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- PDB-6i4r: Crystal structure of the disease-causing R460G mutant of the huma... -

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Basic information

Entry
Database: PDB / ID: 6i4r
TitleCrystal structure of the disease-causing R460G mutant of the human dihydrolipoamide dehydrogenase at 1.44 Angstrom resolution
ComponentsDihydrolipoyl dehydrogenase, mitochondrial
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 / OGDH complex synthesizes succinyl-CoA from 2-OG / OADH complex synthesizes glutaryl-CoA from 2-OA / oxoadipate dehydrogenase complex / Glycine degradation / branched-chain alpha-ketoacid dehydrogenase complex / BCKDH synthesizes BCAA-CoA from KIC, KMVA, KIV / Loss-of-function mutations in DBT cause MSUD2 / Loss-of-function mutations in DLD cause MSUD3/DLDD ...acetyltransferase complex / acrosomal matrix / OGDH complex synthesizes succinyl-CoA from 2-OG / OADH complex synthesizes glutaryl-CoA from 2-OA / oxoadipate dehydrogenase complex / Glycine degradation / branched-chain alpha-ketoacid dehydrogenase complex / BCKDH synthesizes BCAA-CoA from KIC, KMVA, KIV / Loss-of-function mutations in DBT cause MSUD2 / Loss-of-function mutations in DLD cause MSUD3/DLDD / H139Hfs13* PPM1K causes a mild variant of MSUD / PDH complex synthesizes acetyl-CoA from PYR / Branched-chain ketoacid dehydrogenase kinase deficiency / dihydrolipoyl dehydrogenase / dihydrolipoyl dehydrogenase (NADH) activity / Regulation of pyruvate dehydrogenase (PDH) complex / pyruvate decarboxylation to acetyl-CoA / branched-chain amino acid catabolic process / pyruvate dehydrogenase complex / oxoglutarate dehydrogenase complex / Branched-chain amino acid catabolism / 2-oxoglutarate metabolic process / motile cilium / Signaling by Retinoic Acid / pyruvate metabolic process / sperm capacitation / mitochondrial electron transport, NADH to ubiquinone / gastrulation / Mitochondrial protein degradation / regulation of membrane potential / flavin adenine dinucleotide binding / mitochondrial matrix / mitochondrion / proteolysis / nucleus
Similarity search - Function
: / Dihydrolipoamide dehydrogenase / Pyridine nucleotide-disulphide oxidoreductase, class I / Pyridine nucleotide-disulphide oxidoreductase, class I, active site / Pyridine nucleotide-disulphide oxidoreductases class-I active site. / FAD/NAD-linked reductase, C-terminal dimerisation domain / Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain / Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain / FAD/NAD-linked reductase, dimerisation domain superfamily / FAD/NAD(P)-binding domain ...: / Dihydrolipoamide dehydrogenase / Pyridine nucleotide-disulphide oxidoreductase, class I / Pyridine nucleotide-disulphide oxidoreductase, class I, active site / Pyridine nucleotide-disulphide oxidoreductases class-I active site. / FAD/NAD-linked reductase, C-terminal dimerisation domain / 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: 1.439 Å
AuthorsSzabo, E. / Wilk, P. / Bui, D. / 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
Revision 1.3Oct 23, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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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)107,92815
Polymers105,0742
Non-polymers2,85413
Water16,394910
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, mass spectrometry, equilibrium centrifugation
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12970 Å2
ΔGint-157 kcal/mol
Surface area35690 Å2
MethodPISA
Unit cell
Length a, b, c (Å)118.380, 169.641, 62.009
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 3 through 5 or resid 7...
21(chain B and (resid 3 through 5 or resid 7...

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111(chain A and (resid 3 through 5 or resid 7...A3 - 5
121(chain A and (resid 3 through 5 or resid 7...A7 - 44
131(chain A and (resid 3 through 5 or resid 7...A47 - 49
141(chain A and (resid 3 through 5 or resid 7...A59 - 64
151(chain A and (resid 3 through 5 or resid 7...A66 - 104
161(chain A and (resid 3 through 5 or resid 7...A106 - 190
171(chain A and (resid 3 through 5 or resid 7...A192 - 219
181(chain A and (resid 3 through 5 or resid 7...A221 - 227
191(chain A and (resid 3 through 5 or resid 7...A229 - 276
1101(chain A and (resid 3 through 5 or resid 7...A278 - 3347
1111(chain A and (resid 3 through 5 or resid 7...A349 - 396
1121(chain A and (resid 3 through 5 or resid 7...A398396
1131(chain A and (resid 3 through 5 or resid 7...A398 - 402
1141(chain A and (resid 3 through 5 or resid 7...A404 - 436
1151(chain A and (resid 3 through 5 or resid 7...A438 - 444
1161(chain A and (resid 3 through 5 or resid 7...A446 - 448
1171(chain A and (resid 3 through 5 or resid 7...A450 - 470
1181(chain A and (resid 3 through 5 or resid 7...A472 - 474
211(chain B and (resid 3 through 5 or resid 7...B3 - 5
221(chain B and (resid 3 through 5 or resid 7...B7 - 44
231(chain B and (resid 3 through 5 or resid 7...B47 - 49
241(chain B and (resid 3 through 5 or resid 7...B51 - 57
251(chain B and (resid 3 through 5 or resid 7...B4
261(chain B and (resid 3 through 5 or resid 7...B66 - 104
271(chain B and (resid 3 through 5 or resid 7...B-6 - 474
281(chain B and (resid 3 through 5 or resid 7...B221 - 227
291(chain B and (resid 3 through 5 or resid 7...B334 - 342
2101(chain B and (resid 3 through 5 or resid 7...B334 - 347
2111(chain B and (resid 3 through 5 or resid 7...B349 - 396
2121(chain B and (resid 3 through 5 or resid 7...B398 - 402
2131(chain B and (resid 3 through 5 or resid 7...B438 - 446
2141(chain B and (resid 3 through 5 or resid 7...B446 - 444
2151(chain B and (resid 3 through 5 or resid 7...B450 - 478
2161(chain B and (resid 3 through 5 or resid 7...B450 - 470
2171(chain B and (resid 3 through 5 or resid 7...B472 - 474

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Components

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


Mass: 52537.027 Da / Num. of mol.: 2 / Mutation: R460G
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


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


Mass: 96.063 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-BTB / 2-[BIS-(2-HYDROXY-ETHYL)-AMINO]-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / BIS-TRIS BUFFER


Mass: 209.240 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H19NO5 / Comment: pH buffer*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 910 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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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: 3.06 Å3/Da / Density % sol: 59.83 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 2 M ammonium sulfate, 2 (v/v)% PEG 400, 0.1 M Bis-Tris (pH 6.5)

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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: Aug 26, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9184 Å / Relative weight: 1
ReflectionResolution: 1.43→46.106 Å / Num. obs: 225933 / % possible obs: 99 % / Redundancy: 6.584 % / Biso Wilson estimate: 21.76 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.076 / Rrim(I) all: 0.082 / Χ2: 1.163 / Net I/σ(I): 12.12
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
1.43-1.526.4812.3520.62349830.5972.55395.5
1.52-1.636.5631.1041.4343610.8541.198100
1.63-1.766.7980.5462.95320450.9490.591100
1.76-1.926.490.2965.46293930.9810.32199.4
1.92-2.156.8320.14611.48266850.9940.15899.3
2.15-2.486.4010.08918.57235170.9970.09798.9
2.48-3.046.8710.05529.34201580.9990.06100
3.04-4.296.2660.03941.24157490.9990.04299.8
4.29-46.1066.2660.03347.6190420.9990.03699.7

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Phasing

PhasingMethod: molecular replacement
Phasing MRR rigid body: 0.609
Highest resolutionLowest resolution
Rotation48.54 Å1.89 Å

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Processing

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

1zmd


Resolution: 1.439→46.106 Å / SU ML: 0.21 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 24.08
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
Rfree0.1888 2348 1.05 %
Rwork0.1714 --
obs0.1716 223502 98.83 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 131.92 Å2 / Biso mean: 33.2347 Å2 / Biso min: 15.34 Å2
Refinement stepCycle: final / Resolution: 1.439→46.106 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7063 0 279 913 8255
Biso mean--32.11 37.53 -
Num. residues----953
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A4018X-RAY DIFFRACTION5.348TORSIONAL
12B4018X-RAY DIFFRACTION5.348TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 17

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.4385-1.46790.51581260.4932115681169489
1.4679-1.49980.39051350.3989129371307299
1.4998-1.53470.33191380.3431129281306699
1.5347-1.57310.30971380.3067129751311399
1.5731-1.61560.27961370.25641299513132100
1.6156-1.66320.24911380.23381304413182100
1.6632-1.71690.25421380.21481307113209100
1.7169-1.77820.22131390.19771304113180100
1.7782-1.84940.23921380.18281302613164100
1.8494-1.93360.22881370.2101129501308799
1.9336-2.03550.18821380.1731129411307999
2.0355-2.16310.17371390.15371314613285100
2.1631-2.33010.18481370.1662129311306898
2.3301-2.56460.16631410.1451322513366100
2.5646-2.93560.17071410.15131324513386100
2.9356-3.69830.151410.15531336513506100
3.6983-46.12950.1671470.14061376613913100
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)
12.19451.05541.41731.84050.77511.50750.0797-0.09460.07210.1458-0.01780.16320.0119-0.1407-0.02860.19150.0040.05690.19570.01520.178534.44349.271624.0027
20.6373-0.07650.16171.753-0.26210.08110.0475-0.06830.0729-0.07130.00260.5450.0314-0.1963-0.00560.2345-0.01970.06690.30770.04970.353619.3515-0.827121.0414
31.02480.40140.78031.56330.38261.3394-0.0545-0.04170.2482-0.01650.00510.1848-0.1144-0.07810.06290.17810.02980.05080.17880.01580.231333.937119.384817.0107
41.5837-1.03530.43042.6632-0.77410.8760.14550.14370.1343-0.5553-0.02290.16450.1817-0.0032-0.11370.32270.0003-0.0450.21560.0290.198128.13172.9898-2.555
51.9930.27860.81671.08750.18491.214-0.01940.160.1049-0.19590.0520.0469-0.0870.04380.00940.22680.0030.04280.16320.0530.18840.29517.28135.9694
60.47830.41430.25691.91120.24660.38810.01190.0481-0.0703-0.15330.0441-0.15390.01770.0256-0.07610.1986-0.00060.03440.2023-0.00070.209145.7665-8.742512.1855
70.9999-0.7308-0.40281.89640.20330.2890.03950.1099-0.075-0.19180.05180.23930.0474-0.0875-0.07120.2644-0.0211-0.05330.26370.01440.29128.4112-32.003612.3825
81.30680.1053-0.58271.89180.53461.6106-0.08640.0621-0.318-0.01710.0738-0.09970.2410.04860.00920.33660.0005-0.03630.2276-0.02470.338440.0278-51.651712.5723
91.20690.5088-0.03841.3149-0.32020.97190.1201-0.156-0.05980.2060.03410.35990.0287-0.1368-0.10730.2492-0.02760.06080.23820.06390.329619.7686-30.466335.5453
102.401-0.1049-1.00081.27060.25521.485-0.0374-0.2469-0.16390.15590.11350.00550.25690.1598-0.05990.25980.0205-0.05870.19370.02530.223138.9746-41.55827.7187
110.2608-0.3566-0.84332.35961.39412.73760.0402-0.0073-0.17940.13250.2187-0.23540.14150.409-0.21540.1846-0.0041-0.01260.2027-0.02730.226142.0272-24.860526.9202
121.27340.0293-0.1793.0516-0.12730.90940.0333-0.1333-0.01960.306-0.0161-0.1155-0.04310.0067-0.01810.2123-0.01530.0040.17650.00810.1742.1558-9.937231.186
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 307 )A261 - 307
6X-RAY DIFFRACTION6chain 'A' and (resid 308 through 474 )A308 - 474
7X-RAY DIFFRACTION7chain 'B' and (resid -6 through 110 )B-6 - 110
8X-RAY DIFFRACTION8chain 'B' and (resid 111 through 152 )B111 - 152
9X-RAY DIFFRACTION9chain 'B' and (resid 153 through 260 )B153 - 260
10X-RAY DIFFRACTION10chain 'B' and (resid 261 through 342 )B261 - 342
11X-RAY DIFFRACTION11chain 'B' and (resid 343 through 370 )B343 - 370
12X-RAY DIFFRACTION12chain 'B' and (resid 371 through 474 )B371 - 474

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