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- PDB-4xfp: Crystal Structure of Highly Active Mutant of Bacillus sp. TB-90 U... -

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Basic information

Entry
Database: PDB / ID: 4xfp
TitleCrystal Structure of Highly Active Mutant of Bacillus sp. TB-90 Urate Oxidase
ComponentsUrate oxidase
KeywordsOXIDOREDUCTASE / enzyme / protein engineering / flexible loop
Function / homology
Function and homology information


2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase activity / 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase / factor-independent urate hydroxylase / urate oxidase activity / allantoin metabolic process / urate catabolic process / purine nucleobase metabolic process / peroxisome
Similarity search - Function
2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase, type 1 / Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase / Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase superfamily / OHCU decarboxylase / Urate Oxidase / Urate Oxidase; / Uricase, conserved site / Uricase signature. / Uricase / Uricase ...2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase, type 1 / Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase / Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase superfamily / OHCU decarboxylase / Urate Oxidase / Urate Oxidase; / Uricase, conserved site / Uricase signature. / Uricase / Uricase / Roll / Alpha Beta
Similarity search - Domain/homology
8-AZAXANTHINE / Uric acid degradation bifunctional protein
Similarity search - Component
Biological speciesBacillus TB-90 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.66 Å
AuthorsHibi, T. / Hayashi, Y. / Kawamura, A. / Itoh, T.
Citation
Journal: To be published
Title: Glycine Substitution of Surface Proline 287 Involves Entropic Enhancement of Bacillus sp. TB-90 Uricase Activity
Authors: Hibi, T. / Hayashi, Y. / Kawamura, A. / Itoh, T. / Fukada, H. / Kishimoto, T. / Nishiya, Y.
#1: Journal: Biochemistry / Year: 2014
Title: Intersubunit salt bridges with a sulfate anion control subunit dissociation and thermal stabilization of Bacillus sp. TB-90 urate oxidase
Authors: Hibi, T. / Hayashi, Y. / Fukada, H. / Itoh, T. / Nago, T. / Nishiya, Y.
History
DepositionDec 28, 2014Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Jan 13, 2016Provider: repository / Type: Initial release
Revision 1.1Feb 5, 2020Group: Data collection / Derived calculations
Category: diffrn_source / pdbx_prerelease_seq / pdbx_struct_oper_list
Item: _diffrn_source.pdbx_synchrotron_site / _pdbx_struct_oper_list.symmetry_operation
Revision 1.2Nov 8, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
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: Urate oxidase
B: Urate oxidase
C: Urate oxidase
D: Urate oxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,88914
Polymers144,9434
Non-polymers94610
Water20,2671125
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area23820 Å2
ΔGint-155 kcal/mol
Surface area41360 Å2
MethodPISA
Unit cell
Length a, b, c (Å)134.135, 145.195, 71.141
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212
Detailsbiological unit is the same as asym.

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Components

#1: Protein
Urate oxidase


Mass: 36235.684 Da / Num. of mol.: 4 / Fragment: UNP residues 178-494 / Mutation: P287G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus TB-90 (bacteria) / Strain: TB-90 / Gene: uao / Production host: Escherichia coli DH5alpha (bacteria) / Strain (production host): DH5alpha
References: UniProt: Q45697, factor-independent urate hydroxylase
#2: Chemical
ChemComp-AZA / 8-AZAXANTHINE


Mass: 153.099 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C4H3N5O2
#3: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-SO4 / SULFATE ION


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


Mass: 18.015 Da / Num. of mol.: 1125 / 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: 2.43 Å3/Da / Density % sol: 49.4 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8 / Details: 15% PEG 8000, 0.1M TRIS-HCl, 0.07M K2SO4,

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL26B2 / Wavelength: 1 Å
DetectorType: RAYONIX MX-225 / Detector: CCD / Date: Nov 27, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.65→30 Å / Num. obs: 157977 / % possible obs: 94.3 % / Redundancy: 6 % / Biso Wilson estimate: 19.03 Å2 / Rmerge(I) obs: 0.074 / Rpim(I) all: 0.031 / Rrim(I) all: 0.08 / Χ2: 1.664 / Net I/av σ(I): 29.423 / Net I/σ(I): 9.9 / Num. measured all: 944905
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.65-1.686.10.81675560.7830.3420.8870.74491.6
1.68-1.716.10.70975930.8210.2980.7710.75891.3
1.71-1.7460.62776450.8540.2630.6820.78491.8
1.74-1.7860.47976260.9050.2010.5210.81292.1
1.78-1.8260.42476800.9170.1780.4610.8492.5
1.82-1.8660.34576440.9420.1440.3750.88292.5
1.86-1.95.90.29977290.9580.1260.3260.9592.7
1.9-1.965.90.22776800.9680.0950.2471.0692.5
1.96-2.015.90.19877020.9780.0830.2151.1292.6
2.01-2.085.80.16977900.9820.0710.1841.28293
2.08-2.155.70.14277650.9870.060.1541.35793.3
2.15-2.245.60.12378100.9890.0520.1341.47193.6
2.24-2.345.60.11577910.990.0490.1251.49693.4
2.34-2.465.60.178250.9910.0430.1091.61393.2
2.46-2.625.50.09278720.9930.0390.11.87294.1
2.62-2.825.50.08981050.9920.0390.0972.83296.6
2.82-3.15.60.08883790.9930.0380.0964.13399
3.1-3.556.40.06684590.9960.0280.0723.82999.9
3.55-4.477.20.04285590.9990.0170.0462.635100
4.47-307.10.03487670.9990.0140.0371.88799.1

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Processing

Software
NameVersionClassification
PHENIX1.9-1692refinement
SCALEPACKdata reduction
PHASER2.5.6phasing
PDB_EXTRACT3.15data extraction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3WLV
Resolution: 1.66→29.818 Å / SU ML: 0.15 / Cross valid method: FREE R-VALUE / σ(F): 1.97 / Phase error: 18.25 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.1841 7662 4.95 %Random selection
Rwork0.1558 147073 --
obs0.1571 154735 94.26 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 80.9 Å2 / Biso mean: 25.001 Å2 / Biso min: 10.48 Å2
Refinement stepCycle: final / Resolution: 1.66→29.818 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9368 0 70 1125 10563
Biso mean--22.56 33.14 -
Num. residues----1175
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0069859
X-RAY DIFFRACTIONf_angle_d1.0313428
X-RAY DIFFRACTIONf_chiral_restr0.0411499
X-RAY DIFFRACTIONf_plane_restr0.0051726
X-RAY DIFFRACTIONf_dihedral_angle_d11.4553543
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.66-1.67890.26032530.22134730498392
1.6789-1.69860.24012560.21274676493291
1.6986-1.71930.25162530.21774731498492
1.7193-1.74110.25862420.20754760500292
1.7411-1.7640.23872540.19034707496192
1.764-1.78820.21152700.17554721499192
1.7882-1.81370.21772740.18094757503192
1.8137-1.84080.2272320.17444757498992
1.8408-1.86950.19812590.17424737499692
1.8695-1.90020.21122740.17214775504993
1.9002-1.93290.2162490.17094766501592
1.9329-1.96810.22262490.16594794504393
1.9681-2.00590.19112650.16334759502493
2.0059-2.04690.21092380.16114849508793
2.0469-2.09130.19622480.15784800504893
2.0913-2.140.19282560.14964859511594
2.14-2.19350.17542700.14974778504893
2.1935-2.25280.15862330.14824875510893
2.2528-2.3190.19672450.15424846509193
2.319-2.39390.1972310.15164842507393
2.3939-2.47940.20572530.15364867512093
2.4794-2.57860.18742480.15874879512794
2.5786-2.69590.21422130.15815025523896
2.6959-2.83790.17442410.16225084532597
2.8379-3.01560.19532590.16495141540099
3.0156-3.24810.18283230.157751925515100
3.2481-3.57450.16772810.151852315512100
3.5745-4.09060.15092670.135653135580100
4.0906-5.14940.12492670.114453595626100
5.1494-29.82280.19082590.17015463572298
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)
11.0567-0.52330.21262.4584-0.34340.81460.01570.0756-0.0557-0.0732-0.00610.07830.1231-0.0753-0.01420.1382-0.0366-0.00820.18670.00740.1444113.716928.104377.3289
24.7426-2.6272.0021.96591.89291.9573-0.06870.006-0.43180.0053-0.1059-0.2690.7350.09260.26180.29270.00410.02830.18970.00010.1924128.110210.554496.0099
30.940.2436-0.28860.70750.02360.4946-0.00060.1153-0.0096-0.0921-0.02480.08730.0723-0.07210.02460.14120.006-0.01220.1176-0.01070.1006131.436725.54774.0627
42.1460.98110.24331.85720.28060.5112-0.0330.1219-0.1095-0.11930.0166-0.03140.1077-0.02140.02060.18080.01250.01130.1473-0.00140.0857139.613421.355571.1385
50.8582-1.2991-0.42183.40270.15340.28260.0222-0.0190.1041-0.0778-0.0363-0.1483-0.00920.02170.01970.12580.00160.00010.14410.00470.1429141.509456.981273.243
61.5350.46850.24253.63961.26641.4871-0.02440.1938-0.0592-0.32540.0025-0.01010.087-0.05820.02390.16290.00750.01580.14520.02270.0681146.945634.932563.7026
73.0071-1.0007-0.54974.05390.49491.9726-0.00210.08110.1031-0.2458-0.0087-0.13720.03540.09770.01460.09520.00720.01020.1150.00810.0759147.774547.058964.9436
83.30343.69763.36439.47362.15091.9889-0.0844-0.21070.5297-0.2037-0.09390.0839-0.4097-0.08940.06590.16650.00610.02620.2208-0.06970.2997144.391665.293690.2722
90.9938-0.1676-0.21940.5266-0.11030.32950.00260.08310.014-0.07-0.0169-0.02440.0546-0.05670.00120.1335-0.0069-0.01220.13180.01290.1152129.381450.057174.3329
100.51890.9039-0.67551.6126-1.40981.89670.011-0.1464-0.0640.1064-0.128-0.0095-0.02310.05520.1480.1284-0.0181-0.02190.1594-0.00460.1501116.790950.430681.1079
116.8580.66043.08637.94534.51097.1660.15230.86940.1581-0.87850.1705-0.6064-0.38760.3998-0.29990.22440.0260.0760.20890.02440.1948135.925362.009662.5142
124.18442.8179-2.96833.0955-1.01042.95920.06880.05280.0717-0.03370.04130.1596-0.1058-0.1981-0.1140.11230.024-0.01840.13330.03060.1532118.697459.913776.053
130.50120.5356-0.5721.5093-1.33582.4417-0.0330.05990.0308-0.06360.07360.03870.128-0.2224-0.04930.1321-0.0047-0.01420.19080.01930.1866119.120349.238879.4839
140.58220.6142-0.10272.9929-0.45580.3403-0.0034-0.05990.00740.26460.04530.07440.0171-0.0419-0.03410.13930.0050.02190.1328-0.00370.0765125.34247.0421109.0394
151.35630.47350.07164.7421-0.74821.2078-0.019-0.03990.0750.17530.01380.0492-0.0174-0.10790.00710.1266-0.00870.02540.1393-0.00070.1078119.743848.3724112.2711
161.1830.2471-0.03611.1449-0.69422.095-0.0136-0.03250.11620.1714-0.01140.0497-0.08760.02180.05660.1227-0.00820.00030.0837-0.02550.1205127.73352.3862105.3662
170.1041-0.50330.08281.10940.03230.24070.0680.04130.0406-0.0268-0.0921-0.26860.04220.11670.03430.130.0054-0.03140.18180.00330.2279155.724550.245595.4752
184.1724-0.5616-2.39051.49470.20822.67730.1447-0.24230.12390.1664-0.0215-0.1094-0.1110.1521-0.14680.1382-0.0206-0.03750.1015-0.02440.1389144.912560.9721106.0438
190.5086-0.5002-0.57761.34860.89552.2624-0.0364-0.08280.03080.10510.0424-0.07990.12090.2467-0.03540.1318-0.0034-0.03730.1794-0.00940.1906150.058749.592898.173
200.73420.1085-0.03231.91270.13530.5334-0.0067-0.0747-0.04870.1723-0.0059-0.13710.10030.05490.00790.14990.0307-0.02990.15650.01080.1548153.774528.7013100.1076
211.63950.9193-0.40753.39840.51451.0948-0.0019-0.06340.04130.2544-0.0093-0.31730.11070.13650.00120.12160.0278-0.05280.16430.00920.1544159.365429.5883101.6168
221.0568-0.2542-0.30170.5179-0.20670.2505-0.0323-0.0651-0.02930.0594-0.022-0.02730.12330.00380.07710.1667-0.0134-0.01530.1170.01390.1091139.382722.733199.5428
233.5487-1.1548-0.0911.7456-0.16980.4233-0.0239-0.161-0.19460.12080.00880.02070.13250.01980.01820.2127-0.01080.0010.1370.01140.079132.330218.3132107.8635
241.4894-1.28920.25272.3416-0.51210.7913-0.1631-0.19060.01880.21430.1087-0.04670.0063-0.07490.04410.166-0.02370.01750.15420.02220.0771127.508626.3912105.355
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 8 through 119 )A0
2X-RAY DIFFRACTION2chain 'A' and (resid 120 through 135 )A0
3X-RAY DIFFRACTION3chain 'A' and (resid 136 through 192 )A0
4X-RAY DIFFRACTION4chain 'A' and (resid 193 through 310 )A0
5X-RAY DIFFRACTION5chain 'B' and (resid 8 through 43 )B0
6X-RAY DIFFRACTION6chain 'B' and (resid 44 through 72 )B0
7X-RAY DIFFRACTION7chain 'B' and (resid 73 through 119 )B0
8X-RAY DIFFRACTION8chain 'B' and (resid 120 through 135 )B0
9X-RAY DIFFRACTION9chain 'B' and (resid 136 through 192 )B0
10X-RAY DIFFRACTION10chain 'B' and (resid 193 through 213 )B0
11X-RAY DIFFRACTION11chain 'B' and (resid 214 through 229 )B0
12X-RAY DIFFRACTION12chain 'B' and (resid 230 through 263 )B0
13X-RAY DIFFRACTION13chain 'B' and (resid 264 through 310 )B0
14X-RAY DIFFRACTION14chain 'C' and (resid 8 through 72 )C0
15X-RAY DIFFRACTION15chain 'C' and (resid 73 through 106 )C0
16X-RAY DIFFRACTION16chain 'C' and (resid 107 through 172 )C0
17X-RAY DIFFRACTION17chain 'C' and (resid 173 through 213 )C0
18X-RAY DIFFRACTION18chain 'C' and (resid 214 through 263 )C0
19X-RAY DIFFRACTION19chain 'C' and (resid 264 through 310 )C0
20X-RAY DIFFRACTION20chain 'D' and (resid 8 through 72 )D0
21X-RAY DIFFRACTION21chain 'D' and (resid 73 through 119 )D0
22X-RAY DIFFRACTION22chain 'D' and (resid 120 through 192 )D0
23X-RAY DIFFRACTION23chain 'D' and (resid 193 through 263 )D0
24X-RAY DIFFRACTION24chain 'D' and (resid 264 through 310 )D0

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