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- PDB-9bld: crystal structure of thermostable dienelactone hydrolase -

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Basic information

Entry
Database: PDB / ID: 9bld
Titlecrystal structure of thermostable dienelactone hydrolase
ComponentsCarboxymethylenebutenolidase
KeywordsHYDROLASE / Dienelactone hydrolase / esterase activity / PETase / BHETase / thermostable enzyme / PET depolymerization
Function / homology: / Dienelactone hydrolase / Dienelactone hydrolase family / Alpha/Beta hydrolase fold / hydrolase activity / Carboxymethylenebutenolidase
Function and homology information
Biological speciesHydrogenobacter thermophilus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.67 Å
AuthorsMuniz, J.R.C. / Almeida, D. / Brito, V. / Ciancaglini, I. / Sandano, A.L.H. / Squina, F. / Garcia, W.
Funding support Brazil, 25items
OrganizationGrant numberCountry
Sao Paulo Research Foundation (FAPESP)2020/15595-3 Brazil
Sao Paulo Research Foundation (FAPESP)2020/05784-3 Brazil
Sao Paulo Research Foundation (FAPESP)2022/05731-2 Brazil
Sao Paulo Research Foundation (FAPESP)2022/08958-8 Brazil
Sao Paulo Research Foundation (FAPESP)2015/23279-6 Brazil
Sao Paulo Research Foundation (FAPESP)2019/19360-3 Brazil
Sao Paulo Research Foundation (FAPESP)2020/11019-8 Brazil
Sao Paulo Research Foundation (FAPESP)2021/04254-3 Brazil
Sao Paulo Research Foundation (FAPESP)2022/14112-4 Brazil
Sao Paulo Research Foundation (FAPESP)2023/04782-5 Brazil
Sao Paulo Research Foundation (FAPESP)2023/04828-5 Brazil
Sao Paulo Research Foundation (FAPESP)2023/12398-0 Brazil
Sao Paulo Research Foundation (FAPESP)2023/04941-6 Brazil
Sao Paulo Research Foundation (FAPESP)2023/08832-7 Brazil
Brazilian National Council for Scientific and Technological Development (CNPq)305816/2020-9 Brazil
Brazilian National Council for Scientific and Technological Development (CNPq)422132/2018-7 Brazil
Brazilian National Council for Scientific and Technological Development (CNPq)306279/2020-7 Brazil
Brazilian National Council for Scientific and Technological Development (CNPq)315847/2021-2 Brazil
Brazilian National Council for Scientific and Technological Development (CNPq)403844/2023-1 Brazil
Brazilian National Council for Scientific and Technological Development (CNPq)176662/2023-5 Brazil
Brazilian National Council for Scientific and Technological Development (CNPq)304998/2023-0 Brazil
Brazilian National Council for Scientific and Technological Development (CNPq)307199/2023-1 Brazil
Other government88887.625460/2021-00 Brazil
Other government88887.600186/2021-00 Brazil
Other government88887.518696/2020-00 Brazil
CitationJournal: To Be Published
Title: crystal structure of thermostable dienelactone hydrolase
Authors: Muniz, J.R.C. / Almeida, D. / Brito, V. / Ciancaglini, I. / Sandano, A.L.H. / Squina, F. / Garcia, W.
History
DepositionApr 30, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 14, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Carboxymethylenebutenolidase
B: Carboxymethylenebutenolidase
C: Carboxymethylenebutenolidase
D: Carboxymethylenebutenolidase


Theoretical massNumber of molelcules
Total (without water)109,0364
Polymers109,0364
Non-polymers00
Water13,836768
1
A: Carboxymethylenebutenolidase
B: Carboxymethylenebutenolidase


Theoretical massNumber of molelcules
Total (without water)54,5182
Polymers54,5182
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2490 Å2
ΔGint-6 kcal/mol
Surface area18340 Å2
MethodPISA
2
C: Carboxymethylenebutenolidase
D: Carboxymethylenebutenolidase


Theoretical massNumber of molelcules
Total (without water)54,5182
Polymers54,5182
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2340 Å2
ΔGint-6 kcal/mol
Surface area18310 Å2
MethodPISA
Unit cell
Length a, b, c (Å)77.385, 87.298, 143.245
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21A
32A
42A
53A
63A
74A
84A
95A
105A
116A
126A

NCS domain segments:

Auth asym-ID: A / Label asym-ID: A

Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth seq-IDLabel seq-ID
111METMETTHRTHR1 - 2347 - 240
211METMETTHRTHR1 - 2347 - 240
322GLYGLYLEULEU2 - 2338 - 239
422GLYGLYLEULEU2 - 2338 - 239
533METMETTHRTHR1 - 2347 - 240
633METMETTHRTHR1 - 2347 - 240
744GLYGLYLEULEU2 - 2338 - 239
844GLYGLYLEULEU2 - 2338 - 239
955METMETTHRTHR1 - 2347 - 240
1055METMETTHRTHR1 - 2347 - 240
1166GLYGLYLEULEU2 - 2338 - 239
1266GLYGLYLEULEU2 - 2338 - 239

NCS ensembles :
IDDetails
1Local NCS retraints between domains: 1 2
2Local NCS retraints between domains: 3 4
3Local NCS retraints between domains: 5 6
4Local NCS retraints between domains: 7 8
5Local NCS retraints between domains: 9 10
6Local NCS retraints between domains: 11 12

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Components

#1: Protein
Carboxymethylenebutenolidase


Mass: 27259.045 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hydrogenobacter thermophilus (bacteria)
Gene: HTH_1817 / Production host: Escherichia coli (E. coli) / References: UniProt: D3DKA9
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 768 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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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: 2.3 Å3/Da / Density % sol: 46.54 % / Description: long rods
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 0.2 M sodium formate, 0.1 M of bis-tris propane pH 7.5, and 20% w/v of PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: LNLS SIRIUS / Beamline: MANACA / Wavelength: 0.97718 Å
DetectorType: DECTRIS PILATUS 2M / Detector: PIXEL / Date: May 5, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97718 Å / Relative weight: 1
ReflectionResolution: 1.67→74.545 Å / Num. obs: 113000 / % possible obs: 100 % / Redundancy: 13.4 % / CC1/2: 0.995 / Rmerge(I) obs: 0.123 / Rpim(I) all: 0.035 / Rrim(I) all: 0.128 / Net I/σ(I): 11.6
Reflection shellResolution: 1.67→1.699 Å / Redundancy: 12.7 % / Rmerge(I) obs: 3.785 / Mean I/σ(I) obs: 0.8 / Num. unique obs: 5592 / CC1/2: 0.363 / Rpim(I) all: 1.095 / Rrim(I) all: 3.942 / % possible all: 100

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Processing

Software
NameVersionClassification
REFMAC5.8.0425refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.67→74.545 Å / Cor.coef. Fo:Fc: 0.974 / Cor.coef. Fo:Fc free: 0.967 / SU B: 4.785 / SU ML: 0.075 / Cross valid method: FREE R-VALUE / ESU R: 0.093 / ESU R Free: 0.089
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.1891 5771 5.112 %
Rwork0.1641 107121 -
all0.165 --
obs-112892 99.868 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 29.315 Å2
Baniso -1Baniso -2Baniso -3
1-0.446 Å2-0 Å2-0 Å2
2--0.02 Å20 Å2
3----0.466 Å2
Refinement stepCycle: LAST / Resolution: 1.67→74.545 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7410 0 0 768 8178
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.0127734
X-RAY DIFFRACTIONr_bond_other_d0.0010.0167119
X-RAY DIFFRACTIONr_angle_refined_deg1.6021.82710496
X-RAY DIFFRACTIONr_angle_other_deg0.5531.75416442
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.695969
X-RAY DIFFRACTIONr_dihedral_angle_2_deg7.121536
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.447101249
X-RAY DIFFRACTIONr_dihedral_angle_6_deg16.69610367
X-RAY DIFFRACTIONr_chiral_restr0.0810.21094
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.029145
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021843
X-RAY DIFFRACTIONr_nbd_refined0.2090.21365
X-RAY DIFFRACTIONr_symmetry_nbd_other0.190.25985
X-RAY DIFFRACTIONr_nbtor_refined0.1850.23820
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0780.23699
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1590.2626
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.1280.23
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1610.216
X-RAY DIFFRACTIONr_nbd_other0.1490.248
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.1990.217
X-RAY DIFFRACTIONr_xyhbond_nbd_other0.0110.21
X-RAY DIFFRACTIONr_mcbond_it1.9511.8593774
X-RAY DIFFRACTIONr_mcbond_other1.9511.8593774
X-RAY DIFFRACTIONr_mcangle_it2.863.3344720
X-RAY DIFFRACTIONr_mcangle_other2.8623.3344721
X-RAY DIFFRACTIONr_scbond_it3.3052.2813960
X-RAY DIFFRACTIONr_scbond_other3.3052.2813961
X-RAY DIFFRACTIONr_scangle_it5.2373.9965757
X-RAY DIFFRACTIONr_scangle_other5.2373.9965758
X-RAY DIFFRACTIONr_lrange_it7.13222.298936
X-RAY DIFFRACTIONr_lrange_other7.03221.0648724
X-RAY DIFFRACTIONr_ncsr_local_group_10.070.057640
X-RAY DIFFRACTIONr_ncsr_local_group_20.0690.057569
X-RAY DIFFRACTIONr_ncsr_local_group_30.0790.057610
X-RAY DIFFRACTIONr_ncsr_local_group_40.0650.057591
X-RAY DIFFRACTIONr_ncsr_local_group_50.0740.057589
X-RAY DIFFRACTIONr_ncsr_local_group_60.0830.057517
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)Weight position
11AX-RAY DIFFRACTIONLocal ncs0.070420.05009
12AX-RAY DIFFRACTIONLocal ncs0.070420.05009
23AX-RAY DIFFRACTIONLocal ncs0.069480.05009
24AX-RAY DIFFRACTIONLocal ncs0.069480.05009
35AX-RAY DIFFRACTIONLocal ncs0.07890.05009
36AX-RAY DIFFRACTIONLocal ncs0.07890.05009
47AX-RAY DIFFRACTIONLocal ncs0.065390.05009
48AX-RAY DIFFRACTIONLocal ncs0.065390.05009
59AX-RAY DIFFRACTIONLocal ncs0.073950.05009
510AX-RAY DIFFRACTIONLocal ncs0.073950.05009
611AX-RAY DIFFRACTIONLocal ncs0.083460.05009
612AX-RAY DIFFRACTIONLocal ncs0.083460.05009
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
1.67-1.7140.3514110.34677770.34683030.8940.89598.6150.344
1.714-1.760.3084500.30875990.30880520.9340.93399.96270.305
1.76-1.8110.3064160.27774140.27978310.940.94899.98720.27
1.811-1.8670.2664020.24272060.24376080.9530.9611000.226
1.867-1.9280.233780.20970110.2173890.9650.9711000.189
1.928-1.9960.2223570.18967930.1971500.9680.9771000.164
1.996-2.0710.2173440.17765750.17969190.9690.981000.154
2.071-2.1560.1893370.15963300.16166670.9780.9851000.14
2.156-2.2520.1773240.15860640.15963880.9810.9851000.138
2.252-2.3610.1863150.14658210.14861360.9780.9871000.129
2.361-2.4890.172910.14255360.14358280.9830.98899.98280.127
2.489-2.640.1842830.14852230.14955060.980.9871000.137
2.64-2.8220.1942580.1549580.15352170.9770.98699.98080.142
2.822-3.0470.1892430.16146160.16348590.9770.9841000.16
3.047-3.3380.1752220.15542660.15644880.9810.9861000.16
3.338-3.7310.1782050.15338680.15440730.9810.9871000.165
3.731-4.3060.1481980.13834420.13836400.9860.9891000.161
4.306-5.270.1371500.11929380.1230880.9910.9931000.146
5.27-7.4340.193960.16423330.16524290.9780.9861000.193
7.434-74.5450.194910.18413510.18414420.9840.9781000.219
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL112)L122)L132)L222)L232)L332)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T112)T122)T132)T222)T232)T332)Origin x (Å)Origin y (Å)Origin z (Å)
12.0982-2.3553-4.18462.69864.70158.38090.19320.02880.1634-0.25930.0614-0.2203-0.32480.0116-0.25460.1838-0.05460.02780.20080.02860.12720.2865-3.9962-40.0744
21.38530.70070.15171.0440.29622.2526-0.01950.18280.1248-0.04360.0896-0.0037-0.0967-0.123-0.07010.1060.0025-0.02790.14990.04190.046314.5284-2.9652-28.8869
312.6065-0.1966-0.33360.3023-0.32292.022-0.31430.004-0.6806-0.34590.11390.06130.4634-0.16960.20040.4245-0.1172-0.03710.15-0.02230.08969.8987-18.6696-34.3827
40.48440.1428-0.37580.0541-0.14231.4413-0.13570.04860.0648-0.02760.04660.02230.16610.02880.08910.10670.00780.00730.12340.00150.053117.6778-12.2768-18.5257
510.09581.9661-7.22120.7456-0.91626.620.0863-0.30380.35470.15630.05090.01310.2390.2262-0.13720.1795-0.01310.00230.1608-0.03750.071217.5494-1.653726.1264
60.7217-0.716-0.01362.7072-0.63741.83510.0416-0.05550.12840.12440.0224-0.02660.10780.233-0.0640.12420.0063-0.01180.1625-0.00490.035522.4934-4.674614.668
71.683-1.0627-0.65653.13911.51171.19160.0179-0.1869-0.15240.3945-0.11930.27690.33640.00530.10130.2236-0.01320.01460.13160.01270.033312.8756-12.463421.913
80.3164-0.0919-0.33240.30220.29350.9085-0.0125-0.00330.06230.0747-0.03750.03710.15220.0540.050.11680.02460.03510.11090.0210.053513.5553-10.45594.7713
91.8809-0.82622.60650.3935-1.26574.34340.17250.2015-0.2914-0.0610.00220.10670.1130.1187-0.17480.1077-0.0307-0.0370.1973-0.04090.1226-21.8525-1.1587-24.1114
101.61730.16140.01781.7326-0.45781.5071-0.03850.20360.0187-0.01620.0336-0.0599-0.04070.23790.00480.0874-0.01950.00910.155-0.00930.0692-14.72816.3315-17.0809
111.52041.8023-0.09454.5649-0.75890.65130.02790.2401-0.0068-0.0734-0.1309-0.0677-0.1431-0.03690.1030.13810.0059-0.03360.21540.01070.0421-24.784811.4082-23.757
120.67340.06750.00650.26820.38220.6867-0.08920.0158-0.0007-0.01840.07270.0105-0.03750.00460.01650.0669-0.0218-0.00960.1190.03080.0954-21.886611.7903-5.9215
130.67970.2530.42580.5251-0.27510.89920.0898-0.147-0.09670.00130.0152-0.06-0.0183-0.2038-0.10490.0752-0.00910.02140.1540.00390.0658-20.37193.259129.133
140.8614-0.32320.28961.1946-0.40531.19340.1188-0.20940.10460.3118-0.1938-0.0234-0.2193-0.04610.0750.1967-0.06770.00920.1857-0.05280.0401-18.271915.145833.603
150.3760.101-0.0271.436-0.2440.5934-0.0295-0.0677-0.02120.0748-0.0222-0.0405-0.14810.07590.05170.0847-0.0060.00040.1327-0.0170.0734-11.352111.58120.8271
160.81350.05680.4950.1445-0.20220.9113-0.07860.00870.0307-0.05440.07680.0328-0.0447-0.0580.00170.0739-0.0113-0.01150.14-0.00810.0954-22.189614.102911.7911
Refinement TLS group
IDRefine-IDRefine TLS-IDSelectionAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1ALLA1 - 12
2X-RAY DIFFRACTION2ALLA13 - 68
3X-RAY DIFFRACTION3ALLA69 - 92
4X-RAY DIFFRACTION4ALLA93 - 234

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