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- EMDB-7977: Preformed GMPCPP microtubule washed with EB3, class 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-7977
TitlePreformed GMPCPP microtubule washed with EB3, class 2
Map dataPreformed GMPCPP microtubule washed with EB3, class 2
Sample
  • Organelle or cellular component: Preformed GMPCPP microtubule washed with EB3, class 2
    • Organelle or cellular component: microtubule
      • Protein or peptide: alpha tubulin
      • Protein or peptide: beta tubulin
    • Complex: EB3
      • Protein or peptide: EB3
Biological speciesSus scrofa (pig) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsZhang R / Nogales E
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM051487 United States
Howard Hughes Medical Institute (HHMI) United States
National Science Foundation (NSF, United States)1106400 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Separating the effects of nucleotide and EB binding on microtubule structure.
Authors: Rui Zhang / Benjamin LaFrance / Eva Nogales /
Abstract: Microtubules (MTs) are polymers assembled from αβ-tubulin heterodimers that display the hallmark behavior of dynamic instability. MT dynamics are driven by GTP hydrolysis within the MT lattice, and ...Microtubules (MTs) are polymers assembled from αβ-tubulin heterodimers that display the hallmark behavior of dynamic instability. MT dynamics are driven by GTP hydrolysis within the MT lattice, and are highly regulated by a number of MT-associated proteins (MAPs). How MAPs affect MTs is still not fully understood, partly due to a lack of high-resolution structural data on undecorated MTs, which need to serve as a baseline for further comparisons. Here we report three structures of MTs in different nucleotide states (GMPCPP, GDP, and GTPγS) at near-atomic resolution and in the absence of any binding proteins. These structures allowed us to differentiate the effects of nucleotide state versus MAP binding on MT structure. Kinesin binding has a small effect on the extended, GMPCPP-bound lattice, but hardly affects the compacted GDP-MT lattice, while binding of end-binding (EB) proteins can induce lattice compaction (together with lattice twist) in MTs that were initially in an extended and more stable state. We propose a MT lattice-centric model in which the MT lattice serves as a platform that integrates internal tubulin signals, such as nucleotide state, with outside signals, such as binding of MAPs or mechanical forces, resulting in global lattice rearrangements that in turn affect the affinity of other MT partners and result in the exquisite regulation of MT dynamics.
History
DepositionJun 9, 2018-
Header (metadata) releaseJul 4, 2018-
Map releaseJul 4, 2018-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7977.png

Downloads & links

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Map

FileDownload / File: emd_7977.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPreformed GMPCPP microtubule washed with EB3, class 2
Voxel sizeX=Y=Z: 1.33 Å
Density
Contour LevelBy AUTHOR: 4 / Movie #1: 4
Minimum - Maximum-8.535764 - 19.940113
Average (Standard dev.)-0.035690907 (±0.90642476)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 680.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.331.331.33
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z680.960680.960680.960
α/β/γ90.00090.00090.000
start NX/NY/NZ-51-35-11
NX/NY/NZ11110799
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-8.53619.940-0.036

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Supplemental data

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Sample components

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Entire : Preformed GMPCPP microtubule washed with EB3, class 2

EntireName: Preformed GMPCPP microtubule washed with EB3, class 2
Components
  • Organelle or cellular component: Preformed GMPCPP microtubule washed with EB3, class 2
    • Organelle or cellular component: microtubule
      • Protein or peptide: alpha tubulin
      • Protein or peptide: beta tubulin
    • Complex: EB3
      • Protein or peptide: EB3

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Supramolecule #1: Preformed GMPCPP microtubule washed with EB3, class 2

SupramoleculeName: Preformed GMPCPP microtubule washed with EB3, class 2 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: microtubule

SupramoleculeName: microtubule / type: organelle_or_cellular_component / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Sus scrofa (pig)

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Supramolecule #3: EB3

SupramoleculeName: EB3 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria) / Recombinant strain: K-12

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Macromolecule #1: alpha tubulin

MacromoleculeName: alpha tubulin / type: protein_or_peptide / ID: 1 / 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 DYEEVGVDSV EGEGEEEGEE Y

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Macromolecule #2: beta tubulin

MacromoleculeName: beta tubulin / type: protein_or_peptide / ID: 2 / 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 DEQGEFEEEG EEDEA

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Macromolecule #3: EB3

MacromoleculeName: EB3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MAVNVYSTSV TSENLSRHDM LAWVNDSLHL NYTKIEQLCS GAAYCQFMDM LFPGCVHLRK VKFQAKLEHE YIHNFKVLQA AFKKMGVDKI IPVEKLVKGK FQDNFEFIQW FKKFFDANYD GKDYNPLLAR QGQDVAPPPN PGDQIFNKSK KLIGTAVPQR TSPTGPKNMQ ...String:
MAVNVYSTSV TSENLSRHDM LAWVNDSLHL NYTKIEQLCS GAAYCQFMDM LFPGCVHLRK VKFQAKLEHE YIHNFKVLQA AFKKMGVDKI IPVEKLVKGK FQDNFEFIQW FKKFFDANYD GKDYNPLLAR QGQDVAPPPN PGDQIFNKSK KLIGTAVPQR TSPTGPKNMQ TSGRLSNVAP PCILRKNPPS ARNGGHETDA

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration3 mg/mL
BufferpH: 6.8
Details: BRB80 buffer (80 mM PIPES, pH 6.8, 1 mM EGTA, 1 mM MgCl2) supplemented with 1 mM GMPCPP, 1 mM DTT, and 0.05% Nonident P-40
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 4 seconds before plunging..
DetailsPreformed GMPCPP microtubules were washed with EB3

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Electron microscopy

MicroscopeFEI TITAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.7 µm / Calibrated defocus min: 0.8 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-20 / Number grids imaged: 1 / Number real images: 182 / Average exposure time: 6.0 sec. / Average electron dose: 1.44 e/Å2

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Image processing

CTF correctionSoftware - Name: Gctf
Final angle assignmentType: NOT APPLICABLE / Software - Name: FREALIGN
Final reconstructionNumber classes used: 2
Applied symmetry - Helical parameters - Δz: 8.805 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.787 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN / Number images used: 13159

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