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- PDB-8beo: Crystal structure of E. coli glyoxylate carboligase mutant I393A ... -

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Basic information

Entry
Database: PDB / ID: 8beo
TitleCrystal structure of E. coli glyoxylate carboligase mutant I393A with MAP
ComponentsGlyoxylate carboligase
KeywordsLYASE / Complex
Function / homology
Function and homology information


tartronate-semialdehyde synthase / tartronate-semialdehyde synthase activity / glycolate catabolic process / acetolactate synthase complex / glyoxylate catabolic process / L-valine biosynthetic process / isoleucine biosynthetic process / thiamine pyrophosphate binding / FAD binding / flavin adenine dinucleotide binding ...tartronate-semialdehyde synthase / tartronate-semialdehyde synthase activity / glycolate catabolic process / acetolactate synthase complex / glyoxylate catabolic process / L-valine biosynthetic process / isoleucine biosynthetic process / thiamine pyrophosphate binding / FAD binding / flavin adenine dinucleotide binding / magnesium ion binding / identical protein binding
Similarity search - Function
: / Glyoxylate carboligase / Thiamine pyrophosphate enzyme / Thiamine pyrophosphate enzyme, central domain / Thiamine pyrophosphate enzyme, central domain / Thiamine pyrophosphate enzyme, N-terminal TPP-binding domain / Thiamine pyrophosphate enzyme, N-terminal TPP binding domain / Thiamine pyrophosphate enzyme, C-terminal TPP-binding / Thiamine pyrophosphate enzyme, C-terminal TPP binding domain / Thiamin diphosphate-binding fold / DHS-like NAD/FAD-binding domain superfamily
Similarity search - Domain/homology
METHYL HYDROGEN (S)-ACETYLPHOSPHONATE / 2,3-DIHYDROXY-1,4-DITHIOBUTANE / (2R,3S)-1,4-DIMERCAPTOBUTANE-2,3-DIOL / FLAVIN-ADENINE DINUCLEOTIDE / FORMIC ACID / DI(HYDROXYETHYL)ETHER / TRIETHYLENE GLYCOL / Chem-TDK / 2,3-DIMETHOXY-5-METHYL-1,4-BENZOQUINONE / Glyoxylate carboligase
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.96 Å
AuthorsShaanan, B. / Binshtein, E.
Funding support United States, 1items
OrganizationGrant numberCountry
United States - Israel Binational Science Foundation (BSF)2007129 United States
Citation
Journal: To Be Published
Title: Crystal structure of E. coli glyoxylate carboligase mutant I393A with MAP
Authors: Shaanan, B. / Binshtein, E.
#1: Journal: Biochemistry / Year: 2012
Title: Glyoxylate carboligase: a unique thiamin diphosphate-dependent enzyme that can cycle between the 4'-aminopyrimidinium and 1',4'-iminopyrimidine tautomeric forms in the absence of the conserved glutamate.
Authors: Nemeria, N. / Binshtein, E. / Patel, H. / Balakrishnan, A. / Vered, I. / Shaanan, B. / Barak, Z. / Chipman, D. / Jordan, F.
#2: Journal: Nat Chem Biol / Year: 2008
Title: Glyoxylate carboligase lacks the canonical active site glutamate of thiamine-dependent enzymes.
Authors: Kaplun, A. / Binshtein, E. / Vyazmensky, M. / Steinmetz, A. / Barak, Z. / Chipman, D.M. / Tittmann, K. / Shaanan, B.
History
DepositionOct 21, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 8, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glyoxylate carboligase
B: Glyoxylate carboligase
C: Glyoxylate carboligase
D: Glyoxylate carboligase
E: Glyoxylate carboligase
F: Glyoxylate carboligase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)417,086105
Polymers403,4236
Non-polymers13,66299
Water38,1742119
1
A: Glyoxylate carboligase
B: Glyoxylate carboligase
hetero molecules

A: Glyoxylate carboligase
B: Glyoxylate carboligase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)280,21196
Polymers268,9494
Non-polymers11,26292
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation7_555y,x,-z1
2
C: Glyoxylate carboligase
D: Glyoxylate carboligase
E: Glyoxylate carboligase
F: Glyoxylate carboligase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)276,98057
Polymers268,9494
Non-polymers8,03153
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)188.641, 188.641, 246.957
Angle α, β, γ (deg.)90, 90, 90
Int Tables number92
Space group name H-MP41212
Components on special symmetry positions
IDModelComponents
11A-602-

MG

21B-602-

MG

31B-619-

NA

41A-994-

HOH

Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2given(-0.025314, -0.978153, 0.20634), (-0.978055, -0.018466, -0.207526), (0.206803, -0.207065, -0.95622)103.34402, 102.92411, 0.04425
3given(0.00161, 0.999998, -0.000727), (-0.999996, 0.001608, -0.002152), (-0.00215, 0.000731, 0.999997)94.20164, 94.45059, 12.47845
4given(0.978245, -0.027473, 0.205623), (0.016235, -0.978014, -0.207906), (0.206814, 0.206721, -0.956292)11.03935, 6.47993, -11.77843
5given(-0.999992, 0.001855, -0.003508), (0.001851, 0.999998, 0.001221), (0.00351, 0.001214, -0.999993)94.44855, 94.22103, -12.41459
6given(0.021125, -0.977793, -0.208508), (0.977697, -0.023384, 0.208713), (-0.208954, -0.208266, 0.955491)6.30982, 11.24299, 11.83154
7given(-0.028572, -0.977841, 0.207391), (-0.977861, -0.015684, -0.208666), (0.207295, -0.208762, -0.955744)103.58522, 102.69883, 0.14449
8given(0.000596, 0.999999, -0.001554), (-0.999996, 0.000592, -0.002805), (-0.002804, 0.001556, 0.999995)94.27428, 94.38584, 12.57681
9given(0.978297, -0.031632, 0.20478), (0.012314, -0.977658, -0.209841), (0.206843, 0.207808, -0.95605)10.93526, 6.56463, -11.72166
10given(-0.999989, 0.002504, -0.003882), (0.002502, 0.999997, 0.000524), (0.003883, 0.000514, -0.999992)94.47937, 94.1754, -12.50016
11given(0.022034, -0.977141, -0.211445), (0.977743, -0.023077, 0.208534), (-0.208646, -0.211334, 0.954885)6.32294, 11.21402, 11.71794
12given(0.977592, -0.026478, 0.208836), (0.017598, -0.978307, -0.206413), (0.209771, 0.205462, -0.955919)11.10083, 6.50916, -12.00468
13given(0.002366, 0.999997, 0.000521), (-0.999997, 0.002365, 0.000879), (0.000878, -0.000523, 0.999999)94.21255, 94.52508, 12.2611
14given(0.023965, -0.977874, -0.207817), (0.977357, -0.020798, 0.210574), (-0.210237, -0.208158, 0.955233)6.17364, 11.42861, 11.89743
15given(-0.99999, 0.004322, -0.001402), (0.004319, 0.999988, 0.002098), (0.001411, 0.002092, -0.999997)94.55315, 94.17069, -12.27929
16given(0.977274, -0.029057, 0.20998), (0.015398, -0.978213, -0.207032), (0.211421, 0.205561, -0.955534)10.95591, 6.66165, -12.09073
17given(0.002787, 0.999995, 0.001226), (-0.999993, 0.002784, 0.00253), (0.002526, -0.001234, 0.999996)94.19646, 94.58084, 12.18611
18given(0.026988, -0.97784, -0.207607), (0.977, -0.018144, 0.212464), (-0.211523, -0.208566, 0.95486)5.96989, 11.68304, 11.97239
19given(-0.999977, 0.006753, 3.4E-5), (0.006753, 0.999968, 0.004223), (-5.0E-6, 0.004223, -0.999991)94.63847, 94.12711, -12.14373
20given(-0.015104, 0.977522, 0.210292), (0.978421, -0.028895, 0.204592), (0.206069, 0.208845, -0.955991)87.88876, -83.32104, -24.00731
21given(-0.003469, -0.999994, 0.000928), (-0.999985, 0.003465, -0.00423), (0.004227, -0.000943, -0.999991)0.28377, 0.22861, -24.8926
22given(-0.97721, 0.02227, -0.211104), (0.022534, -0.977979, -0.207483), (-0.211076, -0.207512, 0.955189)83.06787, -88.0258, -0.40441
23given(-0.012315, 0.977069, 0.212566), (0.978628, -0.031863, 0.203155), (0.20527, 0.210525, -0.955795)87.83978, -83.42696, -23.92528
24given(-0.003062, -0.999993, 0.002222), (-0.999981, 0.00305, -0.00544), (0.005434, -0.002238, -0.999983)0.27823, 0.16422, -25.07444
25given(-0.97716, 0.023094, -0.211248), (0.022257, -0.977488, -0.209815), (-0.211338, -0.209725, 0.954647)83.12396, -87.9996, -0.49103
26given(-0.977472, 0.018432, -0.210257), (0.026245, -0.977833, -0.207735), (-0.209425, -0.208574, 0.955321)82.89315, -88.227, -0.47556
27given(-0.006565, -0.999974, -0.002921), (-0.999978, 0.006568, -0.000991), (0.00101, 0.002914, -0.999995)0.32992, 0.34792, -24.54829
28given(-0.977467, 0.014654, -0.210579), (0.030176, -0.977641, -0.208104), (-0.20892, -0.209769, 0.95517)82.63487, -88.46791, -0.53027
29given(-0.009643, -0.99993, -0.006805), (-0.999953, 0.009634, 0.001298), (-0.001232, 0.006817, -0.999976)0.39505, 0.47428, -24.32562
30given(-0.020045, 0.977011, 0.212246), (0.97748, -0.025455, 0.209489), (0.210076, 0.211665, -0.954498)88.0696, -83.11198, -24.038
31given(-0.02046, 0.976289, 0.215503), (0.977688, -0.025536, 0.208505), (0.209064, 0.21496, -0.953983)88.03992, -83.16939, -23.91965

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Components

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Protein , 1 types, 6 molecules ABCDEF

#1: Protein
Glyoxylate carboligase / Tartronate-semialdehyde synthase


Mass: 67237.242 Da / Num. of mol.: 6 / Mutation: I393A
Source method: isolated from a genetically manipulated source
Details: E. coli glyoxylatecarboligase (GCL) I393A in complex with metheyl-acetyl-phosphonate (MAP). Cys344 is converted in the structure to CSO because of radiation damage.
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: gcl, b0507, JW0495 / Plasmid: PQE60 / Production host: Escherichia coli (E. coli) / Strain (production host): XL1
References: UniProt: P0AEP7, tartronate-semialdehyde synthase

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Non-polymers , 13 types, 2218 molecules

#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 13 / Source method: obtained synthetically / Formula: Mg
#3: Chemical
ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM
#4: Chemical
ChemComp-TDK / 3-[(4-AMINO-2-METHYLPYRIMIDIN-5-YL)METHYL]-2-{(1S)-1-HYDROXY-1-[(R)-HYDROXY(METHOXY)PHOSPHORYL]ETHYL}-5-(2-{[(S)-HYDROXY(PHOSPHONOOXY)PHOSPHORYL]OXY}ETHYL)-4-METHYL-1,3-THIAZOL-3-IUM / 2-PHOSPHONOLACTYLTHIAMIN DIPHOSPHATE


Mass: 563.373 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C15H26N4O11P3S / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-ALU / METHYL HYDROGEN (S)-ACETYLPHOSPHONATE


Mass: 138.059 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H7O4P
#6: Chemical
ChemComp-UQ0 / 2,3-DIMETHOXY-5-METHYL-1,4-BENZOQUINONE


Mass: 182.173 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C9H10O4
#7: Chemical
ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C4H10O3
#8: Chemical ChemComp-DTT / 2,3-DIHYDROXY-1,4-DITHIOBUTANE / 1,4-DITHIOTHREITOL


Mass: 154.251 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H10O2S2
#9: Chemical...
ChemComp-FMT / FORMIC ACID


Mass: 46.025 Da / Num. of mol.: 35 / Source method: obtained synthetically / Formula: CH2O2
#10: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O4
#11: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: Na
#12: Chemical ChemComp-DTU / (2R,3S)-1,4-DIMERCAPTOBUTANE-2,3-DIOL


Mass: 154.251 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O2S2
#13: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: Cl
#14: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2119 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 2.8 Å3/Da / Density % sol: 56 % / Description: Octahedral
Crystal growTemperature: 296 K / Method: evaporation / pH: 7
Details: 2-4 ul protein (5-15 mg/ml), 100uM ThDP, 10 uM FAD, 1mM MgCl2, 10mM MAP and 10 mM Q0 were mixed with equal volume of resrvoir solution (0.5% PEG 6000, 0.5 M NaCl and 40 mM DTT). Crystals ...Details: 2-4 ul protein (5-15 mg/ml), 100uM ThDP, 10 uM FAD, 1mM MgCl2, 10mM MAP and 10 mM Q0 were mixed with equal volume of resrvoir solution (0.5% PEG 6000, 0.5 M NaCl and 40 mM DTT). Crystals grew to size of roughly 0.15-0.25 mm in all dimensions.
PH range: 6.8-7.2 / Temp details: Room temperature

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID14-1 / Wavelength: 0.9334 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Jul 14, 2009 / Details: monochromator
RadiationMonochromator: as on beamline ESRF_ID14_1 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9334 Å / Relative weight: 1
ReflectionResolution: 1.96→48.35 Å / Num. obs: 277858 / % possible obs: 88.3 % / Redundancy: 7.19 % / Rmerge(I) obs: 0.133 / Net I/σ(I): 9.83
Reflection shellResolution: 1.96→2.06 Å / Redundancy: 5.7 % / Rmerge(I) obs: 0.957 / Mean I/σ(I) obs: 1.73 / Num. unique obs: 13894 / % possible all: 33.5

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Processing

Software
NameVersionClassification
REFMAC5.8.0352refinement
XDS20210120data reduction
XDS20210120data scaling
STARANISO2.3.64data scaling
Aimless0.7.4data scaling
PHASER2.8.3phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2PAN

2pan


Resolution: 1.96→48.35 Å / Cor.coef. Fo:Fc: 0.98 / Cor.coef. Fo:Fc free: 0.972 / Cross valid method: THROUGHOUT
RfactorNum. reflection% reflectionSelection details
Rfree0.204 8395 -RANDOM
Rwork0.176 ---
obs0.177 269463 88.3 %-
Displacement parametersBiso mean: 33.21 Å2
Baniso -1Baniso -2Baniso -3
1--0.14 Å20 Å20 Å2
2---0.14 Å20 Å2
3---0.29 Å2
Refinement stepCycle: LAST / Resolution: 1.96→48.35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms27174 0 864 2119 30157
LS refinement shellResolution: 1.96→2.01 Å /
Rfactor% reflection
Rfree0.372 -
Rwork0.352 -
obs-25.34 %
Refinement TLS params.

Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.62380.1088-0.00520.4328-0.06280.58480.01310.04340.02710.0434-0.0260.06170.02710.06170.01280.04330.01080.00250.03910.02380.058245.91525.111.766
20.58440.085-0.16990.65270.05720.5574-0.0178-0.05730.0331-0.05730.01350.05750.03310.05750.00430.03230.00970.0180.0572-0.00850.057380.00755.165-6.91
30.449-0.08840.05210.6560.00270.5469-0.0060.0605-0.06080.0605-0.01470.0341-0.06080.03410.02070.1389-0.0227-0.01640.1250.00350.125769.302-48.36-0.515
40.643-0.1119-0.03640.5975-0.14540.53850.0222-0.0179-0.0222-0.0179-0.04360.0483-0.02220.04830.02130.162-0.01180.01610.10540.01230.119139.354-14.263-19.198
50.6024-0.1427-0.00480.4850.07380.59310.0217-0.06650.0244-0.0665-0.0461-0.05720.0244-0.05720.02440.1722-0.0064-0.00250.1167-0.0470.153448.487-69.199-24.118
60.5912-0.0912-0.16370.681-0.04960.5753-0.00720.04820.04590.0482-0.0024-0.06470.0459-0.06470.00950.1358-0.0280.02880.1481-0.00570.14514.374-39.06-5.437
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A2 - 962
2X-RAY DIFFRACTION2B2 - 962
3X-RAY DIFFRACTION3C2 - 962
4X-RAY DIFFRACTION4D2 - 962
5X-RAY DIFFRACTION5E2 - 962
6X-RAY DIFFRACTION6F2 - 963

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