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

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Basic information

Entry
Database: PDB / ID: 5m50
TitleMechanism of microtubule minus-end recognition and protection by CAMSAP proteins
Components
  • Calmodulin-regulated spectrin-associated protein 3
  • Tubulin alpha chain
  • Tubulin beta-2B chain
KeywordsSTRUCTURAL PROTEIN / CAMSAP CKK Microtubule Tubulin
Function / homology
Function and homology information


regulation of organelle organization / zonula adherens maintenance / microtubule minus-end / protein transport along microtubule / 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 ...regulation of organelle organization / zonula adherens maintenance / microtubule minus-end / protein transport along microtubule / 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 / microtubule anchoring / regulation of Golgi organization / COPI-dependent Golgi-to-ER retrograde traffic / COPI-independent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / RHO GTPases activate IQGAPs / microtubule minus-end binding / RHO GTPases Activate Formins / establishment or maintenance of microtubule cytoskeleton polarity / MHC class II antigen presentation / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / cilium movement / Aggrephagy / epithelial cell-cell adhesion / The role of GTSE1 in G2/M progression after G2 checkpoint / Separation of Sister Chromatids / zonula adherens / 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 / establishment of epithelial cell apical/basal polarity / embryo development ending in birth or egg hatching / positive regulation of axon guidance / motile cilium / regulation of focal adhesion assembly / spectrin binding / regulation of microtubule polymerization / axoneme / microtubule-based process / cytoplasmic microtubule / cellular response to interleukin-4 / regulation of microtubule cytoskeleton organization / regulation of cell migration / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / neuron projection development / actin filament binding / mitotic cell cycle / double-stranded RNA binding / microtubule cytoskeleton / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / in utero embryonic development / microtubule / calmodulin binding / hydrolase activity / cilium / ciliary basal body / protein heterodimerization activity / GTPase activity / ubiquitin protein ligase binding / centrosome / 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. / 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
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / Tubulin alpha chain / Tubulin alpha-1B chain / Tubulin beta-2B chain / Calmodulin-regulated spectrin-associated protein 3
Similarity search - Component
Biological speciesMus musculus (house mouse)
Bos taurus (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.3 Å
AuthorsAkhmanova, A. / Moores, C.A. / Baldus, M. / Steinmetz, M.O. / Topf, M. / Roberts, A.J. / Grant, B.J. / Scarabelli, G. / Joseph, A.-P. / van Hooff, J.J.E. ...Akhmanova, A. / Moores, C.A. / Baldus, M. / Steinmetz, M.O. / Topf, M. / Roberts, A.J. / Grant, B.J. / Scarabelli, G. / Joseph, A.-P. / van Hooff, J.J.E. / Houben, K. / Hua, S. / Luo, Y. / Stangier, M.M. / Jiang, K. / Atherton, J.
Funding support United Kingdom, United States, Netherlands, Switzerland, 6items
OrganizationGrant numberCountry
European Research CouncilMR/J000973/1 United Kingdom
European Research CouncilMR/M019292/1 United Kingdom
Wellcome Trust104196/Z/14/Z United Kingdom
National Institutes of HealthR01GM070862 United States
NWO718.015.001 Netherlands
Swiss National Science Foundation310030B_138659 Switzerland
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, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 4, 2017Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2017Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed ..._citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Nov 15, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3May 15, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr2_auth_seq_id

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-4154
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Structure viewerMolecule:
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Assembly

Deposited unit
D: Tubulin alpha chain
E: Tubulin beta-2B chain
A: Tubulin alpha chain
B: Tubulin beta-2B chain
C: Calmodulin-regulated spectrin-associated protein 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)210,54213
Polymers206,8535
Non-polymers3,6898
Water1448
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17760 Å2
ΔGint-80 kcal/mol
Surface area68240 Å2
MethodPISA

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Components

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Protein , 3 types, 5 molecules DAEBC

#1: Protein Tubulin alpha chain


Mass: 48769.988 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: F2Z4C1, UniProt: P81947*PLUS
#2: Protein Tubulin beta-2B chain


Mass: 47940.945 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: Q6B856
#3: Protein Calmodulin-regulated spectrin-associated protein 3 / Protein Nezha


Mass: 13431.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Camsap3, Kiaa1543 / Production host: Escherichia coli (E. coli) / References: UniProt: Q80VC9

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Non-polymers , 5 types, 16 molecules

#4: Chemical ChemComp-GTP / GUANOSINE-5'-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


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


Mass: 853.906 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication*YM
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: H2O

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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
1Complex of two tubulin dimers with bound CAMSAP3-CKK domainCOMPLEX#1-#30MULTIPLE SOURCES
2tubulinCOMPLEX#1-#21NATURAL
3CAMSAP3-CKK domainCOMPLEX#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Bos taurus (domestic cattle)9913
23Mus musculus (house mouse)10090
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.8 / Details: BRB80
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 13pf Microtubules
Specimen supportGrid material: COPPER / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen 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 FIELD
Image recordingElectron dose: 25 e/Å2 / Detector mode: INTEGRATING / Film or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Details: 25e-/A2 used in final reconstuctions

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameCategory
9SPIDERinitial Euler assignment
10FREALIGNfinal Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 5.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 6530
Details: Gold-standard noise substitution test used to asses for over fitting (Chen et al., 2013)
Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00415009
ELECTRON MICROSCOPYf_angle_d0.77720402
ELECTRON MICROSCOPYf_dihedral_angle_d3.9979009
ELECTRON MICROSCOPYf_chiral_restr0.0482222
ELECTRON MICROSCOPYf_plane_restr0.0072752

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