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- EMDB-4154: Mechanism of microtubule minus-end recognition and protection by ... -

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Basic information

Entry
Database: EMDB / ID: EMD-4154
TitleMechanism of microtubule minus-end recognition and protection by CAMSAP proteins
Map dataCAMSAP3 CKK decorated 13pf microtubule asymmetric unit
Sample
  • Complex: Complex of two tubulin dimers with bound CAMSAP3-CKK domain
    • Complex: tubulin
      • Protein or peptide: Tubulin alpha chain
      • Protein or peptide: Tubulin beta-2B chain
    • Complex: CAMSAP3-CKK domain
      • Protein or peptide: Calmodulin-regulated spectrin-associated protein 3
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOLPaclitaxel
  • Ligand: water
Function / homology
Function and homology information


regulation of organelle organization / zonula adherens maintenance / microtubule minus-end / microtubule minus-end binding / protein transport along microtubule / regulation of Golgi organization / microtubule anchoring / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Intraflagellar transport ...regulation of organelle organization / zonula adherens maintenance / microtubule minus-end / microtubule minus-end binding / protein transport along microtubule / regulation of Golgi organization / microtubule anchoring / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Intraflagellar transport / Carboxyterminal post-translational modifications of tubulin / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / COPI-dependent Golgi-to-ER retrograde traffic / RHO GTPases activate IQGAPs / COPI-independent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / RHO GTPases Activate Formins / establishment or maintenance of microtubule cytoskeleton polarity / cilium movement / MHC class II antigen presentation / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Aggrephagy / epithelial cell-cell adhesion / The role of GTSE1 in G2/M progression after G2 checkpoint / zonula adherens / Separation of Sister Chromatids / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Anchoring of the basal body to the plasma membrane / AURKA Activation by TPX2 / Recruitment of NuMA to mitotic centrosomes / Regulation of PLK1 Activity at G2/M Transition / Hedgehog 'off' state / negative regulation of microtubule depolymerization / establishment of epithelial cell apical/basal polarity / motile cilium / positive regulation of axon guidance / embryo development ending in birth or egg hatching / regulation of focal adhesion assembly / spectrin binding / regulation of microtubule polymerization / axoneme / cytoplasmic microtubule / microtubule-based process / cytoplasmic microtubule organization / regulation of microtubule cytoskeleton organization / regulation of cell migration / cellular response to interleukin-4 / ciliary basal body / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron projection development / microtubule cytoskeleton / actin filament binding / double-stranded RNA binding / mitotic cell cycle / nervous system development / in utero embryonic development / microtubule / calmodulin binding / hydrolase activity / protein heterodimerization activity / GTPase activity / centrosome / ubiquitin protein ligase binding / GTP binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
CAMSAP, spectrin and Ca2+/calmodulin-binding region / Spectrin-binding region of Ca2+-Calmodulin / CKK domain / Calmodulin-regulated spectrin-associated protein / CKK domain superfamily / Microtubule-binding calmodulin-regulated spectrin-associated / CKK domain profile. / Microtubule-binding calmodulin-regulated spectrin-associated / Calmodulin-regulated spectrin-associated protein-like, Calponin-homology domain / CAMSAP CH domain ...CAMSAP, spectrin and Ca2+/calmodulin-binding region / Spectrin-binding region of Ca2+-Calmodulin / CKK domain / Calmodulin-regulated spectrin-associated protein / CKK domain superfamily / Microtubule-binding calmodulin-regulated spectrin-associated / CKK domain profile. / Microtubule-binding calmodulin-regulated spectrin-associated / Calmodulin-regulated spectrin-associated protein-like, Calponin-homology domain / CAMSAP CH domain / PRC-barrel-like superfamily / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / 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 chain / Tubulin alpha-1B chain / Tubulin beta-2B chain / Calmodulin-regulated spectrin-associated protein 3
Similarity search - Component
Biological speciesBos taurus (cattle) / Mus musculus (house mouse) / Bovine (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.3 Å
AuthorsAkhmanova A / Moores CA / Baldus M / Steinmetz MO / Topf M / Roberts AJ / Grant BJ / Scarabelli G / Joseph A-P / van Hooff JJE ...Akhmanova A / Moores CA / Baldus M / Steinmetz MO / Topf M / Roberts AJ / Grant BJ / Scarabelli G / Joseph A-P / van Hooff JJE / Houben K / Hua S / Luo Y / Stangier MM / Jiang K / Atherton J
CitationJournal: Nat Struct Mol Biol / Year: 2017
Title: A structural model for microtubule minus-end recognition and protection by CAMSAP proteins.
Authors: Joseph Atherton / Kai Jiang / Marcel M Stangier / Yanzhang Luo / Shasha Hua / Klaartje Houben / Jolien J E van Hooff / Agnel-Praveen Joseph / Guido Scarabelli / Barry J Grant / Anthony J ...Authors: Joseph Atherton / Kai Jiang / Marcel M Stangier / Yanzhang Luo / Shasha Hua / Klaartje Houben / Jolien J E van Hooff / Agnel-Praveen Joseph / Guido Scarabelli / Barry J Grant / Anthony J Roberts / Maya Topf / Michel O Steinmetz / Marc Baldus / Carolyn A Moores / Anna Akhmanova /
Abstract: CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize noncentrosomal microtubule networks, which are essential for cell division, polarization ...CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize noncentrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we found that the CAMSAP C-terminal CKK domain is widely present among eukaryotes and autonomously recognizes microtubule minus ends. Through a combination of structural approaches, we uncovered how mammalian CKK binds between two tubulin dimers at the interprotofilament interface on the outer microtubule surface. In vitro reconstitution assays combined with high-resolution fluorescence microscopy and cryo-electron tomography suggested that CKK preferentially associates with the transition zone between curved protofilaments and the regular microtubule lattice. We propose that minus-end-specific features of the interprotofilament interface at this site serve as the basis for CKK's minus-end preference. The steric clash between microtubule-bound CKK and kinesin motors explains how CKK protects microtubule minus ends against kinesin-13-induced depolymerization and thus controls the stability of free microtubule minus ends.
History
DepositionOct 20, 2016-
Header (metadata) releaseNov 30, 2016-
Map releaseOct 4, 2017-
UpdateNov 15, 2017-
Current statusNov 15, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0314
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0314
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5m50
  • Surface level: 0.0314
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4154.png

Downloads & links

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Map

FileDownload / File: emd_4154.map.gz / Format: CCP4 / Size: 2.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCAMSAP3 CKK decorated 13pf microtubule asymmetric unit
Voxel sizeX=Y=Z: 1.534 Å
Density
Contour LevelBy AUTHOR: 0.0314 / Movie #1: 0.0314
Minimum - Maximum-0.039573725 - 0.12910078
Average (Standard dev.)0.0059765955 (±0.016930932)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions888295
Spacing828895
CellA: 125.788 Å / B: 134.992 Å / C: 145.73001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.5341.5341.534
M x/y/z828895
origin x/y/z0.0000.0000.000
length x/y/z125.788134.992145.730
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS828895
D min/max/mean-0.0400.1290.006

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Supplemental data

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Sample components

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Entire : Complex of two tubulin dimers with bound CAMSAP3-CKK domain

EntireName: Complex of two tubulin dimers with bound CAMSAP3-CKK domain
Components
  • Complex: Complex of two tubulin dimers with bound CAMSAP3-CKK domain
    • Complex: tubulin
      • Protein or peptide: Tubulin alpha chain
      • Protein or peptide: Tubulin beta-2B chain
    • Complex: CAMSAP3-CKK domain
      • Protein or peptide: Calmodulin-regulated spectrin-associated protein 3
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOLPaclitaxel
  • Ligand: water

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Supramolecule #1: Complex of two tubulin dimers with bound CAMSAP3-CKK domain

SupramoleculeName: Complex of two tubulin dimers with bound CAMSAP3-CKK domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: tubulin

SupramoleculeName: tubulin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Bos taurus (cattle)

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Supramolecule #3: CAMSAP3-CKK domain

SupramoleculeName: CAMSAP3-CKK domain / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Tubulin alpha chain

MacromoleculeName: Tubulin alpha chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bovine (cattle)
Molecular weightTheoretical: 48.769988 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFSVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFSVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLIGQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRGHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRTIQFVDW CPTGFKVGIN YEPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDS

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Macromolecule #2: Tubulin beta-2B chain

MacromoleculeName: Tubulin beta-2B chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bovine (cattle)
Molecular weightTheoretical: 47.940945 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QD

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Macromolecule #3: Calmodulin-regulated spectrin-associated protein 3

MacromoleculeName: Calmodulin-regulated spectrin-associated protein 3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 13.431391 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
AKSNKFIIHN ALSHCCLAGK VNEPQKNRIL EEIEKSKANH FLILFRDSSC QFRALYTLSG ETEELSRLAG YGPRTVTPAM VEGIYKYNS DRKRFTQIPA KTMSMSVDAF TIQGHLWQS

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 2 / 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: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 2 / 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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Macromolecule #7: TAXOL

MacromoleculeName: TAXOL / type: ligand / ID: 7 / Number of copies: 2 / Formula: TA1
Molecular weightTheoretical: 853.906 Da
Chemical component information

ChemComp-TA1:
TAXOL / medication, chemotherapy*YM / Paclitaxel

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 8 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.8 / Details: BRB80
GridModel: C-flat-2/2 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK III
Details13pf Microtubules

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: INTEGRATING / Average electron dose: 25.0 e/Å2 / Details: 25e-/A2 used in final reconstuctions
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Kinesin decorated microtubule
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: SPIDER
Final angle assignmentType: OTHER / Software - Name: FREALIGN / Details: Projection matching in Fourier space
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.3 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Gold-standard noise substitution test used to asses for over fitting (Chen et al., 2013)
Number images used: 6530

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Atomic model buiding 1

RefinementProtocol: OTHER
Output model

PDB-5m50:
Mechanism of microtubule minus-end recognition and protection by CAMSAP proteins

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