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- EMDB-25156: Undecorated 13pf wildtype microtubule from recombinant human tubulin -

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Basic information

Entry
Database: EMDB / ID: EMD-25156
TitleUndecorated 13pf wildtype microtubule from recombinant human tubulin
Map datarecombinant wildtypehuman tubulin reconstructed with pseudo-helical symmetry
Sample
  • Complex: 13pf wildtype undecorated microtubule from recombinant human tubulin
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta-3 chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
Function / homology
Function and homology information


netrin receptor binding / Post-chaperonin tubulin folding pathway / Carboxyterminal post-translational modifications of tubulin / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / cytoskeleton-dependent intracellular transport / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Gap junction assembly ...netrin receptor binding / Post-chaperonin tubulin folding pathway / Carboxyterminal post-translational modifications of tubulin / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / cytoskeleton-dependent intracellular transport / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Gap junction assembly / Formation of tubulin folding intermediates by CCT/TriC / COPI-independent Golgi-to-ER retrograde traffic / Prefoldin mediated transfer of substrate to CCT/TriC / Kinesins / Assembly and cell surface presentation of NMDA receptors / COPI-dependent Golgi-to-ER retrograde traffic / intercellular bridge / Recycling pathway of L1 / RHOH GTPase cycle / RHO GTPases activate IQGAPs / Hedgehog 'off' state / cytoplasmic microtubule / microtubule-based process / COPI-mediated anterograde transport / Activation of AMPK downstream of NMDARs / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Resolution of Sister Chromatid Cohesion / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / cellular response to interleukin-4 / filopodium / cell periphery / RHO GTPases Activate Formins / Translocation of SLC2A4 (GLUT4) to the plasma membrane / axon guidance / peptide binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / PKR-mediated signaling / structural constituent of cytoskeleton / mitotic spindle / microtubule cytoskeleton organization / Aggrephagy / HCMV Early Events / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / microtubule cytoskeleton / double-stranded RNA binding / mitotic cell cycle / lamellipodium / growth cone / microtubule / cell division / axon / GTPase activity / dendrite / neuronal cell body / ubiquitin protein ligase binding / structural molecule activity / GTP binding / extracellular exosome / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / 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-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / 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 alpha-1B chain / Tubulin beta-3 chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLaFrance BJ / Greber BJ / Zhang R / McCollum C / Nogales E
Funding support United States, 1 items
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 BJ / Roostalu J / Henkin G / Greber BJ / Zhang R / Normanno D / McCollum C / Surrey T / Nogales E
History
DepositionOct 16, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateJan 19, 2022-
Current statusJan 19, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6.14
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 6.14
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7sj7
  • Surface level: 6.14
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7sj7
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25156.png

Downloads & links

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Map

FileDownload / File: emd_25156.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationrecombinant wildtypehuman tubulin reconstructed with pseudo-helical symmetry
Voxel sizeX=Y=Z: 0.92 Å
Density
Contour LevelBy AUTHOR: 6.14 / Movie #1: 6.14
Minimum - Maximum-16.00589 - 25.714348
Average (Standard dev.)3.1482368e-09 (±1.425419)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 471.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.920.920.92
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z471.040471.040471.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-16.00625.7140.000

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Supplemental data

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Sample components

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Entire : 13pf wildtype undecorated microtubule from recombinant human tubulin

EntireName: 13pf wildtype undecorated microtubule from recombinant human tubulin
Components
  • Complex: 13pf wildtype undecorated microtubule from recombinant human tubulin
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta-3 chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: 13pf wildtype undecorated microtubule from recombinant human tubulin

SupramoleculeName: 13pf wildtype undecorated microtubule from recombinant human tubulin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: High-Five Insect Cell / Recombinant plasmid: pFastBacDual
Molecular weightTheoretical: 102 KDa

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Macromolecule #1: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 51.033324 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIHHHHHHGG GDDSFNTFFS ETGAGKHVPR AVFVDLEPTV IDEVRTGTY RQLFHPEQLI TGKEDAANNY ARGHYTIGKE IIDLVLDRIR KLADQCTGLQ GFLVFHSFGG GTGSGFTSLL M ERLSVDYG ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIHHHHHHGG GDDSFNTFFS ETGAGKHVPR AVFVDLEPTV IDEVRTGTY RQLFHPEQLI TGKEDAANNY ARGHYTIGKE IIDLVLDRIR KLADQCTGLQ GFLVFHSFGG GTGSGFTSLL M ERLSVDYG KKSKLEFSIY PAPQVSTAVV EPYNSILTTH TTLEHSDCAF MVDNEAIYDI CRRNLDIERP TYTNLNRLIS QI VSSITAS LRFDGALNVD LTEFQTNLVP YPRIHFPLAT YAPVISAEKA YHEQLSVAEI TNACFEPANQ MVKCDPRHGK YMA CCLLYR GDVVPKDVNA AIATIKTKRS IQFVDWCPTG FKVGINYQPP TVVPGGDLAK VQRAVCMLSN TTAIAEAWAR LDHK FDLMY AKRAFVHWYV GEGMEEGEFS EAREDMAALE KDYEEVGVDS VEGEGEEEGE EY

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Macromolecule #2: Tubulin beta-3 chain

MacromoleculeName: Tubulin beta-3 chain / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 51.276367 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGHLFRPDN FIFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKECENCDC LQGFQLTHSL GGGTGSGMGT LLISKVREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGHLFRPDN FIFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKECENCDC LQGFQLTHSL GGGTGSGMGT LLISKVREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS IHQLVENTDE TYCIDNEALY DICFRTLKLA TPTYGDLNHL VSATMSGVTT SL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTARG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVATVF RGR MSMKEV DEQMLAIQSK NSSYFVEWIP NNVKVAVCDI PPRGLKMSST FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATAEEEGE MYEDDEEESE AQGPKENLYF Q

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Macromolecule #3: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 6 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 6 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM / Guanosine diphosphate

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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

Concentration4.4 mg/mL
BufferpH: 6.8
Details: 80 mM PIPES, 1 mM EGTA, 5 mM MgCl2, 2 mM GTP, pH 6.8
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE
Details44 uM [wildtype tubulin] assembled and pseudo-helical microtubules adsorbed onto the grid

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.6 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionDetails: Microtubules were picked within RELION 3.1 helical picking functionality and extracted along the filament with an 82A sliding box.
CTF correctionSoftware: (Name: CTFFIND (ver. 4), RELION (ver. 3.1))
Details: CTFFind4 and CTFRefine were used within the RELION3.1 framework.
Startup modelType of model: EMDB MAP
EMDB ID:

7973


Details: For the 13pf model, EMD-7973 was low pass filtered to 50A to serve as an initial model. In house reconstructions of 12-15pf at 50A resolution were used to separate by PF.
Final angle assignmentType: NOT APPLICABLE / Software - Name: FREALIGN (ver. 9.11)
Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 81.5 Å
Applied symmetry - Helical parameters - Δ&Phi: 0.05 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Number images used: 19365
DetailsImages collected in super-resolution mode, Motioncorr2 5x5 binning of frames

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Atomic model buiding 1

Initial modelPDB ID:

6dpu

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 115 / Target criteria: Correlation Coefficient
Output model

PDB-7sj7:
Undecorated 13pf wildtype microtubule from recombinant human tubulin

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