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- PDB-5cya: Crystal structure of Arl2 GTPase-activating protein tubulin cofac... -

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Basic information

Entry
Database: PDB / ID: 5cya
TitleCrystal structure of Arl2 GTPase-activating protein tubulin cofactor C (TBCC)
ComponentsTubulin-specific chaperone C
KeywordsCHAPERONE / Tubulin cofactors / mirotubule dynamics / tubulin chaperones / Arl2 GTPase-activating protein TBCC / GAP activity / beta helix / beta-sheets
Function / homology
Function and homology information


post-chaperonin tubulin folding pathway / tubulin complex assembly / GTPase activator activity / cell morphogenesis / protein folding / microtubule / cytoplasm
Similarity search - Function
Tubulin-specific chaperone C / C-CAP/cofactor C-like domain / C-CAP/cofactor C-like domain profile. / Cyclase-associated protein CAP/septum formation inhibitor MinC, C-terminal
Similarity search - Domain/homology
Tubulin-specific chaperone C
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2 Å
AuthorsNithianantham, S. / Le, S. / Seto, E. / Jia, W. / Leary, J. / Corbett, K.D. / Moore, J.K. / Al-Bassam, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM110283, GM08429 United States
CitationJournal: Elife / Year: 2015
Title: Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble αβ-tubulin pool for microtubule dynamics.
Authors: Stanley Nithianantham / Sinh Le / Elbert Seto / Weitao Jia / Julie Leary / Kevin D Corbett / Jeffrey K Moore / Jawdat Al-Bassam /
Abstract: Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into ...Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics.
History
DepositionJul 30, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 12, 2015Provider: repository / Type: Initial release
Revision 1.1Sep 27, 2017Group: Author supporting evidence / Derived calculations / Category: pdbx_audit_support / pdbx_struct_oper_list
Item: _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation
Revision 1.2Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tubulin-specific chaperone C
B: Tubulin-specific chaperone C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)61,6944
Polymers61,5022
Non-polymers1922
Water1,67593
1
A: Tubulin-specific chaperone C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)30,9433
Polymers30,7511
Non-polymers1922
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Tubulin-specific chaperone C


Theoretical massNumber of molelcules
Total (without water)30,7511
Polymers30,7511
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)69.790, 69.790, 78.180
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number78
Space group name H-MP43
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A and segid A
21chain B and segid B

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111chain A and segid AA0
211chain B and segid BB0

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Components

#1: Protein Tubulin-specific chaperone C / Chromosome instability protein 2 / Tubulin-folding cofactor C


Mass: 30750.805 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Missing residues are disordered
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: CIN2, YPL241C, P1043 / Plasmid: pET3A / Production host: Escherichia coli (E. coli) / Strain (production host): SoluBL21 / References: UniProt: P46670
#2: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 93 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 1.55 Å3/Da / Density % sol: 20.54 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5.2
Details: 0.1 M Sodium Citrate, 0.4 M Ammonium Sulfate, 0.7 M Lithium Sulfate pH 5.2
PH range: 5.2-5.6

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 0.9795 Å
DetectorType: MAR CCD 165 mm / Detector: CCD / Date: Jun 18, 2012
RadiationMonochromator: Liquid nitrogen-cooled double crystal / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 2→34.9 Å / Num. all: 25409 / Num. obs: 25341 / % possible obs: 100 % / Redundancy: 7.6 % / Biso Wilson estimate: 33.59 Å2 / Rmerge(I) obs: 0.082 / Rpim(I) all: 0.032 / Rrim(I) all: 0.088 / Rsym value: 0.082 / Net I/av σ(I): 6.107 / Net I/σ(I): 13.4 / Num. measured all: 193620
Reflection shell

Diffraction-ID: 1 / Rejects: 0

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRpim(I) allRsym valueNet I/σ(I) obs% possible all
2-2.117.60.7162.72793536880.2790.7162.7100
2.11-2.247.60.51.52656935020.1950.53.7100
2.24-2.397.60.342.22490832730.1320.345.4100
2.39-2.587.60.2393.12330830550.0930.2397.6100
2.58-2.837.60.1514.82147828150.0590.15111.2100
2.83-3.167.70.0828.41980425780.0320.08218100
3.16-3.657.70.0688.51729922510.0260.06825.5100
3.65-4.477.70.05211.31477819240.020.05232.6100
4.47-6.327.60.04610.91135114850.0170.04633.8100
6.32-34.1057.40.03714.661908380.0150.03734.299.5

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Phasing

PhasingMethod: MAD

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Processing

Software
NameVersionClassification
MOSFLMdata reduction
SCALA3.3.21data scaling
SOLVEphasing
RESOLVEphasing
PHENIX1.9_1692refinement
PDB_EXTRACT3.15data extraction
Cootmodel building
RefinementMethod to determine structure: MAD / Resolution: 2→34.105 Å / Rfactor Rfree error: 0.01 / SU ML: 0.21 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 26.56 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2383 1300 5.13 %Random selection
Rwork0.2011 24041 --
obs0.203 25341 99.84 %-
Solvent computationIon probe radii: 4 Å / Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 190.72 Å2 / ksol: 0.8 e/Å3
Displacement parametersBiso max: 142.85 Å2 / Biso mean: 50.4154 Å2 / Biso min: 27.62 Å2
Baniso -1Baniso -2Baniso -3
1-3.58 Å20 Å20 Å2
2--3.58 Å20 Å2
3----2.65 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.19 Å0.259 Å
Luzzati d res low-3.7 Å
Luzzati sigma a-0.31 Å
Refinement stepCycle: final / Resolution: 2→34.105 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2641 0 10 93 2744
Biso mean--63.14 49.44 -
Num. residues----329
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint function
X-RAY DIFFRACTIONf_bond_d0.0062694SINUSOIDAL
X-RAY DIFFRACTIONf_angle_d1.0013632SINUSOIDAL
X-RAY DIFFRACTIONf_chiral_restr0.043420SINUSOIDAL
X-RAY DIFFRACTIONf_plane_restr0.005463SINUSOIDAL
X-RAY DIFFRACTIONf_dihedral_angle_d11.996986SINUSOIDAL
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A1558X-RAY DIFFRACTION10.302TORSIONAL
12B1558X-RAY DIFFRACTION10.302TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION

Resolution (Å)Rfactor RfreeNum. reflection Rfree% reflection Rfree (%)Rfactor RworkNum. reflection RworkRfactor Rfree error% reflection obs (%)
2.0001-2.08020.29071605.10.252826110.02399
2.0802-2.17480.32361234.50.233826810.027100
2.1748-2.28950.23981294.780.21926680.021100
2.2895-2.43290.2361385.10.208326750.02100
2.4329-2.62060.25731505.60.214526560.021100
2.6206-2.88420.28541615.70.218826440.023100
2.8842-3.30130.22931365.020.215927070.019100
3.3013-4.15810.20811425.80.179626800.017100
4.1581-34.10960.22581615.70.187627190.017100
Refinement TLS params.Method: refined / Origin x: 15.1242 Å / Origin y: 28.3029 Å / Origin z: 33.2249 Å
111213212223313233
T0.3155 Å20.0053 Å20.0173 Å2-0.3585 Å20.0067 Å2--0.3903 Å2
L0.6657 °2-0.945 °21.6899 °2-1.389 °2-2.4075 °2--4.479 °2
S-0.1594 Å °-0.0204 Å °0.0687 Å °0.1294 Å °0.0548 Å °-0.0964 Å °-0.1355 Å °-0.0231 Å °-0.0021 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA101 - 268
2X-RAY DIFFRACTION1allB101 - 268
3X-RAY DIFFRACTION1allB269 - 365

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