|Entry||Database: PDB / ID: 3jas|
|Title||Cryo-EM structure of dynamic GDP-microtubule (14 protofilaments) decorated with kinesin|
|Keywords||STRUCTURAL PROTEIN / microtubule / GDP / kinesin|
|Function / homology|
Function and homology information
cytoplasmic microtubule / microtubule-based process / cellular response to interleukin-4 / structural constituent of cytoskeleton / neuron migration / microtubule cytoskeleton organization / double-stranded RNA binding / mitotic cell cycle / microtubule / GTPase activity ...cytoplasmic microtubule / microtubule-based process / cellular response to interleukin-4 / structural constituent of cytoskeleton / neuron migration / microtubule cytoskeleton organization / double-stranded RNA binding / mitotic cell cycle / microtubule / GTPase activity / GTP binding / ubiquitin protein ligase binding / cytoplasm
Tubulin, conserved site / Tubulin/FtsZ, GTPase domain superfamily / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ, C-terminal domain superfamily / Beta tubulin, autoregulation binding site / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, GTPase domain / Beta tubulin / Alpha tubulin / Tubulin ...Tubulin, conserved site / Tubulin/FtsZ, GTPase domain superfamily / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ, C-terminal domain superfamily / Beta tubulin, autoregulation binding site / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, GTPase domain / Beta tubulin / Alpha tubulin / Tubulin / Tubulin/FtsZ family, GTPase domain / Tubulin, C-terminal / Tubulin C-terminal domain / Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / Helix Hairpins / Rossmann fold / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Tubulin beta chain / Tubulin alpha-1B chain
|Biological species||Sus scrofa (pig)|
|Method||ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.5 Å|
|Authors||Zhang, R. / Nogales, E.|
|Citation||Journal: Cell / Year: 2015|
Title: Mechanistic Origin of Microtubule Dynamic Instability and Its Modulation by EB Proteins.
Authors: Rui Zhang / Gregory M Alushin / Alan Brown / Eva Nogales /
Abstract: Microtubule (MT) dynamic instability is driven by GTP hydrolysis and regulated by microtubule-associated proteins, including the plus-end tracking end-binding protein (EB) family. We report six cryo- ...Microtubule (MT) dynamic instability is driven by GTP hydrolysis and regulated by microtubule-associated proteins, including the plus-end tracking end-binding protein (EB) family. We report six cryo-electron microscopy (cryo-EM) structures of MTs, at 3.5 Å or better resolution, bound to GMPCPP, GTPγS, or GDP, either decorated with kinesin motor domain after polymerization or copolymerized with EB3. Subtle changes around the E-site nucleotide during hydrolysis trigger conformational changes in α-tubulin around an "anchor point," leading to global lattice rearrangements and strain generation. Unlike the extended lattice of the GMPCPP-MT, the EB3-bound GTPγS-MT has a compacted lattice that differs in lattice twist from that of the also compacted GDP-MT. These results and the observation that EB3 promotes rapid hydrolysis of GMPCPP suggest that EB proteins modulate structural transitions at growing MT ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. Our findings explain both EBs end-tracking behavior and their effect on microtubule dynamics.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
Downloads & links
E: Tubulin alpha-1B chain
F: Tubulin beta chain
J: Tubulin alpha-1B chain
G: Tubulin beta chain
C: Tubulin alpha-1B chain
D: Tubulin beta chain
L: Tubulin alpha-1B chain
I: Tubulin beta chain
A: Tubulin alpha-1B chain
B: Tubulin beta chain
K: Tubulin alpha-1B chain
H: Tubulin beta chain
|Symmetry||Helical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 1 / Rise per n subunits: 8.75 Å / Rotation per n subunits: -25.76 °)|
|Noncrystallographic symmetry (NCS)||NCS domain: |
NCS domain segments:
Mass: 50204.445 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Organ: brain / References: UniProt: Q2XVP4
Mass: 49907.770 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Organ: brain / References: UniProt: P02554
Mass: 523.180 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
|Compound details||THOUGH PRESENT IN THE SAMPLE, KINESIN HAS NOT BEEN MODELED.|
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction|
|Buffer solution||Name: BRB80 / pH: 6.8 |
Details: 80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 1 mM DTT, 0.05% Nonidet P-40
|Specimen||Conc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Details: 400 mesh C-flat 1.2/1.3 EM grid, glow discharged in Ar/O2 gas (Solarus, Gatan Inc)|
|Vitrification||Instrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Temp: 90.4 K / Humidity: 95 %|
Details: Blot once for 4 seconds before plunging into liquid ethane (FEI VITROBOT MARK II).
Method: Blot once for 4 seconds before plunging.
-Electron microscopy imaging
|Microscopy||Model: FEI TITAN / Date: May 7, 2014|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 27500 X / Calibrated magnification: 27500 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm|
Astigmatism: Objective lens astigmatism was corrected at 27,500 times magnification.
|Specimen holder||Model: GATAN LIQUID NITROGEN / Specimen holder type: Gatan 626 holder / Temperature: 90 K|
|Image recording||Electron dose: 27.6 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)|
Details: The camera was operated in counting mode with a dose rate of ~8 electrons/pixel/s on the camera. A total exposure time of 6 seconds was fractionated into 20 movie frames.
|Image scans||Num. digital images: 327|
|CTF correction||Details: CTFFIND4, each particle|
|Helical symmerty||Angular rotation/subunit: 25.76 ° / Axial rise/subunit: 8.75 Å / Axial symmetry: C1|
|3D reconstruction||Method: projection matching / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Nominal pixel size: 1.32 Å / Actual pixel size: 1.32 Å|
Details: IHRSR algorithm with microtubule-specific pseudo-helical symmetry applied
Symmetry type: HELICAL
|Refinement||Resolution: 3.5→211.2 Å / Cor.coef. Fo:Fc: 0.841 / SU B: 42.965 / SU ML: 0.644 / σ(F): 0 |
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
|Solvent computation||Solvent model: NONE|
|Displacement parameters||Biso max: 143.07 Å2 / Biso mean: 47.605 Å2 / Biso min: 9.86 Å2|
|Refinement step||Cycle: LAST / Resolution: 3.5→211.2 Å|
|Refine LS restraints|
|Refine LS restraints NCS|
Refinement-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Rms dev position: 0 Å / Weight position: 0.05
|LS refinement shell||Resolution: 3.5→3.591 Å / Total num. of bins used: 20 |
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