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- PDB-5ksw: DHODB-I74D mutant -

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ID or keywords:

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Basic information

Entry
Database: PDB / ID: 5ksw
TitleDHODB-I74D mutant
Components(Dihydroorotate ...) x 2
KeywordsOXIDOREDUCTASE / orotate / pyrimidine bisynthesis FeS cluster / electron transfer / dehydrogenase / reductase / protein engineering
Function / homology
Function and homology information


dihydroorotate dehydrogenase (NAD+) / dihydroorotate dehydrogenase (NAD+) activity / 'de novo' UMP biosynthetic process / 'de novo' pyrimidine nucleobase biosynthetic process / 2 iron, 2 sulfur cluster binding / flavin adenine dinucleotide binding / electron transfer activity / metal ion binding / cytoplasm
Similarity search - Function
Dihydroorotate dehydrogenase B (pyrk subunit); domain 3 / Dihydroorotate dehydrogenase, electron transfer subunit / Dihydroorotate dehydrogenase electron transfer subunit / Dihydroorotate dehydrogenase, class 1B / Dihydroorotate dehydrogenase, electron transfer subunit, Fe-S binding domain superfamily / Cytochrome-c3 hydrogenase, gamma subunit / Dihydroorotate dehydrogenase, electron transfer subunit, iron-sulphur cluster binding domain / Iron-sulfur cluster binding domain of dihydroorotate dehydrogenase B / Dihydroorotate dehydrogenase, class 1 / Dihydroorotate dehydrogenase, class 1/ 2 ...Dihydroorotate dehydrogenase B (pyrk subunit); domain 3 / Dihydroorotate dehydrogenase, electron transfer subunit / Dihydroorotate dehydrogenase electron transfer subunit / Dihydroorotate dehydrogenase, class 1B / Dihydroorotate dehydrogenase, electron transfer subunit, Fe-S binding domain superfamily / Cytochrome-c3 hydrogenase, gamma subunit / Dihydroorotate dehydrogenase, electron transfer subunit, iron-sulphur cluster binding domain / Iron-sulfur cluster binding domain of dihydroorotate dehydrogenase B / Dihydroorotate dehydrogenase, class 1 / Dihydroorotate dehydrogenase, class 1/ 2 / Dihydroorotate dehydrogenase signature 1. / Dihydroorotate dehydrogenase signature 2. / Dihydroorotate dehydrogenase, conserved site / Nucleotide-binding domain of ferredoxin-NADP reductase (FNR) module / Dihydroorotate dehydrogenase domain / Dihydroorotate dehydrogenase / Translation factors / Elongation Factor Tu (Ef-tu); domain 3 / Oxidoreductase FAD/NAD(P)-binding / Oxidoreductase NAD-binding domain / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / Riboflavin synthase-like beta-barrel / Ribbon / Aldolase class I / Aldolase-type TIM barrel / TIM Barrel / Alpha-Beta Barrel / Beta Barrel / Rossmann fold / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / FE2/S2 (INORGANIC) CLUSTER / FLAVIN MONONUCLEOTIDE / : / : / Dihydroorotate dehydrogenase B (NAD(+)), electron transfer subunit / Dihydroorotate dehydrogenase B (NAD(+)), catalytic subunit
Similarity search - Component
Biological speciesLactococcus lactis (lactic acid bacteria)
Lactococcus lactis subsp. lactis (lactic acid bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.47 Å
AuthorsPompeu, Y.A. / Stewart, J.D.
CitationJournal: To Be Published
Title: Isoleucine 74 Plays a Key Role in Controlling Electron Transfer Between Lactococcus lactis Dihydroorotate Dehydrogenase 1B Subunits
Authors: Pompeu, Y.A. / Stewart, J.D.
History
DepositionJul 10, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 3, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 10, 2016Group: Data collection
Revision 1.2Apr 26, 2017Group: Structure summary
Revision 1.3Sep 27, 2017Group: Author supporting evidence / Category: pdbx_audit_support
Revision 1.4Nov 1, 2017Group: Author supporting evidence / Category: pdbx_struct_assembly_auth_evidence
Revision 1.5Oct 4, 2023Group: 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 / struct_ncs_dom_lim / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id / _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dihydroorotate dehydrogenase
B: Dihydroorotate dehydrogenase B (NAD(+)), electron transfer subunit
D: Dihydroorotate dehydrogenase B (NAD(+)), electron transfer subunit
C: Dihydroorotate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,44514
Polymers122,4144
Non-polymers3,03010
Water7,008389
1
A: Dihydroorotate dehydrogenase
B: Dihydroorotate dehydrogenase B (NAD(+)), electron transfer subunit
hetero molecules

A: Dihydroorotate dehydrogenase
B: Dihydroorotate dehydrogenase B (NAD(+)), electron transfer subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,51616
Polymers122,4144
Non-polymers3,10112
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_555x,-y,-z1
Buried area14840 Å2
ΔGint-145 kcal/mol
Surface area40130 Å2
MethodPISA
2
D: Dihydroorotate dehydrogenase B (NAD(+)), electron transfer subunit
C: Dihydroorotate dehydrogenase
hetero molecules

D: Dihydroorotate dehydrogenase B (NAD(+)), electron transfer subunit
C: Dihydroorotate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,37412
Polymers122,4144
Non-polymers2,9608
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation3_654-x+1,y,-z-1/21
Buried area14560 Å2
ΔGint-111 kcal/mol
Surface area38730 Å2
MethodPISA
Unit cell
Length a, b, c (Å)79.990, 151.720, 214.310
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Components on special symmetry positions
IDModelComponents
11B-485-

HOH

21D-473-

HOH

31C-503-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resseq 5:9 or resseq 11:81 or resseq...
21(chain C and (resseq 5:9 or resseq 11:81 or resseq...
12(chain C and (resseq 402))
22(chain A and (resseq 403))
13(chain D and (resseq 2 or (resid 3 and (name...
23(chain B and (resseq 2 or (resid 3 and (name...
14(chain D and (resseq 301))
24(chain B and (resseq 301))
15(chain D and (resseq 302))
25(chain B and (resseq 302))
16(chain A and (resseq 401))
26(chain C and (resseq 401))

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
111ASNASNVALVAL(chain A and (resseq 5:9 or resseq 11:81 or resseq...AA5 - 91 - 5
121LEULEULEULEU(chain A and (resseq 5:9 or resseq 11:81 or resseq...AA11 - 817 - 77
131VALVALPROPRO(chain A and (resseq 5:9 or resseq 11:81 or resseq...AA83 - 9779 - 93
141ASPASPASPASP(chain A and (resseq 5:9 or resseq 11:81 or resseq...AA9894
151ASNASNLYSLYS(chain A and (resseq 5:9 or resseq 11:81 or resseq...AA5 - 3101 - 306
161ASNASNLYSLYS(chain A and (resseq 5:9 or resseq 11:81 or resseq...AA5 - 3101 - 306
171ASNASNLYSLYS(chain A and (resseq 5:9 or resseq 11:81 or resseq...AA5 - 3101 - 306
181ASNASNLYSLYS(chain A and (resseq 5:9 or resseq 11:81 or resseq...AA5 - 3101 - 306
191ASNASNLYSLYS(chain A and (resseq 5:9 or resseq 11:81 or resseq...AA5 - 3101 - 306
211ASNASNVALVAL(chain C and (resseq 5:9 or resseq 11:81 or resseq...CD5 - 91 - 5
221LEULEULEULEU(chain C and (resseq 5:9 or resseq 11:81 or resseq...CD11 - 817 - 77
231VALVALPROPRO(chain C and (resseq 5:9 or resseq 11:81 or resseq...CD83 - 9779 - 93
241ASPASPASPASP(chain C and (resseq 5:9 or resseq 11:81 or resseq...CD9894
251ASNASNSERSER(chain C and (resseq 5:9 or resseq 11:81 or resseq...CD5 - 3091 - 305
261ASNASNSERSER(chain C and (resseq 5:9 or resseq 11:81 or resseq...CD5 - 3091 - 305
271ASNASNSERSER(chain C and (resseq 5:9 or resseq 11:81 or resseq...CD5 - 3091 - 305
281ASNASNSERSER(chain C and (resseq 5:9 or resseq 11:81 or resseq...CD5 - 3091 - 305
291ASNASNSERSER(chain C and (resseq 5:9 or resseq 11:81 or resseq...CD5 - 3091 - 305
112EDOEDOEDOEDO(chain C and (resseq 402))CN402
212EDOEDOEDOEDO(chain A and (resseq 403))AG403
113PROPROPROPRO(chain D and (resseq 2 or (resid 3 and (name...DC22
123LYSLYSLYSLYS(chain D and (resseq 2 or (resid 3 and (name...DC33
133METMETLEULEU(chain D and (resseq 2 or (resid 3 and (name...DC1 - 2621 - 262
143METMETLEULEU(chain D and (resseq 2 or (resid 3 and (name...DC1 - 2621 - 262
153METMETLEULEU(chain D and (resseq 2 or (resid 3 and (name...DC1 - 2621 - 262
163METMETLEULEU(chain D and (resseq 2 or (resid 3 and (name...DC1 - 2621 - 262
173METMETLEULEU(chain D and (resseq 2 or (resid 3 and (name...DC1 - 2621 - 262
183METMETLEULEU(chain D and (resseq 2 or (resid 3 and (name...DC1 - 2621 - 262
213PROPROPROPRO(chain B and (resseq 2 or (resid 3 and (name...BB22
223LYSLYSLYSLYS(chain B and (resseq 2 or (resid 3 and (name...BB33
233METMETLEULEU(chain B and (resseq 2 or (resid 3 and (name...BB1 - 2621 - 262
243METMETLEULEU(chain B and (resseq 2 or (resid 3 and (name...BB1 - 2621 - 262
253METMETLEULEU(chain B and (resseq 2 or (resid 3 and (name...BB1 - 2621 - 262
263METMETLEULEU(chain B and (resseq 2 or (resid 3 and (name...BB1 - 2621 - 262
273METMETLEULEU(chain B and (resseq 2 or (resid 3 and (name...BB1 - 2621 - 262
283METMETLEULEU(chain B and (resseq 2 or (resid 3 and (name...BB1 - 2621 - 262
114FADFADFADFAD(chain D and (resseq 301))DK301
214FADFADFADFAD(chain B and (resseq 301))BH301
115FESFESFESFES(chain D and (resseq 302))DL302
215FESFESFESFES(chain B and (resseq 302))BI302
116FMNFMNFMNFMN(chain A and (resseq 401))AE401
216FMNFMNFMNFMN(chain C and (resseq 401))CM401

NCS ensembles :
ID
1
2
3
4
5
6
DetailsTetramer as determined by gel filtration

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Components

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Dihydroorotate ... , 2 types, 4 molecules ACBD

#1: Protein Dihydroorotate dehydrogenase / DHOdehase


Mass: 32344.492 Da / Num. of mol.: 2 / Fragment: residues 5-310 / Mutation: I74D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis (lactic acid bacteria)
Gene: pyrD, LG36_1039 / Plasmid: pET-22(b)+ / Production host: Escherichia coli (E. coli)
References: UniProt: A0A089ZD72, UniProt: Q9CFW8*PLUS, dihydroorotate dehydrogenase (NAD+)
#2: Protein Dihydroorotate dehydrogenase B (NAD(+)), electron transfer subunit / Dihydroorotate oxidase B / electron transfer subunit


Mass: 28862.615 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: pyrK, NCDO895_2366 / Plasmid: pET-22(b)+ / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0V8EGX5, UniProt: Q9CFW7*PLUS

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Non-polymers , 6 types, 399 molecules

#3: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21N4O9P
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#5: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6O2
#6: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM
#7: Chemical ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER


Mass: 175.820 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe2S2
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 389 / 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: 2.66 Å3/Da / Density % sol: 53.69 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 4
Details: 0.1 M sodium acetate pH 4.0, PEG 4,000 12% w/v, 3% v/v glycerol

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X6A / Type: OTHER / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 270 / Detector: CCD / Date: Oct 15, 2012 / Details: Oxford Danfysik toroidal focusing mirror
RadiationMonochromator: Si(111) channel cut monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.3→100 Å / Num. obs: 47302 / % possible obs: 99.9 % / Observed criterion σ(I): -3 / Redundancy: 4.2 % / Biso Wilson estimate: 41.2 Å2 / Rmerge(I) obs: 0.088 / Net I/σ(I): 12.2
Reflection shellRmerge(I) obs: 0.55 / Mean I/σ(I) obs: 2.8

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX1.10.1_2155refinement
HKL-2000data scaling
PHASERphasing
PDB_EXTRACT3.2data extraction
iMOSFLMdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1EP1

1ep1


Resolution: 2.47→41.921 Å / SU ML: 0.36 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 26.28
RfactorNum. reflection% reflection
Rfree0.2443 2369 5.02 %
Rwork0.1758 --
obs0.1793 47171 99.99 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 116.14 Å2 / Biso mean: 48.8451 Å2 / Biso min: 12.63 Å2
Refinement stepCycle: final / Resolution: 2.47→41.921 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8473 0 186 389 9048
Biso mean--39.42 45.74 -
Num. residues----1135
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0088820
X-RAY DIFFRACTIONf_angle_d1.12911978
X-RAY DIFFRACTIONf_chiral_restr0.0581391
X-RAY DIFFRACTIONf_plane_restr0.0071516
X-RAY DIFFRACTIONf_dihedral_angle_d13.6865362
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2676X-RAY DIFFRACTION7.903TORSIONAL
12C2676X-RAY DIFFRACTION7.903TORSIONAL
21C2X-RAY DIFFRACTION7.903TORSIONAL
22A2X-RAY DIFFRACTION7.903TORSIONAL
31D2298X-RAY DIFFRACTION7.903TORSIONAL
32B2298X-RAY DIFFRACTION7.903TORSIONAL
41D14X-RAY DIFFRACTION7.903TORSIONAL
42B14X-RAY DIFFRACTION7.903TORSIONAL
51D6X-RAY DIFFRACTION7.903TORSIONAL
52B6X-RAY DIFFRACTION7.903TORSIONAL
61A14X-RAY DIFFRACTION7.903TORSIONAL
62C14X-RAY DIFFRACTION7.903TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 17 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all
2.47-2.52040.32881210.250426632784
2.5204-2.57520.34481380.252425802718
2.5752-2.63510.32451360.233525952731
2.6351-2.7010.32821440.229925862730
2.701-2.7740.36151430.227126272770
2.774-2.85560.31121420.224626382780
2.8556-2.94780.3121290.227625842713
2.9478-3.05310.31741520.212525972749
3.0531-3.17530.29011280.212526392767
3.1753-3.31980.30691450.191625992744
3.3198-3.49470.26081340.190726242758
3.4947-3.71350.24421480.171826282776
3.7135-4.00010.18641310.157926462777
4.0001-4.40220.19481470.141226312778
4.4022-5.03830.20021350.131326882823
5.0383-6.34420.22041390.152426852824
6.3442-41.92660.18011570.150327922949

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