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- PDB-5n5n: Cryo-EM structure of tsA201 cell alpha1B and betaI and betaIVb mi... -

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Basic information

Entry
Database: PDB / ID: 5n5n
TitleCryo-EM structure of tsA201 cell alpha1B and betaI and betaIVb microtubules
DescriptorTubulin beta chain, Tubulin alpha-1B chain
KeywordsSTRUCTURAL PROTEIN / Microtubules Dynamics Tubulin Isoform / structural protein
Specimen sourceHomo sapiens / human
MethodElectron microscopy (4.2 Å resolution / Filament / Single particle)
AuthorsVemu, A. / Atherton, J. / Spector, J.O. / Moores, C.A. / Roll-Mecak, A.
CitationMol. Biol. Cell, 2017

Mol. Biol. Cell, 2017 Yorodumi Papers
Tubulin isoform composition tunes microtubule dynamics.
Annapurna Vemu / Joseph Atherton / Jeffrey O Spector / Carolyn A Moores / Antonina Roll-Mecak

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 14, 2017 / Release: Nov 1, 2017

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Assembly

Deposited unit
B: Tubulin beta chain
K: Tubulin alpha-1B chain
G: Tubulin alpha-1B chain
H: Tubulin alpha-1B chain
I: Tubulin alpha-1B chain
J: Tubulin alpha-1B chain
L: Tubulin alpha-1B chain
A: Tubulin beta chain
C: Tubulin beta chain
D: Tubulin beta chain
E: Tubulin beta chain
F: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)584,96336
Polyers578,40512
Non-polymers6,55824
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)54520
ΔGint (kcal/M)-306
Surface area (Å2)176740
MethodPISA

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Components

#1: Polypeptide(L)
Tubulin beta chain / Tubulin beta-5 chain


Mass: 47735.793 Da / Num. of mol.: 6 / Details: GMPCPP (GTP-analogue) bound / Source: (gene. exp.) Homo sapiens / human / References: UniProt: P07437
#2: Polypeptide(L)
Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 48665.027 Da / Num. of mol.: 6 / Details: GTP-bound / Source: (gene. exp.) Homo sapiens / human / References: UniProt: P68363
#3: Chemical
ChemComp-G2P / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GMP-CPP (energy-carrying molecule analogue) *YM


Mass: 521.208 Da / Num. of mol.: 6 / Formula: C11H18N5O13P3
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Formula: Mg
#5: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / GTP *YM


Mass: 523.180 Da / Num. of mol.: 6 / Formula: C10H16N5O14P3

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Human tsA201 cell tubulin microtubules / Type: COMPLEX
Details: Microtubules formed from tsA201 cell tubulin (14pf) with bound GTP analogue GMPCPP. Tubulin was purified via TOG affinity.
Entity ID: 1, 2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens
Source (recombinant)Organism: Homo sapiens
Buffer solutionDetails: BRB80 with GMPCPP (80mM PIPES, 2mM MgCl2, 1mM EGTA, 1mM DTT, 1mM GMPCPP)
pH: 6.8
SpecimenConc.: 2.5 mg/ml
Details: Microtubules polymerised at 25mg/ml in BRB80 with GMPCPP and diluted to a final concentration of 2.5mg/ml
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyMicroscope model: 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)
Image scansSampling size: 1.221 microns

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Processing

SoftwareName: REFMAC / Version: 5.8.0135 / Classification: refinement
EM software
IDNameCategoryImage processing IDImaging IDFitting ID
1EMANPARTICLE SELECTION1
2SerialEMIMAGE ACQUISITION1
7UCSF ChimeraMODEL FITTING1
10FREALIGNFINAL EULER ASSIGNMENT1
12FREALIGNRECONSTRUCTION1
13CootMODEL REFINEMENT1
14PHENIXMODEL REFINEMENT
15REFMACMODEL REFINEMENT
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 13000
Details: Gold-standard high-resolution noise substitution test employed in resolution estimation (Chen et al., 2013)
Symmetry type: POINT
Atomic model buildingRef protocol: OTHER / Ref space: REAL
Refine
Refine IDB iso meanAniso B11Aniso B12Aniso B13Aniso B22Aniso B23Aniso B33Correlation coeff Fo to FcDetailsR factor R workR factor obsHighest resolutionLowest resolutionNumber reflection obsPercent reflection obsOverall SU BOverall SU MLSolvent ion probe radiiSolvent shrinkage radiiSolvent vdw probe radiiStereochemistry target valuesSolvent model details
166.218-5.481.19-0.851.441.474.040.812HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS0.440920.440924.00214.90102758100.0081.8630.9560.800.801.20MAXIMUM LIKELIHOOD WITH PHASESMASK
ELECTRON MICROSCOPY
Number of atoms included #1Total: 40470
Refine LS restraints
Refine IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01941382
ELECTRON MICROSCOPYr_bond_other_d0.0010.02038220
ELECTRON MICROSCOPYr_angle_refined_deg1.1351.95956220
ELECTRON MICROSCOPYr_angle_other_deg0.8673.00087870
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.9425.0005094
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.96624.1231950
ELECTRON MICROSCOPYr_dihedral_angle_3_deg11.09715.0006684
ELECTRON MICROSCOPYr_dihedral_angle_4_deg9.64215.000252
ELECTRON MICROSCOPYr_chiral_restr0.0650.2006174
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.02147094
ELECTRON MICROSCOPYr_gen_planes_other0.0010.0209738
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it0.7116.70220466
ELECTRON MICROSCOPYr_mcbond_other0.7116.70220465
ELECTRON MICROSCOPYr_mcangle_it1.34910.05225530
ELECTRON MICROSCOPYr_mcangle_other1.34910.05225531
ELECTRON MICROSCOPYr_scbond_it0.3276.73120916
ELECTRON MICROSCOPYr_scbond_other0.3276.73120909
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other0.71710.05630681
ELECTRON MICROSCOPYr_long_range_B_refined2.97956.28657454
ELECTRON MICROSCOPYr_long_range_B_other2.97856.28757451
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints ncs

Refine ID: ELECTRON MICROSCOPY / Type: interatomic distance / Rms dev position: 0 Å / Weight position: 0.05

Dom IDAuth asym IDEns IDNumber
1B149120
2A149120
1B249118
2C249118
1B349116
2D349116
1B449120
2E449120
1B549120
2F549120
1K649634
2G649634
1K749634
2H749634
1K849634
2I849634
1K949634
2J949634
1K1049634
2L1049634
1G1149636
2H1149636
1G1249634
2I1249634
1G1349634
2J1349634
1G1449634
2L1449634
1H1549634
2I1549634
1H1649634
2J1649634
1H1749634
2L1749634
1I1849634
2J1849634
1I1949634
2L1949634
1J2049634
2L2049634
1A2149122
2C2149122
1A2249118
2D2249118
1A2349120
2E2349120
1A2449124
2F2449124
1C2549118
2D2549118
1C2649118
2E2649118
1C2749122
2F2749122
1D2849116
2E2849116
1D2949118
2F2949118
1E3049120
2F3049120
Refine LS shellHighest resolution: 4 Å / R factor R free: 0 / R factor R work: 0.548 / Lowest resolution: 4.104 Å / Number reflection R free: 0 / Number reflection R work: 7593 / Total number of bins used: 20 / Percent reflection obs: 1

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