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- PDB-6i4t: Crystal structure of the disease-causing I445M mutant of the huma... -

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Basic information

Entry
Database: PDB / ID: 6i4t
TitleCrystal structure of the disease-causing I445M 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 / 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: 1.823 Å
AuthorsSzabo, E. / Wilk, P. / Zambo, Z. / 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)107,88813
Polymers105,3102
Non-polymers2,57811
Water7,404411
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12750 Å2
ΔGint-160 kcal/mol
Surface area34770 Å2
MethodPISA
Unit cell
Length a, b, c (Å)118.915, 169.004, 60.831
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 44 or resid 47...
21(chain B and (resid 3 through 44 or resid 47...

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111(chain A and (resid 3 through 44 or resid 47...A3 - 44
121(chain A and (resid 3 through 44 or resid 47...A47 - 49
131(chain A and (resid 3 through 44 or resid 47...A3 - 474
141(chain A and (resid 3 through 44 or resid 47...A0
151(chain A and (resid 3 through 44 or resid 47...A91 - 6
161(chain A and (resid 3 through 44 or resid 47...A106 - 325
171(chain A and (resid 3 through 44 or resid 47...A32725
181(chain A and (resid 3 through 44 or resid 47...A327 - 347
191(chain A and (resid 3 through 44 or resid 47...A349 - 396
1101(chain A and (resid 3 through 44 or resid 47...A398 - 436
1111(chain A and (resid 3 through 44 or resid 47...A438 - 474
1121(chain A and (resid 3 through 44 or resid 47...A500
211(chain B and (resid 3 through 44 or resid 47...B3 - 44
221(chain B and (resid 3 through 44 or resid 47...B47 - 49
231(chain B and (resid 3 through 44 or resid 47...B51 - 57
241(chain B and (resid 3 through 44 or resid 47...B-6 - 474
251(chain B and (resid 3 through 44 or resid 47...B91 - 104
261(chain B and (resid 3 through 44 or resid 47...B-6 - 474
271(chain B and (resid 3 through 44 or resid 47...B-6 - 474
281(chain B and (resid 3 through 44 or resid 47...B349 - 396
291(chain B and (resid 3 through 44 or resid 47...B438 - 474
2101(chain B and (resid 3 through 44 or resid 47...B500

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Components

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


Mass: 52655.211 Da / Num. of mol.: 2 / Mutation: I445M
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.: 8 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-1PE / PENTAETHYLENE GLYCOL / PEG400 / Polyethylene glycol


Mass: 238.278 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H22O6 / Comment: precipitant*YM
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 411 / 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 Å3/Da / Density % sol: 58.99 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.5
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: Sep 24, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9184 Å / Relative weight: 1
ReflectionResolution: 1.82→45.596 Å / Num. obs: 108394 / % possible obs: 98.5 % / Redundancy: 6.607 % / Biso Wilson estimate: 28.88 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.094 / Rrim(I) all: 0.102 / Χ2: 0.972 / Net I/σ(I): 11.05
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
1.82-1.936.6391.1821.39166520.841.28194.5
1.93-2.076.4510.7992.01163690.9160.86999.1
2.07-2.236.9050.4223.82152560.9750.45799
2.23-2.456.5320.2666.19140470.9830.28998.7
2.45-2.736.7370.14910.1128580.9940.16299.5
2.73-3.156.6640.08916.42114140.9970.09799.6
3.15-3.866.5960.05326.8997300.9980.05799.8
3.86-5.446.3270.04234.0776400.9980.04699.8
5.44-45.5966.2490.0435.5944280.9990.04399.3

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Phasing

PhasingMethod: molecular replacement
Phasing MRR rigid body: 0.604
Highest resolutionLowest resolution
Rotation48.63 Å2.26 Å

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Processing

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

1zmd


Resolution: 1.823→45.596 Å / SU ML: 0.2 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 26.73
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
Rfree0.2103 2093 1.94 %
Rwork0.1909 --
obs0.1913 108085 98.25 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 142.15 Å2 / Biso mean: 48.0385 Å2 / Biso min: 21.71 Å2
Refinement stepCycle: final / Resolution: 1.823→45.596 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7077 0 246 411 7734
Biso mean--43.44 40.73 -
Num. residues----953
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A4177X-RAY DIFFRACTION8.572TORSIONAL
12B4177X-RAY DIFFRACTION8.572TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 15

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.8235-1.86590.35351210.36216169629087
1.8659-1.91260.39311400.37667050719099
1.9126-1.96430.41681370.39747025716299
1.9643-2.02210.29731380.2837006714499
2.0221-2.08730.28381390.25257040717999
2.0873-2.16190.27471390.227032717199
2.1619-2.24850.25581390.25346999713898
2.2485-2.35080.25681380.22827004714298
2.3508-2.47480.21531400.17967097723799
2.4748-2.62980.19131410.17727123726499
2.6298-2.83280.2191420.187871727314100
2.8328-3.11780.19831420.188571667308100
3.1178-3.56880.21041430.181672397382100
3.5688-4.49570.14531440.138372927436100
4.4957-45.61020.17411500.156175787728100
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)
11.53190.31080.92211.55470.22041.43260.1522-0.21580.34980.427-0.12010.4555-0.0417-0.2150.02230.3430.00550.16850.2365-0.00680.370634.74588.877523.9057
20.5676-0.3525-0.23941.6071-0.52440.42960.1682-0.15960.25070.1649-0.02150.8437-0.0084-0.2586-0.06560.3194-0.01080.18880.32380.0390.583920.1593-0.889221.5607
31.18080.46480.40451.9335-0.11990.7833-0.0772-0.05380.60820.08190.03760.391-0.21-0.11980.04720.31220.04230.12890.26370.02930.538333.77319.210416.8047
42.1731-1.24280.7173.7616-1.01341.4230.06980.39220.276-0.6679-0.00690.4340.0939-0.0307-0.03830.4269-0.0356-0.03740.33920.08770.346527.69533.0329-2.4143
52.95430.19971.73391.482-0.03741.56110.02190.2420.5075-0.1438-0.01050.2778-0.03340.013-0.0090.33750.01250.09220.29190.12840.455840.015617.21335.8999
61.52010.83470.35972.23750.10830.72830.02170.1466-0.1957-0.07910.0938-0.23440.0730.0017-0.11260.27940.00920.08370.2523-0.01820.30545.9766-8.602511.5648
71.36260.26050.45243.59570.74811.824-0.09330.4335-0.4012-0.84870.3554-0.129-0.25010.2855-0.24770.4879-0.08280.00330.3651-0.13640.578242.819-45.12536.3693
83.4845-2.7734-0.88225.42620.48821.53610.0561-0.1386-0.1188-0.02270.23240.42690.0494-0.0231-0.2170.3126-0.05050.0210.30050.03580.534624.2264-28.013117.9451
92.0618-1.5336-0.38164.38880.43240.16990.25680.2209-0.3239-0.4867-0.04120.963-0.0877-0.1984-0.18680.4587-0.0472-0.08480.41890.08250.589720.1402-23.56512.4974
101.80371.0946-0.09043.41150.87692.2323-0.02620.3013-0.7609-0.28240.1692-0.43550.11840.2393-0.15170.4357-0.0023-0.01510.3315-0.12330.687840.6789-51.710611.8243
111.72861.7628-0.55821.8544-0.33781.20840.1065-0.1512-0.20020.5311-0.00430.6877-0.0017-0.1967-0.1020.4223-0.0580.14260.31040.08490.713518.6137-34.474529.021
123.8831.0382-0.3981.1702-0.0061.92450.1992-0.5973-0.23460.7573-0.03970.3125-0.25170.1412-0.15120.5922-0.05690.21360.31640.05350.416126.6742-23.0537.8115
131.73330.5618-1.36782.4550.38151.470.3437-0.4533-0.32530.6618-0.16241.00990.0993-0.3972-0.18620.6568-0.10490.22680.46380.18930.791115.6738-35.771340.9182
142.32890.0365-0.85392.29230.4531.61320.0735-0.1449-0.45340.29950.0932-0.12160.15770.1692-0.10450.4057-0.0206-0.06360.28520.0370.54639.709-42.030727.0362
150.0434-0.4422-0.10744.79281.64982.1840.114-0.1043-0.31620.29050.3357-0.5191-0.04740.2956-0.44870.3465-0.05210.01020.2996-0.04530.411942.768-25.219426.3094
162.0814-0.0322-0.34133.3705-0.37290.98590.1167-0.26750.02240.6586-0.0926-0.0806-0.07170.0242-0.03080.4412-0.03560.04270.2573-0.00490.256142.9023-10.428530.4064
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 110 )A68 - 110
3X-RAY DIFFRACTION3chain 'A' and (resid 111 through 187 )A111 - 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 36 )B-6 - 36
8X-RAY DIFFRACTION8chain 'B' and (resid 37 through 67 )B37 - 67
9X-RAY DIFFRACTION9chain 'B' and (resid 68 through 110 )B68 - 110
10X-RAY DIFFRACTION10chain 'B' and (resid 111 through 152 )B111 - 152
11X-RAY DIFFRACTION11chain 'B' and (resid 153 through 199 )B153 - 199
12X-RAY DIFFRACTION12chain 'B' and (resid 200 through 233 )B200 - 233
13X-RAY DIFFRACTION13chain 'B' and (resid 234 through 260 )B234 - 260
14X-RAY DIFFRACTION14chain 'B' and (resid 261 through 342 )B261 - 342
15X-RAY DIFFRACTION15chain 'B' and (resid 343 through 370 )B343 - 370
16X-RAY DIFFRACTION16chain 'B' and (resid 371 through 474 )B371 - 474

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