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- PDB-6qus: HsCKK (human CAMSAP1) decorated 13pf taxol-GDP microtubule -

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Entry
Database: PDB / ID: 6qus
TitleHsCKK (human CAMSAP1) decorated 13pf taxol-GDP microtubule
Components
  • Calmodulin-regulated spectrin-associated protein 1
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / Microtubule CAMSAP Calmodulin-regulated spectrum-associated proteins CKK Cryo-EM Cryo-Electron Microscopy
Function / homology
Function and homology information


microtubule minus-end binding / odontoblast differentiation / Post-chaperonin tubulin folding pathway / Carboxyterminal post-translational modifications of tubulin / 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 ...microtubule minus-end binding / odontoblast differentiation / Post-chaperonin tubulin folding pathway / Carboxyterminal post-translational modifications of tubulin / 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 / natural killer cell mediated cytotoxicity / COPI-independent Golgi-to-ER retrograde traffic / Kinesins / Assembly and cell surface presentation of NMDA receptors / regulation of cell morphogenesis / GTPase activating protein binding / COPI-dependent Golgi-to-ER retrograde traffic / microtubule organizing center / spectrin binding / regulation of synapse organization / intercellular bridge / nuclear envelope lumen / regulation of microtubule polymerization / Recycling pathway of L1 / RHOH GTPase cycle / spindle assembly / RHO GTPases activate IQGAPs / MHC class I protein binding / Hedgehog 'off' state / cytoplasmic microtubule / microtubule-based process / COPI-mediated anterograde transport / Activation of AMPK downstream of NMDARs / Mitotic Prometaphase / cytoskeleton organization / EML4 and NUDC in mitotic spindle formation / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of mitotic centrosome proteins and complexes / Resolution of Sister Chromatid Cohesion / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Anchoring of the basal body to the plasma membrane / MHC class II antigen presentation / cellular response to interleukin-4 / AURKA Activation by TPX2 / RHO GTPases Activate Formins / Translocation of SLC2A4 (GLUT4) to the plasma membrane / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / PKR-mediated signaling / structural constituent of cytoskeleton / cytoplasmic ribonucleoprotein granule / mitotic spindle / microtubule cytoskeleton organization / Aggrephagy / HCMV Early Events / neuron projection development / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / microtubule cytoskeleton / azurophil granule lumen / Regulation of PLK1 Activity at G2/M Transition / double-stranded RNA binding / mitotic cell cycle / cell body / microtubule binding / microtubule / Potential therapeutics for SARS / cytoskeleton / calmodulin binding / membrane raft / cell division / protein domain specific binding / GTPase activity / ubiquitin protein ligase binding / Neutrophil degranulation / protein-containing complex binding / structural molecule activity / GTP binding / protein-containing complex / extracellular exosome / extracellular region / metal ion binding / nucleus / 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 / Helix hairpin bin / PRC-barrel-like superfamily / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / 60s Ribosomal Protein L30; Chain: A; / 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 / Helix Hairpins / Rossmann fold / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / Tubulin beta chain / Tubulin alpha-1B chain / Calmodulin-regulated spectrin-associated protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsAtherton, J.M. / Luo, Y. / Xiang, S. / Yang, C. / Jiang, K. / Stangier, M. / Vemu, A. / Cook, A. / Wang, S. / Roll-Mecak, A. ...Atherton, J.M. / Luo, Y. / Xiang, S. / Yang, C. / Jiang, K. / Stangier, M. / Vemu, A. / Cook, A. / Wang, S. / Roll-Mecak, A. / Steinmetz, M.O. / Akhmanova, A. / Baldus, M. / Moores, C.A.
Funding support United Kingdom, Netherlands, Switzerland, 6items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MR/R000352/1 United Kingdom
722.016.002 Netherlands
175.010.2009.002 Netherlands
718.015.001 Netherlands
184.032.207 Netherlands
Swiss National Science Foundation31003A_166608 Switzerland
CitationJournal: Nat Commun / Year: 2019
Title: Structural determinants of microtubule minus end preference in CAMSAP CKK domains.
Authors: Joseph Atherton / Yanzhang Luo / Shengqi Xiang / Chao Yang / Ankit Rai / Kai Jiang / Marcel Stangier / Annapurna Vemu / Alexander D Cook / Su Wang / Antonina Roll-Mecak / Michel O Steinmetz ...Authors: Joseph Atherton / Yanzhang Luo / Shengqi Xiang / Chao Yang / Ankit Rai / Kai Jiang / Marcel Stangier / Annapurna Vemu / Alexander D Cook / Su Wang / Antonina Roll-Mecak / Michel O Steinmetz / Anna Akhmanova / Marc Baldus / Carolyn A Moores /
Abstract: CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To ...CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which lacks minus-end specificity. Here we report near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes, which show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit different footprints on microtubules. NMR experiments show that both HsCKK and NgCKK are remarkably rigid. However, whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. Thus, in contrast to many MAPs, the HsCKK domain can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition.
History
DepositionFeb 28, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 27, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
X: Tubulin alpha-1B chain
O: Tubulin alpha-1B chain
I: Calmodulin-regulated spectrin-associated protein 1
U: Tubulin beta chain
S: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)223,16213
Polymers219,4735
Non-polymers3,6898
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18950 Å2
ΔGint-93 kcal/mol
Surface area66900 Å2
MethodPISA

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Components

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Protein , 3 types, 5 molecules XOIUS

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


Mass: 50204.445 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: GTP / Source: (natural) Homo sapiens (human) / Cell line: tsa201 / Tissue: embryonic kidney / References: UniProt: P68363
#2: Protein Calmodulin-regulated spectrin-associated protein 1


Mass: 19628.666 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CAMSAP1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5T5Y3
#3: Protein Tubulin beta chain / Tubulin beta-5 chain


Mass: 49717.629 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: GDP Taxol / Source: (natural) Homo sapiens (human) / Cell line: tsa201 / Tissue: embryonic kidney / References: UniProt: P07437

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Non-polymers , 4 types, 8 molecules

#4: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#7: Chemical ChemComp-TA1 / TAXOL / Paclitaxel


Mass: 853.906 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1HsCKK (human CAMSAP1) decorated 13pf taxol-GDP microtubuleCOMPLEX#1-#30MULTIPLE SOURCES
2tubulinCOMPLEX#1, #31NATURAL
3Calmodulin-regulated spectrin-associated protein 1COMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.8 / Details: BRB20
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 42 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Details: dose weighted images used in final reconstructions

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameCategory
7Cootmodel fitting
10RELIONfinal Euler assignment
11RELIONclassification
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 36342 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL

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