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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-7977 | ||||||||||||
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Title | Preformed GMPCPP microtubule washed with EB3, class 2 | ||||||||||||
![]() | Preformed GMPCPP microtubule washed with EB3, class 2 | ||||||||||||
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Biological species | ![]() ![]() ![]() | ||||||||||||
Method | helical reconstruction / cryo EM / Resolution: 4.1 Å | ||||||||||||
![]() | Zhang R / Nogales E | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 152.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 15.9 KB 15.9 KB | Display Display | ![]() |
Images | ![]() | 160.1 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7973C ![]() 7974C ![]() 7975C ![]() 7976C ![]() 6dpuC ![]() 6dpvC ![]() 6dpwC C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Preformed GMPCPP microtubule washed with EB3, class 2 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.33 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Preformed GMPCPP microtubule washed with EB3, class 2
Entire | Name: Preformed GMPCPP microtubule washed with EB3, class 2 |
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Components |
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-Supramolecule #1: Preformed GMPCPP microtubule washed with EB3, class 2
Supramolecule | Name: 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
Supramolecule | Name: microtubule / type: organelle_or_cellular_component / ID: 2 / Parent: 1 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: ![]() ![]() |
-Supramolecule #3: EB3
Supramolecule | Name: EB3 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3 |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #1: alpha tubulin
Macromolecule | Name: alpha tubulin / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #2: beta tubulin
Macromolecule | Name: beta tubulin / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #3: EB3
Macromolecule | Name: EB3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | helical reconstruction |
Aggregation state | helical array |
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Sample preparation
Concentration | 3 mg/mL |
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Buffer | pH: 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 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 4 seconds before plunging.. |
Details | Preformed GMPCPP microtubules were washed with EB3 |
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Electron microscopy
Microscope | FEI TITAN |
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Image recording | Film 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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated defocus max: 2.7 µm / Calibrated defocus min: 0.8 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 stage | Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER Cooling holder cryogen: NITROGEN |
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Image processing
Final reconstruction | Number 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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CTF correction | Software - Name: Gctf |
Final angle assignment | Type: NOT APPLICABLE / Software - Name: FREALIGN |