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- EMDB-25159: 13pf E254A microtubule from recombinant human tubulin decorated w... -

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Basic information

Entry
Database: EMDB / ID: EMD-25159
Title13pf E254A microtubule from recombinant human tubulin decorated with EB3
Map data13pf E254A Microtubule decorated with EB3 from human recombinant tubulin
Sample
  • Complex: 13pf E254A microtubule decorated with EB3
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta-3 chain
    • Protein or peptide: Microtubule-associated protein RP/EB family member 3
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
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 / EB1, C-terminal domain superfamily / EB1, C-terminal / Microtubule-associated protein RP/EB / EB1-C terminal (EB1-C) domain profile. / EB1-like C-terminal motif / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. ...EB3 / EB1, C-terminal domain superfamily / EB1, C-terminal / Microtubule-associated protein RP/EB / EB1-C terminal (EB1-C) domain profile. / EB1-like C-terminal motif / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / 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, 2-layer sandwich domain / Tubulin/FtsZ, C-terminal / 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 / Microtubule-associated protein RP/EB family member 3
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: 3.8
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 3.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7sj9
  • Surface level: 3.8
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7sj9
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_25159.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation13pf E254A Microtubule decorated with EB3 from human recombinant tubulin
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.92 Å/pix.
x 512 pix.
= 471.04 Å
0.92 Å/pix.
x 512 pix.
= 471.04 Å
0.92 Å/pix.
x 512 pix.
= 471.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.92 Å
Density
Contour LevelBy AUTHOR: 3.8 / Movie #1: 3.8
Minimum - Maximum-7.1691055 - 15.033943
Average (Standard dev.)4.1641823e-09 (±1.0)
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-7.16915.0340.000

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Supplemental data

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Sample components

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Entire : 13pf E254A microtubule decorated with EB3

EntireName: 13pf E254A microtubule decorated with EB3
Components
  • Complex: 13pf E254A microtubule decorated with EB3
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta-3 chain
    • Protein or peptide: Microtubule-associated protein RP/EB family member 3
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION

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Supramolecule #1: 13pf E254A microtubule decorated with EB3

SupramoleculeName: 13pf E254A microtubule decorated with EB3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
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: 50.975289 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 LTAFQTNLVP 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: Microtubule-associated protein RP/EB family member 3

MacromoleculeName: Microtubule-associated protein RP/EB family member 3 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 32.021166 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MAVNVYSTSV TSENLSRHDM LAWVNDSLHL NYTKIEQLCS GAAYCQFMDM LFPGCVHLRK VKFQAKLEHE YIHNFKVLQA AFKKMGVDK IIPVEKLVKG KFQDNFEFIQ WFKKFFDANY DGKDYNPLLA RQGQDVAPPP NPGDQIFNKS KKLIGTAVPQ R TSPTGPKN ...String:
MAVNVYSTSV TSENLSRHDM LAWVNDSLHL NYTKIEQLCS GAAYCQFMDM LFPGCVHLRK VKFQAKLEHE YIHNFKVLQA AFKKMGVDK IIPVEKLVKG KFQDNFEFIQ WFKKFFDANY DGKDYNPLLA RQGQDVAPPP NPGDQIFNKS KKLIGTAVPQ R TSPTGPKN MQTSGRLSNV APPCILRKNP PSARNGGHET DAQILELNQQ LVDLKLTVDG LEKERDFYFS KLRDIELICQ EH ESENSPV ISGIIGILYA TEEGFAPPED DEIEEHQQED QDEY

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 12 / 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 #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 12 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration5.5 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
Details55 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:

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: 81.64 Å
Applied symmetry - Helical parameters - Δ&Phi: -0.19 °
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: 10008
DetailsImages collected in super-resolution mode, Motioncorr2 5x5 binning of frames

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Atomic model buiding 1

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

PDB-7sj9:
13pf E254A microtubule from recombinant human tubulin decorated with EB3

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