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- PDB-1tvk: The binding mode of epothilone A on a,b-tubulin by electron cryst... -

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Basic information

Entry
Database: PDB / ID: 1tvk
TitleThe binding mode of epothilone A on a,b-tubulin by electron crystallography
Components
  • Tubulin alpha chain
  • Tubulin beta chain
KeywordsCELL CYCLE / STRUCTURAL PROTEIN / epothilone / taxol / ligand interactions
Function / homology
Function and homology information


positive regulation of axon guidance / microtubule-based process / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / nervous system development / microtubule / hydrolase activity ...positive regulation of axon guidance / microtubule-based process / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / nervous system development / microtubule / hydrolase activity / protein heterodimerization activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal ...Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 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
EPOTHILONE A / GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Tubulin alpha-1A chain / Tubulin beta chain / Tubulin alpha-1D chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesBos taurus (cattle)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 2.89 Å
AuthorsNettles, J.H. / Li, H. / Cornett, B. / Krahn, J.M. / Snyder, J.P. / Downing, K.H.
CitationJournal: Science / Year: 2004
Title: The binding mode of epothilone A on alpha,beta-tubulin by electron crystallography.
Authors: James H Nettles / Huilin Li / Ben Cornett / Joseph M Krahn / James P Snyder / Kenneth H Downing /
Abstract: The structure of epothilone A, bound to alpha,beta-tubulin in zinc-stabilized sheets, was determined by a combination of electron crystallography at 2.89 angstrom resolution and nuclear magnetic ...The structure of epothilone A, bound to alpha,beta-tubulin in zinc-stabilized sheets, was determined by a combination of electron crystallography at 2.89 angstrom resolution and nuclear magnetic resonance-based conformational analysis. The complex explains both the broad-based epothilone structure-activity relationship and the known mutational resistance profile. Comparison with Taxol shows that the longstanding expectation of a common pharmacophore is not met, because each ligand exploits the tubulin-binding pocket in a unique and independent manner.
History
DepositionJun 29, 2004Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 14, 2004Provider: repository / Type: Initial release
Revision 1.1Apr 30, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Jul 18, 2018Group: Author supporting evidence / Data collection
Category: em_image_scans / em_single_particle_entity / em_software
Item: _em_software.image_processing_id / _em_software.name
Revision 1.4Aug 23, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Remark 240 EXPERIMENT TYPE : ELECTRON DIFFRACTION ELECTRON MICROSCOPE SAMPLE SAMPLE AGGREGATION STATE : TWO- ... EXPERIMENT TYPE : ELECTRON DIFFRACTION ELECTRON MICROSCOPE SAMPLE SAMPLE AGGREGATION STATE : TWO-DIMENSIONAL NAME OF SAMPLE : CRYSTAL TUBULIN- : EPOTHILONE A COMPLEX SAMPLE CONCENTRATION : 2MG/ML SAMPLE SUPPORT DETAILS : CONTINUOUS CARBON FILM SAMPLE VITRIFICATION DETAILS : TANNIC ACID AND GLUCOSE : EMBEDDING AND THEN : LIQUID NITROGEN : FREEZING SAMPLE BUFFER : 60mM MES, pH5.3, : 150mM NaCl, 2.5mM GTP, : 1mM MgSO4, 1mM ZnSO4, : 0.02mg/ml Pepstatin. PH : PH=5.3 SAMPLE DETAILS: NULL DATA ACQUISITION DATE OF EXPERIMENT : 1999-2000 NUMBER OF MICROGRAPHS-IMAGES : NULL TEMPERATURE (KELVIN) : 100.00 MICROSCOPE MODEL : JEOL 4000EX DETECTOR TYPE : GATAN 794 MINIMUM DEFOCUS (NM) : NULL MAXIMUM DEFOCUS (NM) : NULL MINIMUM TILT ANGLE (DEGREES) : 15.00 MAXIMUM TILT ANGLE (DEGREES) : 55.00 NOMINAL CS : NULL IMAGING MODE : DIFFRACTION ELECTRON DOSE (ELECTRONS NM**-2) : 1000.00 ILLUMINATION MODE : LOW-DOSE NOMINAL MAGNIFICATION : NULL CAMERA LENGTH : 150 CM CALIBRATED MAGNIFICATION : NULL SOURCE : LaB6 ACCELERATION VOLTAGE (KV) : 400 IMAGING DETAILS: A WEAK ELECTRON BEAM AND LONG EXPOSURE TIME (40-60S) WERE USED TO MINIMIZE THE VERTICAL BLOOMING STREAK IN THE DIFFRACTION PATTERN RECORDED WITH THE CCD CAMERA. A PATENT APPLICATION IS PENDING WITH RESPECT TO THESE COORDINATES. CONTACT EMORY UNIVERSITY OFFICE OF TECHNOLOGY TRANSFER FOR COMMERCIAL APPLICATIONS OR LICENSING OPPORTUNIES HTTP://WWW.OTT.EMORY.EDU CONTACT JHN FOR QUESTIONS REGARDING COORDINATES AND PROCESSING JHN@WELLYES.COM
Remark 400 COMPOUND THE MODEL OF THE A,B-TUBULIN/EPOTHILONE A COMPLEX WAS DERIVED USING HIGH RESOLUTION ... COMPOUND THE MODEL OF THE A,B-TUBULIN/EPOTHILONE A COMPLEX WAS DERIVED USING HIGH RESOLUTION ELECTRON DIFFRACTIONS FROM TWO DIMENSIONAL CRYSTALS OF TUBULIN INDUCED BY THE PRESENCE OF ZN++ IONS. DEPOSITED ARE COORDINATES FOR EPOTHILONE A BOUND TO AB-TUBULIN DIMER IN THE ZINC-INDUCED SHEETS. THE LIGAND MODEL WAS FIT INTO A DENSITY MAP FOR WHICH THE RESOLUTION IN THE PLANE OF THE SHEET WAS 2.89 ANGSTROMS AND THAT PERPENDICULAR TO THE SHEET WAS ABOUT 4.2 ANGSTROMS AS DESCRIBED IN THE SUPPLEMENTAL TEXT. PROTEIN MODEL HAS NOT BEEN OPTIMIZED AT THE RESOLUTION REPORTED IN REMARK 3 - SEE WWW.ORGANIC.EMORY.EDU/EPO R FREE REPORTED BY THE SOFTWARE ABOVE IS NOT RELEVANT. PHASES WERE DERIVED FROM A PREVIOUS MODEL OF ALPHA/BETA TUBULIN COMPLEXED WITH TAXOL (PDB ID 1JFF). "SHAKING", HIGH TEMPERATURE ANNEALING, AND MODEL AVERAGING WERE COMBINED TO PRODUCE AN OMIT MAP OF THE BOUND EPOTHILONE THAT MINIMIZED SYSTEMATIC BIAS. A NUMBER OF CONFORMATIONAL MODELS WERE FLEXIBLY FITTED INTO THE INITIAL MAP AND TESTED AGAINST THE DIFFRACTIONS BY DIFFERENCE MAP REFINEMENT AS DESCRIBED IN THE PRIMARY REFERENCE. REFINEMENT TO THE FINAL STRUCTURE WAS LIMITED WITHIN AN 8 A RADIUS OF THE LIGAND. ALTHOUGH THE REST OF THE PROTEIN SHOWS LITTLE DEVIATION FROM THAT SEEN IN 1JFF, CERTAIN RESIDUES DO NOT FULLY CONFORM TO RAMACHANDRAN CHARACTERISTICS AND SHOULD BE REGARDED WITH CAUTION. THE FINAL MAPS ASSOCIATED WITH THE PRESENT MODEL WERE DERIVED BY RIGID FITTING OF THE MODELED COMPLEX. AS SUCH, FREE R REPORTED BY THE SOFTWARE ABOVE IS NOT RELEVANT.

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Assembly

Deposited unit
A: Tubulin alpha chain
B: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)98,2705
Polymers96,8102
Non-polymers1,4603
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5900 Å2
ΔGint-27 kcal/mol
Surface area29710 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.200, 93.500, 90.000
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Tubulin alpha chain


Mass: 48869.117 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: see REMARK 400 / Source: (natural) Bos taurus (cattle) / Organ: Brain / References: UniProt: P02550, UniProt: Q2HJ86*PLUS
#2: Protein Tubulin beta chain


Mass: 47940.945 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: see REMARK 400 / Source: (natural) Bos taurus (cattle) / Organ: Brain / References: UniProt: P02554, UniProt: Q6B856*PLUS
#3: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#4: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#5: Chemical ChemComp-EP / EPOTHILONE A


Mass: 493.656 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C26H39NO6S

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: tubulin 2D crystal / Type: COMPLEX
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
CrystalDensity % sol: 66.11 %

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Data collection

MicroscopyModel: JEOL 4000EX
Details: A weak electron beam and long exposure time (40-60s) were used to minimize the vertical blooming streak in the diffraction pattern recorded with the CCD camera.
Electron gunAccelerating voltage: 400 kV / Illumination mode: SPOT SCAN
Electron lensMode: DIFFRACTION
Specimen holderTemperature: 100 K / Tilt angle max: 55 ° / Tilt angle min: 15 °
Image recordingElectron dose: 100 e/Å2 / Film or detector model: GENERIC GATAN / Details: 2K CCD
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: electron
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategoryDetails
1CNS1.1model fitting
2QUANTAmodel fittingX-ligand module
3REFMAC5model fitting
4MRC3D reconstructionimage2000
3D reconstructionResolution: 2.89 Å / Symmetry type: 2D CRYSTAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: RECIPROCAL
Target criteria: Analysis of comparitive difference densities
Details: METHOD--annealing, rigid refining REFINEMENT PROTOCOL--rigid body DETAILS--THE MODEL WAS DERIVED USING HIGH RESOLUTION ELECTRON DIFFRACTIONS FROM TWO DIMENSIONAL CRYSTALS OF TUBULIN INDUCED ...Details: METHOD--annealing, rigid refining REFINEMENT PROTOCOL--rigid body DETAILS--THE MODEL WAS DERIVED USING HIGH RESOLUTION ELECTRON DIFFRACTIONS FROM TWO DIMENSIONAL CRYSTALS OF TUBULIN INDUCED BY THE PRESENCE OF ZN++ IONS. WHAT FOLLOWS ARE THE COORDINATES FOR EPOLTHILONE-A BOUND TO AB-TUBULIN DIMER IN THE ZINC-INDUCED SHEETS. THE LIGAND MODEL WAS FIT INTO A DENSITY MAP FOR WHICH THE RESOLUTION IN THE PLANE OF THE SHEET WAS 2.89 ANGSTROMS AND THAT PERPENDICULAR TO THE SHEET WAS ABOUT 4.2 ANGSTROMS AS DESCRIBED IN THE SUPPLEMENTARY MATERIAL. PHASES WERE DERIVED FROM A PREVIOUS MODEL OF ALPHA/BETA TUBULIN COMPLEXED WITH TAXOL (1JFF). SHAKING, HIGH TEMPERATURE ANANEALING, AND MODEL WERE COMBINED TO PRODUCE AN OMIT MAP OF THE BOUND EPOTHILONE THAT SYSTEMATIC BIAS.A NUMBER OF CONFORMATIONAL MODELS WERE FLEXIBLY FITTED INITIAL MAP AND TESTED AGAINST THE DIFFRACTIONS BY DIFFERENCE MAP AS DESCRIBED IN THE PRIMARY REFERENCE. REFINEMENT TO THE FINAL STAGE WAS LIMITED WITHIN AN 8A RADIUS OF THE LIGAND. ALTHOUGH THE REMAINING PART OF THE PROTEIN SHOWS LITTLE DEVIATION FROM THAT SEEN IN 1JFF, CER NOT FULLY CONFORM WITH RAMACHANDRAN CHARACTERISTICS. AND SHOULD B THE FINAL MAPS ASSOCIATED WITH THE PRESENT MODEL WERE DERIVED BY COMPLEX. AS SUCH, FREE R REPORTED BY THE SOFTWARE BELOW IS NOT RE
Atomic model buildingPDB-ID: 1JFF

1jff

RefinementStarting model: PDB ID 1JFF

1jff


Resolution: 2.89→91.29 Å / SU ML: 0.846 / SU R Cruickshank DPI: 0.846 / SU Rfree: 0.861 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.675 / ESU R Free: 0.651 / Stereochemistry target values: Engh & Huber / Details: SEE REMARK 400, COMPOUND
RfactorNum. reflection% reflectionSelection details
Rfree0.32096 967 5 %RANDOM
Rwork0.33275 ---
all0.33214 18321 --
obs0.33214 18321 67.03 %-
Displacement parametersBiso mean: 79.8 Å2
Baniso -1Baniso -2Baniso -3
1--5.48 Å20 Å20.41 Å2
2---3.03 Å20 Å2
3---8.5 Å2
Refinement stepCycle: LAST / Resolution: 2.89→91.29 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6578 0 94 0 6672
LS refinement shellResolution: 2.89→3.046 Å / Total num. of bins used: 10 /
RfactorNum. reflection
Rfree0.469 66
Rwork0.519 1467

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