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- EMDB-76717: In situ cryo-EM structure of axonal microtubules from ghost neuro... -

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Basic information

Entry
Database: EMDB / ID: EMD-76717
TitleIn situ cryo-EM structure of axonal microtubules from ghost neurons (expanded lattice)
Map datasharpened map from local refinement
Sample
  • Organelle or cellular component: Microtubules, axonal
    • Protein or peptide: Detyrosinated tubulin alpha-1A chain
    • Protein or peptide: Tubulin beta-3 chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
Keywordscytoskeleton / neuron / microtubules / axon / STRUCTURAL PROTEIN
Function / homology
Function and homology information


Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cargo trafficking to the periciliary membrane / Sealing of the nuclear envelope (NE) by ESCRT-III / netrin-activated signaling pathway / Carboxyterminal post-translational modifications of tubulin / Intraflagellar transport / netrin receptor binding / COPI-independent Golgi-to-ER retrograde traffic / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / pyramidal neuron differentiation ...Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cargo trafficking to the periciliary membrane / Sealing of the nuclear envelope (NE) by ESCRT-III / netrin-activated signaling pathway / Carboxyterminal post-translational modifications of tubulin / Intraflagellar transport / netrin receptor binding / COPI-independent Golgi-to-ER retrograde traffic / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / pyramidal neuron differentiation / COPI-mediated anterograde transport / Kinesins / PKR-mediated signaling / Aggrephagy / RHO GTPases activate IQGAPs / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / COPI-dependent Golgi-to-ER retrograde traffic / glial cell differentiation / Resolution of Sister Chromatid Cohesion / dorsal root ganglion development / The role of GTSE1 in G2/M progression after G2 checkpoint / Recycling pathway of L1 / dentate gyrus development / axonemal microtubule / organelle transport along microtubule / Hedgehog 'off' state / RHO GTPases Activate Formins / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Separation of Sister Chromatids / Anchoring of the basal body to the plasma membrane / forebrain morphogenesis / Recruitment of NuMA to mitotic centrosomes / AURKA Activation by TPX2 / cerebellar cortex morphogenesis / Regulation of PLK1 Activity at G2/M Transition / MHC class II antigen presentation / neuron projection arborization / smoothened signaling pathway / homeostasis of number of cells within a tissue / motor behavior / adult behavior / response to L-glutamate / centrosome cycle / sperm principal piece / startle response / 'de novo' protein folding / intercellular bridge / flagellated sperm motility / regulation of synapse organization / sperm end piece / locomotory exploration behavior / microtubule polymerization / ciliary tip / response to tumor necrosis factor / response to mechanical stimulus / neuron apoptotic process / neurogenesis / sperm flagellum / adult locomotory behavior / cytoplasmic microtubule / condensed chromosome / peptide binding / cellular response to calcium ion / gene expression / visual learning / axon guidance / locomotory behavior / hippocampus development / cell periphery / filopodium / neuromuscular junction / myelin sheath / neuron migration / memory / cerebral cortex development / intracellular protein transport / synapse organization / microtubule cytoskeleton organization / recycling endosome / mitotic spindle / structural constituent of cytoskeleton / cytoplasmic ribonucleoprotein granule / microtubule cytoskeleton / cilium / neuron differentiation / mitotic cell cycle / lamellipodium / growth cone / protein-folding chaperone binding / microtubule binding / microtubule / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / protein stabilization / membrane raft / protein heterodimerization activity / protein domain specific binding / axon
Similarity search - Function
Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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 ...Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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-1A chain / Tubulin beta-3 chain
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodhelical reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsBodakuntla S / Marelli J / Vasquez-Montes V / Biertumpfel C / Mizuno N
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)1ZIAHL006264 United States
CitationJournal: To Be Published
Title: Ghost neuron unlocks in situ high-resolution structural mapping of intracellular architecture in neurons
Authors: Bodakuntla S / Marelli J / Vasquez-Montes V / Biertumpfel C / Mizuno N
History
DepositionApr 15, 2026-
Header (metadata) releaseAug 26, 2026-
Map releaseAug 26, 2026-
UpdateAug 26, 2026-
Current statusAug 26, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_76717.png

Downloads & links

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Map

FileDownload / File: emd_76717.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map from local refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 600 pix.
= 494.4 Å		0.82 Å/pix.
x 600 pix.
= 494.4 Å		0.82 Å/pix.
x 600 pix.
= 494.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.824 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.22786205 - 0.36910364
Average (Standard dev.)0.00077378564 (±0.009214164)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 494.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_76717_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map from local refinement

Fileemd_76717_half_map_1.map
Annotationhalf map from local refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map from local refinement

Fileemd_76717_half_map_2.map
Annotationhalf map from local refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Microtubules, axonal

EntireName: Microtubules, axonal
Components
  • Organelle or cellular component: Microtubules, axonal
    • Protein or peptide: Detyrosinated tubulin alpha-1A chain
    • Protein or peptide: Tubulin beta-3 chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Microtubules, axonal

SupramoleculeName: Microtubules, axonal / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: explant axon from thalamus primary mouse embryo E15.5 tissue after ghost preparation (hypotonic treatment and mechanical plasma membrane removal)
Source (natural)Organism: Mus musculus (house mouse) / Strain: CD-1 / Organ: Brain / Tissue: Thalamus / Location in cell: Axon

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Macromolecule #1: Detyrosinated tubulin alpha-1A chain

MacromoleculeName: Detyrosinated tubulin alpha-1A chain / type: protein_or_peptide / ID: 1 / Details: TUBA1A component of microtubules / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: CD-1 / Organ: Brain / Tissue: Thalamus
Molecular weightTheoretical: 50.188441 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLIGQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRTIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

UniProtKB: Tubulin alpha-1A chain

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Macromolecule #2: Tubulin beta-3 chain

MacromoleculeName: Tubulin beta-3 chain / type: protein_or_peptide / ID: 2 / Details: TUBB3 component of microtubules / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: CD-1 / Organ: Brain / Tissue: Thalamus
Molecular weightTheoretical: 50.467492 KDa
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 MYEDDDEESE AQGPK

UniProtKB: Tubulin beta-3 chain

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Macromolecule #3: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

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

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / 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: 2 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

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

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.2 / Details: Gibco Neurobasal Media (diluted 1:3 to 160 mOsm)
GridModel: Quantifoil R2/4 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 3.8000000000000003 kPa / Details: coated with poly-L-lysine and laminin
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 303 K / Instrument: FEI VITROBOT MARK IV / Details: blot force 4, blot time 4 s.
Detailsghost neurons prepared by hypotonic and mechanical treatment

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 10 / Number real images: 4172 / Average electron dose: 52.2 e/Å2 / Details: curated image number
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 81.23 Å
Applied symmetry - Helical parameters - Δ&Phi: 0 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.0) / Number images used: 48459
CTF correctionSoftware - Name: cryoSPARC (ver. 4.7.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Segment selectionNumber selected: 158108 / Software - Name: cryoSPARC (ver. 4.7.0)
Startup modelType of model: OTHER / Details: 13-protofilament
Final angle assignmentType: NOT APPLICABLE / Software - Name: FREALIGN (ver. 9.11) / Details: seam detection
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

9pnd


Chain - Source name: PDB / Chain - Initial model type: experimental model
Detailsiterative rounds of manual building and automated real-space refinement
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-12ru:
In situ cryo-EM structure of axonal microtubules from ghost neurons (expanded lattice)

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