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- PDB-6hg8: Crystal structure of the R460G disease-causing mutant of the huma... -

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Basic information

Entry
Database: PDB / ID: 6hg8
TitleCrystal structure of the R460G disease-causing mutant of the human dihydrolipoamide dehydrogenase.
ComponentsDihydrolipoyl dehydrogenase, mitochondrial
KeywordsOXIDOREDUCTASE / lipoamide dehydrogenase (E3) component / disease-causing mutant / E3 deficiency
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 / oxoglutarate dehydrogenase complex / pyruvate decarboxylation to acetyl-CoA / pyruvate dehydrogenase complex / branched-chain amino acid catabolic process / Branched-chain amino acid catabolism / 2-oxoglutarate metabolic process / pyruvate metabolic process / Signaling by Retinoic Acid / motile cilium / 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 / 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 ...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 / Resolution: 1.76 Å
AuthorsAmbrus, A. / Szabo, E. / Weichsel, A. / Bui, D. / Wilk, P. / Torocsik, B. / Weiss, M.S. / Montfort, W.R. / Jordan, F. / Adam-Vizi, V.
Funding support Hungary, Germany, 8items
OrganizationGrant numberCountry
Hungarian Academy of Sciences (MTA), grant#: 02001 [to V.A.-V.] Hungary
Hungarian Scientific Research Fund (OTKA), grant#: 112230 [to V.A.-V.] Hungary
Hungarian Brain Research Program (NAP), grant#: 2017-1.2.1-NKP2017-00002 [to V.A.-V.] Hungary
European Molecular Biology OrganizationShort-term Fellowship [to A.A.] Germany
Hungarian Academy of Sciences, Bolyai Fellowship [to A.A] Hungary
Semmelweis University, Young Investigator Research Grant [to A.A.] Hungary
Gedeon Richter Pharmaceuticals PIc., Young Investigator Research Grant [to A.A.] Hungary
European Union, Horizon 2020 Research and Innovation Programme grant [to A.A.] Germany
CitationJournal: To Be Published
Title: Crystal structure of the R460G disease-causing mutant of the human dihydrolipoamide dehydrogenase.
Authors: Ambrus, A. / Szabo, E. / Weichsel, A. / Bui, D. / Torocsik, B. / Montfort, W.R. / Jordan, F. / Adam-Vizi, V.
History
DepositionAug 22, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 4, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 17, 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 1.2Nov 20, 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)107,82416
Polymers105,0742
Non-polymers2,75014
Water16,286904
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: equilibrium centrifugation, gel filtration, mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12810 Å2
ΔGint-198 kcal/mol
Surface area35550 Å2
MethodPISA
Unit cell
Length a, b, c (Å)117.913, 169.497, 61.401
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212

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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: Crystallized in 2 M (NH4)2SO4, 2 v/v% PEG-400, 0.1 M HEPES (pH 6.9).
Source: (gene. exp.) Homo sapiens (human) / Gene: DLD, GCSL, LAD, PHE3 / Plasmid: pET52b+
Details (production host): N-terminal Strep-tag (for affinity purification)
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.: 11 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 904 / 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: 2.92 Å3/Da / Density % sol: 57.87 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 6.9
Details: protein in: 100 mM Tris,150 mM NaCl, 1 mM EDTA, pH 8.0 reservoir solution: 2 M (NH4)2SO4, 2 v/v% PEG-400, 0.1 M HEPES (pH 6.9) volume ratio: 1
Temp details: 20 oC incubator

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.91841 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Aug 26, 2017
RadiationMonochromator: Si-111 crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.91841 Å / Relative weight: 1
ReflectionResolution: 1.76→48.4 Å / Num. obs: 114691 / % possible obs: 93 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 6.8 % / CC1/2: 0.998 / Rmerge(I) obs: 0.141 / Net I/σ(I): 9.5
Reflection shellResolution: 1.76→1.86 Å / Redundancy: 6.7 % / Rmerge(I) obs: 0.255 / Mean I/σ(I) obs: 0.59 / Num. unique obs: 14032 / CC1/2: 0.365 / % possible all: 71.2

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Processing

Software
NameVersionClassification
REFMAC5.8.0158refinement
MxCuBEdata collection
XDSdata scaling
XDSdata reduction
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1ZMD

1zmd


Resolution: 1.76→30 Å / Cor.coef. Fo:Fc: 0.97 / Cor.coef. Fo:Fc free: 0.952 / SU B: 4.215 / SU ML: 0.118 / Cross valid method: THROUGHOUT / ESU R: 0.111 / ESU R Free: 0.118 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2374 2083 1.8 %RANDOM
Rwork0.18737 ---
obs0.1883 111625 92.19 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 33.417 Å2
Baniso -1Baniso -2Baniso -3
1-0.87 Å2-0 Å2-0 Å2
2---2.95 Å20 Å2
3---2.08 Å2
Refinement stepCycle: 1 / Resolution: 1.76→30 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7072 0 169 904 8145
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0190.0197515
X-RAY DIFFRACTIONr_bond_other_d0.0060.027082
X-RAY DIFFRACTIONr_angle_refined_deg1.8931.98810195
X-RAY DIFFRACTIONr_angle_other_deg1.036316492
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.6365986
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.67825.429280
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.39151297
X-RAY DIFFRACTIONr_dihedral_angle_4_deg11.8471525
X-RAY DIFFRACTIONr_chiral_restr0.1140.21169
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.028361
X-RAY DIFFRACTIONr_gen_planes_other0.0030.021370
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.6323.0473900
X-RAY DIFFRACTIONr_mcbond_other2.633.0463898
X-RAY DIFFRACTIONr_mcangle_it3.4574.5544894
X-RAY DIFFRACTIONr_mcangle_other3.4574.5544895
X-RAY DIFFRACTIONr_scbond_it3.9273.5223615
X-RAY DIFFRACTIONr_scbond_other3.9263.5223615
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other5.7795.0815300
X-RAY DIFFRACTIONr_long_range_B_refined7.40737.18503
X-RAY DIFFRACTIONr_long_range_B_other7.40637.1078504
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.756→1.802 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.464 99 -
Rwork0.441 5335 -
obs--60.51 %

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