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- PDB-6qjm: Cryo-EM structure of heparin-induced 2N4R tau twister filaments -

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Basic information

Entry
Database: PDB / ID: 6qjm
TitleCryo-EM structure of heparin-induced 2N4R tau twister filaments
ComponentsMicrotubule-associated protein tauTau protein
KeywordsPROTEIN FIBRIL / Recombinant tau protein / heparin / filament / cross-beta structure
Function / homology
Function and homology information


plus-end-directed organelle transport along microtubule / axonal transport / histone-dependent DNA binding / neurofibrillary tangle assembly / positive regulation of diacylglycerol kinase activity / negative regulation of establishment of protein localization to mitochondrion / neurofibrillary tangle / positive regulation of protein localization to synapse / microtubule lateral binding / tubulin complex ...plus-end-directed organelle transport along microtubule / axonal transport / histone-dependent DNA binding / neurofibrillary tangle assembly / positive regulation of diacylglycerol kinase activity / negative regulation of establishment of protein localization to mitochondrion / neurofibrillary tangle / positive regulation of protein localization to synapse / microtubule lateral binding / tubulin complex / phosphatidylinositol bisphosphate binding / main axon / regulation of long-term synaptic depression / negative regulation of kinase activity / negative regulation of tubulin deacetylation / generation of neurons / regulation of chromosome organization / positive regulation of protein localization / rRNA metabolic process / internal protein amino acid acetylation / regulation of mitochondrial fission / lipoprotein particle binding / intracellular distribution of mitochondria / axonal transport of mitochondrion / axon development / central nervous system neuron development / regulation of microtubule polymerization / microtubule polymerization / minor groove of adenine-thymine-rich DNA binding / negative regulation of mitochondrial membrane potential / dynactin binding / glial cell projection / apolipoprotein binding / protein polymerization / negative regulation of mitochondrial fission / axolemma / Caspase-mediated cleavage of cytoskeletal proteins / regulation of microtubule polymerization or depolymerization / positive regulation of axon extension / supramolecular fiber organization / Activation of AMPK downstream of NMDARs / regulation of microtubule cytoskeleton organization / stress granule assembly / cytoplasmic microtubule organization / regulation of cellular response to heat / regulation of calcium-mediated signaling / axon cytoplasm / positive regulation of microtubule polymerization / cellular response to brain-derived neurotrophic factor stimulus / somatodendritic compartment / synapse assembly / phosphatidylinositol binding / nuclear periphery / cellular response to nerve growth factor stimulus / positive regulation of superoxide anion generation / protein phosphatase 2A binding / regulation of autophagy / astrocyte activation / synapse organization / microglial cell activation / response to lead ion / Hsp90 protein binding / regulation of synaptic plasticity / PKR-mediated signaling / protein homooligomerization / cytoplasmic ribonucleoprotein granule / memory / microtubule cytoskeleton organization / cellular response to reactive oxygen species / SH3 domain binding / neuron projection development / activation of cysteine-type endopeptidase activity involved in apoptotic process / microtubule cytoskeleton / protein-macromolecule adaptor activity / single-stranded DNA binding / cell-cell signaling / cellular response to heat / cell body / actin binding / growth cone / protein-folding chaperone binding / double-stranded DNA binding / microtubule binding / microtubule / amyloid fibril formation / sequence-specific DNA binding / dendritic spine / learning or memory / neuron projection / nuclear speck / membrane raft / axon / negative regulation of gene expression / dendrite / neuronal cell body / DNA damage response / protein kinase binding / enzyme binding / mitochondrion / DNA binding
Similarity search - Function
: / Microtubule associated protein, tubulin-binding repeat / Microtubule-associated protein Tau / Tau and MAP protein, tubulin-binding repeat / Tau and MAP proteins tubulin-binding repeat signature. / Tau and MAP proteins tubulin-binding repeat profile.
Similarity search - Domain/homology
Microtubule-associated protein tau
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsZhang, W. / Falcon, B. / Murzin, A.G. / Fan, J. / Crowther, R.A. / Goedert, M. / Scheres, S.H.W.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_A025_1013 United Kingdom
Medical Research Council (United Kingdom)MC_U105184291 United Kingdom
CitationJournal: Elife / Year: 2019
Title: Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer's and Pick's diseases.
Authors: Wenjuan Zhang / Benjamin Falcon / Alexey G Murzin / Juan Fan / R Anthony Crowther / Michel Goedert / Sjors Hw Scheres /
Abstract: 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 ...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.
History
DepositionJan 24, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 27, 2019Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Microtubule-associated protein tau
B: Microtubule-associated protein tau
C: Microtubule-associated protein tau


Theoretical massNumber of molelcules
Total (without water)15,5673
Polymers15,5673
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: assay for oligomerization, We used Thioflavin T fluorescence to check the kinetic assays of the filament assembly., microscopy, We used negative-stain electron microscopy to check the ...Evidence: assay for oligomerization, We used Thioflavin T fluorescence to check the kinetic assays of the filament assembly., microscopy, We used negative-stain electron microscopy to check the morphology of assembled filaments.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7080 Å2
ΔGint-19 kcal/mol
Surface area7620 Å2
MethodPISA

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Components

#1: Protein/peptide Microtubule-associated protein tau / Tau protein / Neurofibrillary tangle protein / Paired helical filament-tau / PHF-tau


Mass: 5189.081 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAPT, MAPTL, MTBT1, TAU / Plasmid: pRK172 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P10636

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: heparin-induced 2N4R tau twister filaments / Type: COMPLEX
Details: Recombinant tau protein was induced into filaments by incubation with heparin at 37 C for 3 days
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 45.85 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: PRK172
Buffer solutionpH: 7.4 / Details: 20 mM Tris, pH 7.4, 100mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
10.02 mol/Ltris(hydroxymethyl)aminomethaneTris1
20.1 mol/Lsodium chlorideNaClSodium chloride1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Recombinant tau protein was induced into filaments by incubation with heparin at 37 C for 3 days. The filaments were pronase-treated before making Cryo-grids.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blot force: -12 ; Blot time: 4s

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2800 nm / Nominal defocus min: 1700 nm / Cs: 2 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: GATAN LIQUID NITROGEN
Image recordingAverage exposure time: 1 sec. / Electron dose: 48 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 717
Details: Images were collected in movie-mode at 30 frames per second
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2EPU1.5.0.1243RELimage acquisition
4CTFFIND4.1CTF correction
7Coot0.8.9.1model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13REFMAC5.8.0236model refinement
14PHENIXdev-2919-000model refinement
Image processingDetails: Movie frames were gain-corrected, aligned, dose weighted and then summed into a single micrograph using MOTIONCOR2 (Zheng et al., 2017)
CTF correctionDetails: Aligned, non-dose-weighted micrographs were used to estimate the contrast transfer function (CTF) using CTFFIND4.1
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -3.38 ° / Axial rise/subunit: 4.7 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 187555 / Details: Manually picked
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141461 / Algorithm: FOURIER SPACE / Details: we performed two rounds of 3D auto-refinement. / Num. of class averages: 6 / Symmetry type: HELICAL
Atomic model buildingB value: 58.51 / Protocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Fourier shell correlation
Details: A stack of three consecutive monomers was refined to preserve nearest-neighbour interactions for the middle chain. Side-chain clashes were detected using MOLPROBITY, and corrected by ...Details: A stack of three consecutive monomers was refined to preserve nearest-neighbour interactions for the middle chain. Side-chain clashes were detected using MOLPROBITY, and corrected by iterative cycles of real-space refinement in COOT and Fourier-space refinement in REFMAC and PHENIX. For each refined structure, separate model refinements were performed against a single half-map, and the resulting model was compared to the other half-map to confirm the absence of overfitting.

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