or 4R recombinant tau expressed in Escherichia coli led to the formation of polymorphic filaments, with structures that are unlike those in disease (Zhang et al., 2019). Below, we showed that the addition of RNA or phosphoserine to full-length recombinant 4R tau also led to filament structures that are different from those observed in disease thus far. The mechanisms of templated seeding, where filaments provide a template for the incorporation of tau monomers, thus resulting in filament growth, underlies the hypothesis of prion-like spreading of tau pathology. It is therefore possible that in vitro assembly of disease-relevant structures may also require the presence of a seed. Protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT-QuIC) are commonly used (Soto et al., 2002; Saijo et al., 2020). These techniques use protein aggregates from brain as a template to seed the assembly of filaments from recombinant proteins. However, they may not always amplify the predominant filamentous assemblies. Therefore, structural verification of seeds and seeded aggregates is required. For α-synuclein and immunoglobulin light-chain, cryo-EM has shown that using similar amplification methods, the structures of seeded filaments differed from those of the seeds under the conditions used (Burger et al., 2021; Lövestam et al., 2021; Radamaker et al., 2021). Full-length human tau is highly soluble, but truncated proteins encompassing the repeat region readily assemble into filaments that resemble AD PHFs by negative staining. A fragment consisting of residues 250–378, excluding R2, formed filaments when using the hanging drop approach, a method commonly used in protein crystallography (Crowther et al., 1992). The same was true of K11 and K12 constructs (tau residues 244–394 for K11, and the same sequences, excluding R2, for K12) (Wille et al., 1992). Moreover, proteins comprising the ordered cores of tau filaments from AD [residues 306–378], PiD [residues 254–378, but excluding R2], and CBD [residues 274–380] have been reported to assemble into filaments (Carlomagno et al., 2021). It remains to be established if the structures of these filaments resembled those from diseased brain. Multiple studies have also used constructs K18 and K19, which comprise the microtubule-binding repeats and four amino acids after the repeats (residues 244–372 for K18, and the same sequences, excluding R2, for K19); they spontaneously assemble into filaments (Gustke et al., 1994; Mukrasch et al., 2005; von Bergen et al., 2006; Li et al., 2009; Yu et al., 2012; Shammas, 2015) and are seeding-active by RT-QuIC (Saijo et al., 2020). However, since all known tau structures from human brain include residues beyond 372, tau filament structures of K18 and K19 cannot be the same as those in disease. Another fragment that has been used for in vitro assembly studies is dGAE, which comprises residues 297–391 of 4R tau, and was identified as the proteolytically stable core of PHFs from tangle fragments of AD (Wischik et al., 1988b). It assembles into filaments with similar morphologies to AD PHFs by negative staining EM and atomic force microscopy (Novak et al., 1993; Al-Hilaly et al., 2017; Al-Hilaly et al., 2020; Lutter et al., 2022). Here, we report conditions that lead to the formation of AD PHFs from purified tau (297–391) expressed in E. coli. We show that the same construct can also be used to form type II filaments of CTE, and demonstrate how different cations affect the differences between the structures of the two diseases. In addition, we describe to what extent the tau (297–391) fragment can be extended, as well as shortened, while still forming PHFs. We report 76 cryo-EM structures, including those of 27 previously unobserved filaments. Table 1 gives an overview of assembly conditions (numbered) and filament types (indicated with letters). Figure 1 and Figure 1—figure supplements 1–7 describe the cryo-EM structures that were determined. Our results illustrate how high-throughput cryo-EM structure determination can guide the quest for understanding the molecular mechanisms of amyloid filament formation. Figure 1 with 8 supplements see all Download asset Open asset New electron cryo-microscopy (cryo-EM) structures. Backbone traces for filaments with previously unobserved structures. Residues 244–274 (R1) are shown in purple; residues 275–305 (R2) are shown in blue; residues 306–336 (R3) are shown in green; residues 337–368 (R4) are shown in yellow; residues 369–441 (C-terminal domain) are shown in orange. The filament types (as defined in Table 1) are shown at the top left of each structure. Table 1 In vitro assembly conditions for all filament types. Filament typesConstruct (residues)BufferShaking (rpm)Time(hr)Fold1a297–39110 mM PB 10 mM DTT pH 7.470048New2a–d297–39110 mM PB 10 mM DTT pH 7.420048AD3a297–39110 mM PB 10 mM DTT pH 7.40.1 μg /ml dextran sulphate20048AD4a297–39110 mM PB 10 mM DTT pH 7.4 200 mM MgCl220048AD5a297–39110 mM PB 10 mM DTT pH 7.4 20 mM CaCl220048AD6a–c266/297–391*10 mM PB 10 mM DTT pH 7.420048AD7a–b266–273 –39110 mM PB 10 mM DTT pH 7.4 200 mM MgCl220048AD8a–b266/297–39110 mM PB pH 7.4 10 mM DTT 200 mM NaCl20048CTE9a–b266/297–39110 mM PB pH 7.4 10 mM DTT 200 mM LiCl20048New10a–b266/297–39110 mM PB pH 7.4 10 mM DTT 200 mM KCl20048New11a266/297–39110 mM PB pH 7.4 10 mM DTT 100 μM ZnCl220048New12a266/297–39110 mM PB pH 7.4 10 mM DTT 200 μM CuCl220048New13a266/297–39110 mM PB pH 7.4 10 mM DTT 20 mM MgCl2 50 mM KCl 50 mM NaCl20048New14a–b266/297–39110 mM PB pH 7.4 10 mM DTT 20 mM MgCl2 100 mM NaCl20048New15a–d266/297–39110 mM PB pH 7.4 10 mM DTT 10 mM MgSO4 100 mM NaCl20048CTE16a–b266/297–39110 mM PB pH 7.4 10 mM DTT 10 mM NaHCO3 100 mM NaCl20048New17a–c266/297–39110 mM PB pH 7.4 10 mM DTT 500 mM NaCl20048New18a244–39150 mM PB pH 7.4 10 mM DTT 20 mM MgCl220076New19a244–39150 mM PB pH 7.4 10 mM DTT 200 mM NaCl20076New20a244–39110 mM PB 10 mM DTT 5 mM Na4P2O720076New21a–b258–39110 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New22a266–39110 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New23a–c266–391PBS pH 7.4 10 mM DTT20048CTE24a287–39110 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD25a300–39110 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD26a303–39110 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD27a305–37910 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New28a297–42110 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New29a297–41210 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New30a297–40210 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New31a297–39610 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New32a–b297–39410 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD33a297–38410 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD34a–b297–39410 mM PB pH 7.4 10 mM DTT70048AD35a–d297–394PBS pH 7.4 10 mM DTT70048New36a–c300–391PBS pH 7.4 10 mM DTT70048New37a303–391PBS pH 7.4 10 mM DTT70048New38a258–39110 mM PB pH 7.4 10 mM DTT70048GGT39a–b258–39110 mM PB pH 7.4 10 mM DTT 5 mM phosphoglycerate70048GGT40a258–39110 mM PB pH 7.4 10 mM DTT 300 ug/ul heparan sulphate70048GGT41a258–39110 mM PB pH 7.4 10 mM DTT 0.1% NaN370048New42a–b297–408 4-pmm*10 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD43a297–441 4-pmm10 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD44a266–391 S356D10 mM PB pH 7.4 10 mM DTT 200 mM KCl20048New45a266–391 S356D10 mM PB pH 7.4 10 mM DTT 200 mM NaCl20048New46a0N4RPBS pH 7.4 5 mM TCEP 50 ug/mL polyA RNA20096New47a0N4RPBS pH 7.4 5 mM TCEP 5 mM L-phosphoserine20096New DTT: 1,4-dithiothreitol PB: Na2HPO4, NaH2PO4; Dextran sulphate: molecular weight 7–20 kDa (9011-18-1, Sigma-Aldrich); Heparan sulphate: 50–200 disaccharide units (57459-72-0, Sigma-Aldrich); Poly-A RNA (26763-19-9, Sigma-Aldrich); PBS: Phospho-buffered saline; TCEP: Tris(2-carboxyethyl) phosphine; *4-pmm: four-phospho mimetic mutations: S396D S400D T403D S404D *PB: Na2HPO4, NaH2PO4;*266/297–391: 50:50 ratio of 266LKHQ269 (3 R) and 297IKHV300 (4 R) –391. Fold: AD: Alzheimer’s Disease protofilament fold, CTE: chronic traumatic encephalopathy protofilament fold and GGT: globular glial tauopathy-like fold, New: new tau fold. Results In vitro assembly of tau (297–391) into PHFs We first performed in vitro assembly of tau (297–391) in 10 mM phosphate buffer (PB) containing 10 mM dithiothreitol (DTT), with shaking at 700 rpm, as described (Al-Hilaly et al., 2017). We will refer to this as assembly condition 1. Filaments formed within 4 hr, as indicated by ThT fluorescence. Cryo-EM imaging after 48 hr revealed a single type of filament comprising two protofilaments that were related by pseudo-21 helical screw symmetry. Although the extent and topology of the ordered cores resembled those of the protofilaments of AD and CTE, the protofilament cores were extended, rather than C-shaped (Figure 2A–C; filament type 1a). Figure 2 with 1 supplement see all Download asset Open asset Assembly of recombinant tau into filaments like Alzheimer’s disease paired helical filaments (AD PHFs). (A) Schematic of 2N4R tau sequence with domains highlighted. The regions 1N (44–73), 2N (74–102), P1 (151–197), and P2 (198–243) are shown in increasingly lighter greys; R1 (244–274) is shown in purple; R2 (275–305) is shown in blue; R3 (306–336) is shown in green; R4 (337–368) is shown in yellow; the C-terminal domain (369-441) is shown in orange. (B) Amino acid sequence of residues 244–441 of tau, with the same colour scheme as in A. (C–E:) Projected slices, with a thickness of approximately 4.7 Å, orthogonal to the helical axis for several cryo-microscopy (cryo-EM) reconstructions. The filament types (as defined in Table 1) are shown at the bottom left and the percentages of types for each cryo-EM data set are given at the top right of the images. (C) Conditions of Al-Hilaly et al., 2017, with shaking at 700 rpm. (D) Our adapted protocol, using 200 rpm shaking. From left to right; paired helical filament (PHF), triple helical filament, quadruple helical filament type 1, and quadruple helical filament type 2. (E) Our optimised conditions for in vitro assembly of relatively pure PHFs.( F) Cryo-EM density map (grey transparent) of in vitro assembled tau filaments of type 4a and the atomic model colour coded according as in A. (G) Backbone ribbon of in vitro PHF (grey) overlaid with AD PHF (blue). We then reduced the shaking speed to 200 rpm. This resulted in a slower assembly reaction, with filaments appearing after 6 hr. Cryo-EM structure determination after 48 hr showed that the filaments were polymorphic. They shared the AD protofilament fold, spanning residues 305–378. However, besides AD PHFs, we also observed filaments comprising three or four protofilaments, which we called triple helical filaments (THFs) and quadruple helical filaments (QHFs) (Figure 2D; filament types 2b–d). THFs and QHFs have not been observed in brain extracts from individuals with AD (Fitzpatrick et al., 2017; Falcon et al., 2018b), possibly because the presence of the fuzzy coat would hinder their formation. SFs were not seen. THFs consist of a PHF and an additional single protofilament with the AD fold, whereas QHFs are made of two stacked PHFs that come in two different arrangements (types 1 and 2). The cryo-EM maps of QHF type 1 filaments were of sufficient quality to build atomic models. QHF type 1 and the THF share a common interface, where one protofilament forms a salt bridge at E342 with K343 from the third, adjoining protofilament. This protofilament remains on its own in THFs, whereas it forms a typical PHF interface with a fourth protofilament in QHFs. Although the cryo-EM reconstruction of the QHF type 2 filament was of insufficient resolution for atomic modelling, the cross-section perpendicular to the helical axis suggested that a salt bridge was present between E342 from one PHF and K321 from another (Figure 1—figure supplement 1; filament types 2b–d). Next, we sought to optimise the assembly conditions. Since THFs and QHFs have not been observed in AD, their formation would confound the use of this assembly assay for screens or to model diseases. In addition, filaments from the above experiments tended to stick together, which complicated their cryo-EM imaging, and could interfere with their use. Over longer incubation periods (>76 hr), filaments tended to precipitate, resulting in cloudy solutions. To assemble tau (297–391) into pure PHFs and reduce stickiness, we explored the addition of salts and crowding reagents. In particular, the addition of 200 mM MgCl2, 20 mM CaCl2, or 0.1 μg/ml dextran sulphate resulted in purer populations of PHFs (~95%) (Figure 2E; filament types 3–5). Cryo-EM structure determination of filaments made using these conditions confirmed that their ordered cores were identical, with a root mean square deviation (r.m.s.d) of all non-hydrogen atoms of 1.3 Å, to those of AD PHFs (Figure 2F–G). Tau (297–391) comprises the C-terminal 9 residues of R2, the whole of R3 and R4, as well as 23 amino acids after R4. The equivalent 3R tau construct, which lacks R2, begins with the C-terminal 9 residues of R1, the first four of which (266LKHQ269) are different from R2. We also assembled tau (266–391), excluding R2, in the presence of 200 mM MgCl2, as well as a 50:50 mixture of the 3R/4R tau constructs, in the absence of MgCl2. We observed PHFs, THFs, and QHFs in the absence of MgCl2, whereas assembly in the presence of 200 mM MgCl2 gave rise to AD PHFs with a purity greater than 94% (Figure 1—figure supplement 1; filament types 6 and 7). These findings show that bona fide PHFs can be formed from only 3R or 4R, albeit truncated, tau. In AD and CTE, all six isoforms, each full-length, are present in tau filaments (Goedert et al., 1992; Schmidt et al., 2001). It remains to be determined if there are PHFs in human diseases that are made of only 3R or 4R tau. The cryo-EM structures of in vitro assembled PHFs and of AD PHFs shared the same left-handed twist and pseudo-21 helical screw symmetry. Moreover, there were similar additional densities in front of lysine residues 317 and 321, and on the inside of the protofilament’s C, as we previously observed for AD PHFs (Figure 2—figure supplement 1). The assembly buffers contained only Na2HPO4, NaH2PO4, MgCl2, and DTT. Although we cannot exclude the possibility that negatively charged co-factors may have purified together with recombinant tau, it appears more likely that the additional densities arose from phosphate ions in the buffer. The phosphates’ negative charges may have counteracted the positive charges of stacked lysines. It remains to be established if similar densities in AD PHFs also correspond to phosphate ions, or if other negatively charged co-factors or parts of the fuzzy coat may play a role. The fuzzy coat, which consists of only a few residues on either side of the ordered core, is not visible in cryo-EM micrographs of in vitro assembled tau (297–391) (Figure 2—figure supplement 1). The effects of salts on tau filament assembly During optimisation of assembly, we noticed that different cations in the buffer caused the formation of filaments with distinct protofilament folds. Besides MgCl2 and CaCl2, which led to the formation of AD PHFs, we also explored the effects of ZnCl2, CuCl2, NaCl, LiCl, and KCl (Figure 3a). Addition of ZnCl2 resulted in the same fold as observed for filaments assembled using condition 1, whereas addition of CuCl2 led to folds with little resemblance to previously observed tau folds (Figure 1 and Figure 1—figure supplement 1; filament types 11 and 12). Cu2+ ions led to the formation of intermolecular disulphide bonds that were part of the ordered cores of these filaments. Figure 3 with 3 supplements see all Download asset Open asset Assembly of recombinant tau into filaments like chronic traumatic encephalopathy (CTE) type II filaments. (A) Projected slices, with a thickness of approximately 4.7 Å, orthogonal to the helical axis are shown for different assembly conditions and filament types (as defined in Table 1), which are indicated in the bottom left. The percentages of types are shown in the top right of each panel. ( B) Cryo-EM density map (grey transparent) of filament type 8a and the corresponding atomic model with the same colour scheme as in Figure 1. (C–E) Backbone ribbon views of protofilament and filament folds. (C) In vitro NaCl filament type 8a (grey) overlaid with CTE type II (orange). (D) Extended and C-shaped protofilaments aligned at residues 338–354 for LiCl, filament types 9a and 9b (left) and NaCl, filament types 8a and 8b (right). (E) Filament types 8b (NaCl), 9a (LiCl), 10a (KCl), and 4a (MgCl2) aligned at residues 356–364. (F) Atomic view of residues 334–358. The distance between the Cα of L344 and I354 is indicated. Filament types 8a, 8b, 9a, 9b, and 10a are shown in light purple, dark purple, dark orange, light orange, and blue, respectively. Monovalent cations modulated the formation of protofilament folds that were similar or identical to AD and CTE folds. The CTE fold is similar to the AD fold, in that it also comprises a double-layered arrangement of residues 274/305–379; however, it adopts a more open C-shaped conformation and comprises a larger cavity at the tip of the C (residues 338–354), which is filled with an additional, unknown density (Falcon et al., 2019). We first describe how different monovalent cations led to the formation of both C-shaped and more extended protofilament folds. We then present the effects of cations on the additional density in the cavity and the conformations of the surrounding residues. Addition of 200 mM NaCl led to the formation of two types of filaments. The first type was identical, with an all-atom r.m.s.d. of 1.4 Å to CTE type II filaments (Figure 3A–C; filament type 8a); in the second type (filament type 8b), two identical protofilaments with a previously unobserved, extended protofilament fold packed against each other with pseudo-21 helical symmetry. This fold resembled the extended fold observed when using condition 1. The extended fold concurred with a flipping of the side chains of residues 322–330, which were alternatively buried in the core or solvent-exposed in opposite manner to the CTE fold. Side chains before and after 364PGGG367 had the same orientations, but formed a 90° turn in the CTE fold and adopted a straight conformation in the extended fold. Residues 338–354 had identical conformations, with an all-atom r.m.s.d for these residues of 1.3 Å, at the tips of the C-shaped and extended folds (Figure 1; Figure 3—figure supplement 1). When adding 200 mM LiCl, we observed two types of filaments, with either C-shaped or more extended protofilament folds (Figure 3A; filament types 10a and 10b). In the first type, two C-shaped protofilaments packed against each other in an asymmetrical manner. In the second type, two protofilaments with an extended conformation packed against each other with pseudo-21 helical symmetry. As observed for the filaments obtained with NaCl, the side chain orientations of residues 322–330 differed between folds. However, whereas the side chain of H330 was buried in the core of the C-shaped protofilament formed with NaCl, it was solvent-exposed in the C-shaped protofilament formed with LiCl. This suggests that the conformation of the 364PGGG367 motif defines the extended or C-shaped conformation (Figure 3—figure supplement 2). Addition of 200 mM KCl also led to two different filaments with either extended or C-shaped protofilament folds. However, in this case, low numbers of filaments with extended protofilaments resulted in poor cryo-EM reconstructions. The filaments with C-shaped folds comprised three protofilaments, which packed against each other with C3 symmetry (Figure 1; Figure 3A; filament type 10a). For each monovalent cation, residues 338–354 adopted identical conformations when comparing extended and C-shaped protofilament folds (Figure 3D). These residues surrounded the cavity at the tip of the fold, which was filled with an additional density in the CTE fold. Additional densities were also observed in filaments formed in the presence of NaCl, KCl, and in the extended filaments formed with LiCl. The cryo-EM reconstructions of the threefold symmetric filaments formed with KCl, with a resolution of 1.9 Å, showed multiple additional spherical densities inside the protofilament core. Besides additional densities for what were probably water molecules in front of several asparagines and glutamines, the cavity at the tip of the fold contained two larger, separate spherical densities per β-rung, which were 3.1 Å apart, and at approximately 3.0–4.5 Å distance from S341 and S352, the only polar residues in the cavity. Another pair of additional densities, similar in size to those inside the cavity, was present at a distance of 2.6 Å from the carbonyl of G335 (Figure 3—figure supplement 3). Below, we will argue that these densities corresponded to pairs of K+ and Cl− ions. Reconstructions for the filaments formed with NaCl were at resolutions of 2.8 and 3.3 Å. The additional density in these maps was not separated into two spheres, but was present as one larger blob per rung, with separation between blobs along the helical axis. Filaments formed with LiCl were resolved to resolutions of 3.1 and 3.4 Å. No additional densities were present inside the cavity of the C-shaped fold, but the cavity in the extended fold contained a spherical density that was smaller than the densities observed for NaCl and" @default.
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• W4225585148 title "Author response: Assembly of recombinant tau into filaments identical to those of Alzheimer’s disease and chronic traumatic encephalopathy" @default.
• W4225585148 doi "https://doi.org/10.7554/elife.76494.sa2" @default.
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