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- PDB-7sj9: 13pf E254A microtubule from recombinant human tubulin decorated w... -

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Basic information

Entry
Database: PDB / ID: 7sj9
Title13pf E254A microtubule from recombinant human tubulin decorated with EB3
Components
  • Microtubule-associated protein RP/EB family member 3
  • Tubulin alpha-1B chain
  • Tubulin beta-3 chain
KeywordsSTRUCTURAL PROTEIN / microtubule / cell division / cytoskeleton / GTPase
Function / homology
Function and homology information


netrin receptor binding / mitotic spindle astral microtubule end / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / positive regulation of microtubule plus-end binding / Post-chaperonin tubulin folding pathway / protein localization to microtubule plus-end / Carboxyterminal post-translational modifications of tubulin / Gap junction assembly / dorsal root ganglion development / Cilium Assembly ...netrin receptor binding / mitotic spindle astral microtubule end / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / positive regulation of microtubule plus-end binding / Post-chaperonin tubulin folding pathway / protein localization to microtubule plus-end / Carboxyterminal post-translational modifications of tubulin / Gap junction assembly / dorsal root ganglion development / Cilium Assembly / Formation of tubulin folding intermediates by CCT/TriC / Sealing of the nuclear envelope (NE) by ESCRT-III / Prefoldin mediated transfer of substrate to CCT/TriC / Intraflagellar transport / microtubule plus-end / COPI-independent Golgi-to-ER retrograde traffic / cytoskeleton-dependent intracellular transport / microtubule plus-end binding / Assembly and cell surface presentation of NMDA receptors / Kinesins / COPI-dependent Golgi-to-ER retrograde traffic / regulation of microtubule polymerization / Recycling pathway of L1 / Hedgehog 'off' state / RHOH GTPase cycle / regulation of microtubule polymerization or depolymerization / Activation of AMPK downstream of NMDARs / RHO GTPases activate IQGAPs / spindle assembly / cytoplasmic microtubule / COPI-mediated anterograde transport / spindle midzone / microtubule-based process / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / positive regulation of cyclin-dependent protein serine/threonine kinase activity / positive regulation of protein kinase activity / Resolution of Sister Chromatid Cohesion / MHC class II antigen presentation / Recruitment of NuMA to mitotic centrosomes / cellular response to interleukin-4 / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / filopodium / RHO GTPases Activate Formins / Translocation of SLC2A4 (GLUT4) to the plasma membrane / protein localization / microtubule organizing center / microtubule cytoskeleton organization / axon guidance / HCMV Early Events / structural constituent of cytoskeleton / microtubule cytoskeleton / Aggrephagy / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / midbody / growth cone / double-stranded RNA binding / lamellipodium / mitotic cell cycle / microtubule binding / microtubule / protein C-terminus binding / axon / cell division / GTPase activity / dendrite / GTP binding / ubiquitin protein ligase binding / structural molecule activity / protein kinase binding / perinuclear region of cytoplasm / positive regulation of transcription, DNA-templated / extracellular exosome / identical protein binding / nucleus / cytoplasm
Similarity search - Function
EB3 / Microtubule-associated protein RP/EB / EB1, C-terminal domain superfamily / EB1-C terminal (EB1-C) domain profile. / EB1, C-terminal / EB1-like C-terminal motif / Calponin homology (CH) domain / Calponin homology (CH) domain profile. / CH domain superfamily / Calponin homology domain ...EB3 / Microtubule-associated protein RP/EB / EB1, C-terminal domain superfamily / EB1-C terminal (EB1-C) domain profile. / EB1, C-terminal / EB1-like C-terminal motif / Calponin homology (CH) domain / Calponin homology (CH) domain profile. / CH domain superfamily / Calponin homology domain / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Alpha tubulin / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
Tubulin beta-3 chain / GUANOSINE-5'-TRIPHOSPHATE / Tubulin alpha-1B chain / Microtubule-associated protein RP/EB family member 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLaFrance, B.J. / Greber, B.J. / Zhang, R. / McCollum, C. / Nogales, E.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2022
Title: Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules.
Authors: Benjamin J LaFrance / Johanna Roostalu / Gil Henkin / Basil J Greber / Rui Zhang / Davide Normanno / Chloe O McCollum / Thomas Surrey / Eva Nogales /
Abstract: Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is ...Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.
#1: Journal: BioRxiv / Year: 2021
Title: Structural transitions in the GTP cap visualized by cryo-EM of catalytically inactive microtubules
Authors: LaFrance, B.J. / Roostalu, J. / Henkin, G. / Greber, B.J. / Zhang, R. / Normanno, D. / McCollum, C. / Surrey, T. / Nogales, E.
History
DepositionOct 16, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 19, 2022Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Tubulin alpha-1B chain
B: Tubulin beta-3 chain
C: Tubulin alpha-1B chain
D: Tubulin beta-3 chain
E: Tubulin alpha-1B chain
F: Tubulin beta-3 chain
G: Tubulin beta-3 chain
H: Tubulin beta-3 chain
I: Tubulin beta-3 chain
J: Tubulin alpha-1B chain
K: Tubulin alpha-1B chain
L: Tubulin alpha-1B chain
M: Microtubule-associated protein RP/EB family member 3
N: Microtubule-associated protein RP/EB family member 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)684,12238
Polymers677,55214
Non-polymers6,57024
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 50975.289 Da / Num. of mol.: 6 / Mutation: E254A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBA1B / Plasmid: pFastBacDual / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P68363
#2: Protein
Tubulin beta-3 chain / Tubulin beta-4 chain / Tubulin beta-III


Mass: 51276.367 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBB3, TUBB4 / Plasmid: pFastBacDual / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q13509
#3: Protein Microtubule-associated protein RP/EB family member 3 / EB1 protein family member 3 / EBF3 / End-binding protein 3 / EB3 / RP3


Mass: 32021.166 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAPRE3 / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 RIL / References: UniProt: Q9UPY8
#4: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: 13pf E254A microtubule decorated with EB3 / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.102 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Cell: High-Five Insect Cell / Plasmid: pFastBacDual
Buffer solutionpH: 6.8
Details: 80 mM PIPES, 1 mM EGTA, 5 mM MgCl2, 2 mM GTP, pH 6.8
SpecimenConc.: 5.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 55 uM [wildtype tubulin] assembled and pseudo-helical microtubules adsorbed onto the grid
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 64000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.6 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19_4080: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4CTF correction
5RELION3.1CTF correction
8PHENIXdev-2883model fitting
10RELION3.1initial Euler assignment
11FREALIGN9.11final Euler assignment
12RELION3.1classification
13FREALIGN9.113D reconstruction
14PHENIXdev-2883model refinement
Image processingDetails: Images collected in super-resolution mode, Motioncorr2 5x5 binning of frames
CTF correctionDetails: CTFFind4 and CTFRefine were used within the RELION3.1 framework.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -0.19 ° / Axial rise/subunit: 81.64 Å / Axial symmetry: C1
Particle selectionDetails: Microtubules were picked within RELION 3.1 helical picking functionality and extracted along the filament with an 82A sliding box.
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10008 / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingB value: 115 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation Coefficient
Atomic model buildingPDB-ID: 6DPU
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00344220
ELECTRON MICROSCOPYf_angle_d0.63260022
ELECTRON MICROSCOPYf_dihedral_angle_d11.6666062
ELECTRON MICROSCOPYf_chiral_restr0.0456546
ELECTRON MICROSCOPYf_plane_restr0.0057782

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