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- PDB-6i4s: Crystal structure of the disease-causing R447G mutant of the huma... -

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Basic information

Entry
Database: PDB / ID: 6i4s
TitleCrystal structure of the disease-causing R447G mutant of the human dihydrolipoamide dehydrogenase
ComponentsDihydrolipoyl dehydrogenase, mitochondrialDihydrolipoamide dehydrogenase
KeywordsOXIDOREDUCTASE / Lipoamide dehydrogenase / Pathogenic mutation / E3 deficiency / Alpha-ketoglutarate dehydrogenase complex / 2-oxoglutarate dehydrogenase complex / Pyruvate dehydrogenase complex
Function / homology
Function and homology information


acetyltransferase complex / acrosomal matrix / Glycine degradation / : / dihydrolipoyl dehydrogenase / dihydrolipoyl dehydrogenase activity / oxoglutarate dehydrogenase complex / acetyl-CoA biosynthetic process from pyruvate / pyruvate dehydrogenase complex / : ...acetyltransferase complex / acrosomal matrix / Glycine degradation / : / dihydrolipoyl dehydrogenase / dihydrolipoyl dehydrogenase activity / oxoglutarate dehydrogenase complex / acetyl-CoA biosynthetic process from pyruvate / pyruvate dehydrogenase complex / : / Lysine catabolism / branched-chain amino acid catabolic process / Citric acid cycle (TCA cycle) / Branched-chain amino acid catabolism / Pyruvate metabolism / Glyoxylate metabolism and glycine degradation / Regulation of pyruvate dehydrogenase (PDH) complex / motile cilium / sperm capacitation / Signaling by Retinoic Acid / mitochondrial electron transport, NADH to ubiquinone / Mitochondrial protein degradation / gastrulation / regulation of membrane potential / flavin adenine dinucleotide binding / mitochondrial matrix / mitochondrion / proteolysis / nucleus
Similarity search - Function
Dihydrolipoamide dehydrogenase / Pyridine nucleotide-disulphide oxidoreductase, class I / FAD/NAD-linked reductase, C-terminal dimerisation domain / Pyridine nucleotide-disulphide oxidoreductase, class I, active site / Pyridine nucleotide-disulphide oxidoreductases class-I active site. / Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain / Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain / FAD/NAD-linked reductase, dimerisation domain superfamily / FAD/NAD(P)-binding domain / Pyridine nucleotide-disulphide oxidoreductase ...Dihydrolipoamide dehydrogenase / Pyridine nucleotide-disulphide oxidoreductase, class I / FAD/NAD-linked reductase, C-terminal dimerisation domain / Pyridine nucleotide-disulphide oxidoreductase, class I, active site / Pyridine nucleotide-disulphide oxidoreductases class-I active site. / Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain / Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain / FAD/NAD-linked reductase, dimerisation domain superfamily / FAD/NAD(P)-binding domain / Pyridine nucleotide-disulphide oxidoreductase / Enolase-like; domain 1 / FAD/NAD(P)-binding domain / FAD/NAD(P)-binding domain / 3-Layer(bba) Sandwich / FAD/NAD(P)-binding domain superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / Dihydrolipoyl dehydrogenase, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.75 Å
AuthorsSzabo, E. / Wilk, P. / Hubert, A. / Torocsik, B. / Weiss, M.S. / Adam-Vizi, V. / Ambrus, A.
Funding support Hungary, United States, Germany, 10items
OrganizationGrant numberCountry
Hungarian Academy of Sciences02001 [to A.-V.V.] Hungary
Hungarian Scientific Research Fund, grant#112230 [to A.-V.V.] Hungary
Hungarian Brain Research Program, grant#KTIA_13_NAP_III/6 and 2017-1.2.1-NKP-2017-00002 [to A.-V.V.] Hungary
Hungarian Academy of SciencesBolyai Fellowship [to A.A.] Hungary
European Molecular Biology OrganizationShort-term Fellowship [to A.A.] Hungary
Semmelweis University, Young Investigator Research Grant [to A.A.] Hungary
Gedeon Richter PIc., Young Investigator Research Grant [to A.A.] Hungary
Fulbright Commission, Fulbright Fellowship [to A.A.] United States
European Union and Government of Hungary, grant#EFOP-3.6.3-VEKOP-16-2017-00009 [to S.E.] Hungary
European Union CALIPSOplus, grant#16204087-ST [to S.E. and A.A.] Germany
CitationJournal: Hum.Mol.Genet. / Year: 2019
Title: Underlying molecular alterations in human dihydrolipoamide dehydrogenase deficiency revealed by structural analyses of disease-causing enzyme variants.
Authors: Szabo, E. / Wilk, P. / Nagy, B. / Zambo, Z. / Bui, D. / Weichsel, A. / Arjunan, P. / Torocsik, B. / Hubert, A. / Furey, W. / Montfort, W.R. / Jordan, F. / Weiss, M.S. / Adam-Vizi, V. / Ambrus, A.
History
DepositionNov 10, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 20, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2019Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jan 24, 2024Group: 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: Dihydrolipoyl dehydrogenase, mitochondrial
B: Dihydrolipoyl dehydrogenase, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)107,60614
Polymers105,0742
Non-polymers2,53212
Water13,259736
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry, equilibrium centrifugation
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12210 Å2
ΔGint-178 kcal/mol
Surface area35410 Å2
MethodPISA
Unit cell
Length a, b, c (Å)117.273, 169.043, 60.845
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212
Components on special symmetry positions
IDModelComponents
11A-602-

HOH

21B-946-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 3 through 44 or resid 46...
21(chain B and (resid 3 through 44 or resid 46...

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111(chain A and (resid 3 through 44 or resid 46...A3 - 44
121(chain A and (resid 3 through 44 or resid 46...A46 - 49
131(chain A and (resid 3 through 44 or resid 46...A51 - 57
141(chain A and (resid 3 through 44 or resid 46...A59 - 64
151(chain A and (resid 3 through 44 or resid 46...A66 - 89
161(chain A and (resid 3 through 44 or resid 46...A91 - 104
171(chain A and (resid 3 through 44 or resid 46...A106 - 290
181(chain A and (resid 3 through 44 or resid 46...A292 - 331
191(chain A and (resid 3 through 44 or resid 46...A333 - 347
1101(chain A and (resid 3 through 44 or resid 46...A349 - 396
1111(chain A and (resid 3 through 44 or resid 46...A398 - 4159
1121(chain A and (resid 3 through 44 or resid 46...A461 - 470
1131(chain A and (resid 3 through 44 or resid 46...A472470
1141(chain A and (resid 3 through 44 or resid 46...A472 - 474
211(chain B and (resid 3 through 44 or resid 46...B3 - 44
221(chain B and (resid 3 through 44 or resid 46...B46 - 49
231(chain B and (resid 3 through 44 or resid 46...B51 - 57
241(chain B and (resid 3 through 44 or resid 46...B59 - 64
251(chain B and (resid 3 through 44 or resid 46...B-6 - 474
261(chain B and (resid 3 through 44 or resid 46...B91 - 104
271(chain B and (resid 3 through 44 or resid 46...B-6 - 474
281(chain B and (resid 3 through 44 or resid 46...B333 - 347
291(chain B and (resid 3 through 44 or resid 46...B415 - 453
2101(chain B and (resid 3 through 44 or resid 46...B415 - 459
2111(chain B and (resid 3 through 44 or resid 46...B461 - 470
2121(chain B and (resid 3 through 44 or resid 46...B472 - 474

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Components

#1: Protein Dihydrolipoyl dehydrogenase, mitochondrial / Dihydrolipoamide dehydrogenase / Dihydrolipoamide dehydrogenase / Glycine cleavage system L protein


Mass: 52537.027 Da / Num. of mol.: 2 / Mutation: R447G
Source method: isolated from a genetically manipulated source
Details: sequence of the Strep-tag with linker amino acids: MASWSHPQFEKGALEVLFQGPG
Source: (gene. exp.) Homo sapiens (human) / Gene: DLD, GCSL, LAD, PHE3 / Plasmid: pET52b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P09622, dihydrolipoyl dehydrogenase
#2: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE / Flavin adenine dinucleotide


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM
#3: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 736 / 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: 3.05 Å3/Da / Density % sol: 59.65 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 2 M ammonium sulfate, 2 (v/v)% PEG 400, 0.1 M Hepes (pH 7.5)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.9184 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Feb 29, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9184 Å / Relative weight: 1
ReflectionResolution: 1.75→45.511 Å / Num. obs: 121942 / % possible obs: 99.1 % / Redundancy: 6.566 % / Biso Wilson estimate: 21.91 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.102 / Rrim(I) all: 0.111 / Χ2: 0.991 / Net I/σ(I): 14.49
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
1.75-1.856.7231.1391.49194520.691.23498.9
1.85-1.986.4560.7572.36182330.8440.82498.6
1.98-2.146.5590.3884.39172520.9420.42299.9
2.14-2.346.6330.2517.44154180.9740.27396.7
2.34-2.626.5030.15111.04144240.9910.16499.9
2.62-3.026.8590.08619.02128300.9970.093100
3.02-3.76.4470.04634.5108540.9990.0599.7
3.7-5.226.5180.02952.7785280.9990.03299.8
5.22-45.5115.9370.02555.99495110.02799.2

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Phasing

PhasingMethod: molecular replacement
Phasing MRR rigid body: 0.607
Highest resolutionLowest resolution
Rotation45.98 Å1.96 Å

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Processing

Software
NameVersionClassification
MxCuBEdata collection
XDSdata reduction
XDSdata scaling
MOLREPphasing
PHENIXrefinement
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1ZMD

1zmd


Resolution: 1.75→45.511 Å / SU ML: 0.17 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 20.94
Details: TLS groups, NCS torsion-angle restraints, automatic occupancy refinement and real-space refinement were involved, hydrogen atoms were added to the final model during refinement
RfactorNum. reflection% reflection
Rfree0.1944 2099 1.72 %
Rwork0.1784 --
obs0.1787 121870 99.03 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 148.23 Å2 / Biso mean: 31.2738 Å2 / Biso min: 12.57 Å2
Refinement stepCycle: final / Resolution: 1.75→45.511 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7063 0 218 736 8017
Biso mean--31.08 32.83 -
Num. residues----953
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A4058X-RAY DIFFRACTION5.227TORSIONAL
12B4058X-RAY DIFFRACTION5.227TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
1.75-1.78830.31081360.2812777897
1.7883-1.8330.26151390.2617956100
1.833-1.88260.30411390.25247923100
1.8826-1.9380.43391350.4274770897
1.938-2.00050.28041400.21627988100
2.0005-2.0720.19791390.19577960100
2.072-2.1550.21041400.17957999100
2.155-2.25310.24151370.2294775096
2.2531-2.37180.20871360.1943775797
2.3718-2.52040.17951410.15858058100
2.5204-2.7150.17721410.15778042100
2.715-2.98820.1931420.16488093100
2.9882-3.42050.18371420.16238122100
3.4205-4.30890.14221430.1347816499
4.3089-45.5110.15721490.14988473100
Refinement TLS params.

Method: refined / 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)
12.29631.16211.25341.44050.74941.42790.0656-0.10680.06870.13420.00360.1131-0.0295-0.1243-0.02390.18860.01710.03470.17910.01550.154234.08229.401823.468
20.53350.26130.20891.30830.06350.26890.0302-0.03450.06270.01180.01220.35780.0556-0.11240.01640.2166-0.00260.0420.26560.03830.286418.8752-0.584920.6501
30.94060.45190.42231.29830.30660.9927-0.0569-0.05610.28020.01280.02070.2308-0.1147-0.09020.04570.1720.03260.01860.15720.02840.22833.531119.399116.391
41.09190.0480.2710.895-0.42450.93030.0260.28420.2222-0.29890.05270.23530.1053-0.0795-0.06310.2793-0.015-0.05020.24420.07230.188927.53152.9494-2.8784
51.803-0.01270.68190.47-0.27451.2078-0.05010.2530.0474-0.22850.1380.2337-0.0225-0.08980.01340.2377-0.0024-0.04990.24340.10170.241931.061614.81410.059
60.66350.37410.18211.62770.22680.3664-0.03550.0892-0.0563-0.13760.0797-0.0746-0.00880.0209-0.04990.1528-0.00150.02490.16610.00680.118946.1059-5.213911.932
71.0949-0.3482-0.23981.29810.20280.43340.05190.1386-0.154-0.14350.02340.07330.0223-0.0621-0.05840.1814-0.0182-0.0180.188-0.01960.19733.2836-36.916812.1792
81.1656-0.0099-0.19341.3890.05940.09160.08430.1431-0.092-0.1738-0.00160.3514-0.0346-0.0841-0.04130.2271-0.0095-0.06240.26160.05950.233218.4845-22.929812.7825
91.2866-0.0355-0.34551.00960.29660.8009-0.01240.0133-0.27010.04840.060.07680.1214-0.0015-0.03090.203-0.0238-0.02460.17410.01160.265929.4343-44.708420.1269
101.35160.6991-0.11171.0585-0.22030.32390.1145-0.264-0.04780.2009-0.04740.1979-0.0336-0.0797-0.07280.2397-0.03220.05470.25310.03960.227820.6327-27.448337.5143
111.8165-0.2588-0.41481.00680.35610.86330.0088-0.2207-0.17690.14390.1015-0.05470.14050.1369-0.08060.20230.0103-0.04860.17040.02940.20738.7252-41.436627.4957
120.2954-0.0329-0.60552.4211.71492.3603-0.03720.0293-0.12760.20070.224-0.25620.1650.3125-0.13420.2212-0.0096-0.0140.2147-0.00830.220641.248-24.383726.7838
131.56970.1318-0.42422.3745-0.14981.27690.0407-0.13050.03860.2013-0.0477-0.0527-0.07930.0293-0.0150.2114-0.0179-0.00610.18040.00370.145941.7692-9.73130.6701
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 3 through 67 )A3 - 67
2X-RAY DIFFRACTION2chain 'A' and (resid 68 through 109 )A68 - 109
3X-RAY DIFFRACTION3chain 'A' and (resid 110 through 187 )A110 - 187
4X-RAY DIFFRACTION4chain 'A' and (resid 188 through 260 )A188 - 260
5X-RAY DIFFRACTION5chain 'A' and (resid 261 through 288 )A261 - 288
6X-RAY DIFFRACTION6chain 'A' and (resid 289 through 474 )A289 - 474
7X-RAY DIFFRACTION7chain 'B' and (resid -6 through 67 )B-6 - 67
8X-RAY DIFFRACTION8chain 'B' and (resid 68 through 109 )B68 - 109
9X-RAY DIFFRACTION9chain 'B' and (resid 110 through 187 )B110 - 187
10X-RAY DIFFRACTION10chain 'B' and (resid 188 through 260 )B188 - 260
11X-RAY DIFFRACTION11chain 'B' and (resid 261 through 342 )B261 - 342
12X-RAY DIFFRACTION12chain 'B' and (resid 343 through 370 )B343 - 370
13X-RAY DIFFRACTION13chain 'B' and (resid 371 through 474 )B371 - 474

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