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- EMDB-7520: cryo-EM reconstruction of microtubule-bound 2R-Tau -

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Basic information

Entry
Database: EMDB / ID: EMD-7520
Titlecryo-EM reconstruction of microtubule-bound 2R-Tau
Map datacryo-EM reconstruction of shortened tau construct (WT residues: 197-304,368-400) corresponding to the first two microtubule-binding repeat sequences.
Sample
  • Complex: Ternary complex of alpha-beta tubulin with shortened tau construct
    • Protein or peptide: 2R-tau
    • Protein or peptide: alpha-tubulin
    • Protein or peptide: beta-tubulin
Biological speciesSus scrofa (pig) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 5.6 Å
AuthorsNogales E / Hejab NMA / Kellogg EH
CitationJournal: Science / Year: 2018
Title: Near-atomic model of microtubule-tau interactions.
Authors: Elizabeth H Kellogg / Nisreen M A Hejab / Simon Poepsel / Kenneth H Downing / Frank DiMaio / Eva Nogales /
Abstract: Tau is a developmentally regulated axonal protein that stabilizes and bundles microtubules (MTs). Its hyperphosphorylation is thought to cause detachment from MTs and subsequent aggregation into ...Tau is a developmentally regulated axonal protein that stabilizes and bundles microtubules (MTs). Its hyperphosphorylation is thought to cause detachment from MTs and subsequent aggregation into fibrils implicated in Alzheimer's disease. It is unclear which tau residues are crucial for tau-MT interactions, where tau binds on MTs, and how it stabilizes them. We used cryo-electron microscopy to visualize different tau constructs on MTs and computational approaches to generate atomic models of tau-tubulin interactions. The conserved tubulin-binding repeats within tau adopt similar extended structures along the crest of the protofilament, stabilizing the interface between tubulin dimers. Our structures explain the effect of phosphorylation on MT affinity and lead to a model of tau repeats binding in tandem along protofilaments, tethering together tubulin dimers and stabilizing polymerization interfaces.
History
DepositionMar 5, 2018-
Header (metadata) releaseApr 11, 2018-
Map releaseJan 16, 2019-
UpdateMay 15, 2019-
Current statusMay 15, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7520.png

Downloads & links

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Map

FileDownload / File: emd_7520.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM reconstruction of shortened tau construct (WT residues: 197-304,368-400) corresponding to the first two microtubule-binding repeat sequences.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 512 pix.
= 675.84 Å		1.32 Å/pix.
x 512 pix.
= 675.84 Å		1.32 Å/pix.
x 512 pix.
= 675.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.2 / Movie #1: 4.2
Minimum - Maximum-3.793261 - 14.217280000000001
Average (Standard dev.)0.03442833 (±1.0410348)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 675.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z675.840675.840675.840
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ208208208
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-3.79314.2170.034

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Supplemental data

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Sample components

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Entire : Ternary complex of alpha-beta tubulin with shortened tau construct

EntireName: Ternary complex of alpha-beta tubulin with shortened tau construct
Components
  • Complex: Ternary complex of alpha-beta tubulin with shortened tau construct
    • Protein or peptide: 2R-tau
    • Protein or peptide: alpha-tubulin
    • Protein or peptide: beta-tubulin

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Supramolecule #1: Ternary complex of alpha-beta tubulin with shortened tau construct

SupramoleculeName: Ternary complex of alpha-beta tubulin with shortened tau construct
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Tau construct contains first two microtubule-targeting repeat motifs and portions of projection-domain and R' sequences
Source (natural)Organism: Sus scrofa (pig)

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Macromolecule #1: 2R-tau

MacromoleculeName: 2R-tau / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
YSSPGSPGTP GSRSRTPSLP TPPTREPKKV AVVRTPPKSP SSAKSRLQTA PVPMPDLKNV KSKIGSTENL KHQPGGGKVQ IINKKLDLSN VQSKCGSKDN IKHVPGGGNK KIETHKLTFR ENAKAKTDHG AEIVYKSPVV S

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Macromolecule #2: alpha-tubulin

MacromoleculeName: alpha-tubulin / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHPE QLITGKEDAA NNYARGHYTI GKEIIDLVLD RIRKLADQCT GLQGFLVFHS FGGGTGSGFT SLLMERLSVD YGKKSKLEFS ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHPE QLITGKEDAA NNYARGHYTI GKEIIDLVLD RIRKLADQCT GLQGFLVFHS FGGGTGSGFT SLLMERLSVD YGKKSKLEFS IYPAPQVSTA VVEPYNSILT THTTLEHSDC AFMVDNEAIY DICRRNLDIE RPTYTNLNRL ISQIVSSITA SLRFDGALNV DLTEFQTNLV PYPRIHFPLA TYAPVISAEK AYHEQLSVAE ITNACFEPAN QMVKCDPRHG KYMACCLLYR GDVVPKDVNA AIATIKTKRS IQFVDWCPTG FKVGINYQPP TVVPGGDLAK VQRAVCMLSN TTAIAEAWAR LDHKFDLMYA KRAFVHWYVG EGMEEGEFSE AREDMAALEK DYEEVGVDS

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Macromolecule #3: beta-tubulin

MacromoleculeName: beta-tubulin / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDNF VFGQSGAGNN WAKGHYTEGA ELVDSVLDVV RKESESCDCL QGFQLTHSLG GGTGSGMGTL LISKIREEYP DRIMNTFSVV ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDNF VFGQSGAGNN WAKGHYTEGA ELVDSVLDVV RKESESCDCL QGFQLTHSLG GGTGSGMGTL LISKIREEYP DRIMNTFSVV PSPKVSDTVV EPYNATLSVH QLVENTDETY CIDNEALYDI CFRTLKLTTP TYGDLNHLVS ATMSGVTTCL RFPGQLNADL RKLAVNMVPF PRLHFFMPGF APLTSRGSQQ YRALTVPELT QQMFDAKNMM AACDPRHGRY LTVAAVFRGR MSMKEVDEQM LNVQNKNSSY FVEWIPNNVK TAVCDIPPRG LKMSATFIGN STAIQELFKR ISEQFTAMFR RKAFLHWYTG EGMDEMEFTE AESNMNDLVS EYQQYQDATA D

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 6.8
Component:
ConcentrationFormulaName
80.0 mMC8H18N2O6S2PIPES
1.0 mMC14H24N2O10EGTA
1.0 mMMgCl2Magnesium Chloride
1.0 mMC4H10O2S2DTT
1.0 mMGTPGuanine tri-phosphate
GridModel: C-flat-1.2/1.3 4C / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: upspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 310.15 K / Instrument: FEI VITROBOT MARK IV
Details: blot force 10 pN, 6 second blot time. used C-flat 1.2/1.3 holey grids. First 2 uL of microtubules were adhered to grid for 30 seconds, followed by 2 4 uL washes with 30 second incubation for each wash..
Detailstubulin concentration is 0.5 mg/mL, tau concentration is 1 mg/mL

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Electron microscopy

MicroscopeFEI TITAN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 310 / Average exposure time: 6.0 sec. / Average electron dose: 27.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 27500
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

Detailsused motioncorr to correct for beam-induced motion
Final reconstructionApplied symmetry - Helical parameters - Δz: 8.75 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.76 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.09) / Number images used: 16613
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Segment selectionNumber selected: 26106 / Software - Name: Appion
Details: manually selected helical segments, then filled in with overlapping boxes spaced 80 Angstrom apart.
Startup modelType of model: EMDB MAP
EMDB ID:

5194


Details: used a naked microtubule reconstruction as the initial model, low pass filtered to 20 Angstrom
Final angle assignmentType: NOT APPLICABLE / Software - Name: FREALIGN (ver. 9.09)

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