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- PDB-1q2o: Bovine endothelial nitric oxide synthase N368D mutant heme domain... -

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Basic information

Entry
Database: PDB / ID: 1q2o
TitleBovine endothelial nitric oxide synthase N368D mutant heme domain dimer with L-N(omega)-nitroarginine-2,4-L-diaminobutyramide bound
ComponentsNitric-oxide synthase, endothelialNitric oxide synthase
KeywordsOXIDOREDUCTASE / endothelial nitric oxide synthase / enos / nos III / heme protein
Function / homology
Function and homology information


cellular response to laminar fluid shear stress / positive regulation of guanylate cyclase activity / negative regulation of leukocyte cell-cell adhesion / nitric-oxide synthase (NADPH) / nitric-oxide synthase activity / nitric oxide mediated signal transduction / arginine catabolic process / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / nitric oxide biosynthetic process / negative regulation of blood pressure ...cellular response to laminar fluid shear stress / positive regulation of guanylate cyclase activity / negative regulation of leukocyte cell-cell adhesion / nitric-oxide synthase (NADPH) / nitric-oxide synthase activity / nitric oxide mediated signal transduction / arginine catabolic process / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / nitric oxide biosynthetic process / negative regulation of blood pressure / mitochondrion organization / response to hormone / caveola / blood coagulation / FMN binding / flavin adenine dinucleotide binding / NADP binding / response to lipopolysaccharide / cytoskeleton / calmodulin binding / heme binding / Golgi apparatus / metal ion binding / nucleus / plasma membrane / cytosol
Similarity search - Function
Bovine Endothelial Nitric Oxide Synthase Heme Domain; Chain: A,domain 3 / Nitric Oxide Synthase; Chain A, domain 3 / Nitric Oxide Synthase; Chain A, domain 1 / Nitric Oxide Synthase; Chain A, domain 1 / Nitric Oxide Synthase;Heme Domain; Chain A, domain 2 / Nitric Oxide Synthase;Heme Domain;Chain A domain 2 / Nitric-oxide synthase, eukaryote / Nitric oxide synthase, domain 2 superfamily / Nitric oxide synthase, domain 1 superfamily / Nitric oxide synthase, domain 3 superfamily ...Bovine Endothelial Nitric Oxide Synthase Heme Domain; Chain: A,domain 3 / Nitric Oxide Synthase; Chain A, domain 3 / Nitric Oxide Synthase; Chain A, domain 1 / Nitric Oxide Synthase; Chain A, domain 1 / Nitric Oxide Synthase;Heme Domain; Chain A, domain 2 / Nitric Oxide Synthase;Heme Domain;Chain A domain 2 / Nitric-oxide synthase, eukaryote / Nitric oxide synthase, domain 2 superfamily / Nitric oxide synthase, domain 1 superfamily / Nitric oxide synthase, domain 3 superfamily / Nitric oxide synthase, N-terminal / Nitric oxide synthase, N-terminal domain superfamily / Nitric oxide synthase, oxygenase domain / Nitric oxide synthase (NOS) signature. / Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding / NADPH-cytochrome p450 reductase, FAD-binding, alpha-helical domain superfamily / FAD binding domain / Flavodoxin-like / Flavoprotein pyridine nucleotide cytochrome reductase / Flavodoxin / Flavodoxin-like domain profile. / Flavodoxin/nitric oxide synthase / 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 / Flavoprotein-like superfamily / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
ACETATE ION / CACODYLATE ION / Chem-DP1 / 5,6,7,8-TETRAHYDROBIOPTERIN / PROTOPORPHYRIN IX CONTAINING FE / Nitric oxide synthase 3
Similarity search - Component
Biological speciesBos taurus (cattle)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 1.74 Å
AuthorsFlinspach, M.L. / Li, H. / Jamal, J. / Yang, W. / Huang, H. / Hah, J.M. / Gomez-Vidal, J.A. / Litzinger, E.A. / Silverman, R.B. / Poulos, T.L.
Citation
Journal: Nat.Struct.Mol.Biol. / Year: 2004
Title: Structural basis for dipeptide amide isoform-selective inhibition of neuronal nitric oxide synthase.
Authors: Flinspach, M.L. / Li, H. / Jamal, J. / Yang, W. / Huang, H. / Hah, J.M. / Gomez-Vidal, J.A. / Litzinger, E.A. / Silverman, R.B. / Poulos, T.L.
#1: Journal: Cell(Cambridge,Mass.) / Year: 1998
Title: Crystal Structure of Constitutive Endothelial Nitric Oxide Synthase: A Paradigm for Pterin Function Involving a Novel Metal Center
Authors: Raman, C.S. / Li, H. / Martasek, P. / Kral, V. / Masters, B.S.S. / Poulos, T.L.
History
DepositionJul 25, 2003Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 13, 2004Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Non-polymer description / Version format compliance
Revision 1.3Oct 27, 2021Group: Database references / Derived calculations
Category: database_2 / pdbx_struct_conn_angle ...database_2 / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Feb 14, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Revision 1.5Apr 3, 2024Group: Refinement description / Category: pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nitric-oxide synthase, endothelial
B: Nitric-oxide synthase, endothelial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)96,70415
Polymers93,7102
Non-polymers2,99413
Water10,881604
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12490 Å2
ΔGint-117 kcal/mol
Surface area32020 Å2
MethodPISA
Unit cell
Length a, b, c (Å)57.908, 106.989, 156.989
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Nitric-oxide synthase, endothelial / Nitric oxide synthase / EC-NOS / NOS / type III / NOSIII / Endothelial NOS / eNOS / Constitutive NOS / cNOS


Mass: 46855.012 Da / Num. of mol.: 2 / Fragment: heme domain of enos enzyme / Mutation: N368D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: NOS3 / Plasmid: pCWori / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P29473, nitric-oxide synthase (NADPH)

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Non-polymers , 8 types, 617 molecules

#2: Chemical ChemComp-CAC / CACODYLATE ION / dimethylarsinate / Cacodylic acid


Mass: 136.989 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6AsO2
#3: Chemical ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H3O2
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#6: Chemical ChemComp-H4B / 5,6,7,8-TETRAHYDROBIOPTERIN / Tetrahydrobiopterin


Mass: 241.247 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C9H15N5O3 / Comment: neurotransmitter*YM
#7: Chemical ChemComp-DP1 / L-N(OMEGA)-NITROARGININE-2,4-L-DIAMINOBUTYRIC AMIDE


Mass: 318.333 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H22N8O4
#8: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#9: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 604 / 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: 2.59 Å3/Da / Density % sol: 52.56 %
Crystal growTemperature: 280 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 15% PEG 3350, 200 mM MgOAc, 100 mM Na Cacodylate, 5 mM TCEP, 2 mM imidazole, 3.5 mM L-N(omega)-nitroarginine-L-2,4-diaminobutyramide, pH 7.0, VAPOR DIFFUSION, SITTING DROP, temperature 280K
Crystal grow
*PLUS
Temperature: 5 ℃ / Method: vapor diffusion, sitting drop / Details: Li, H., (2002) Biochemistry, 41, 13868. / PH range low: 6 / PH range high: 5.6
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetails
17-9 mg/mlprotein1drop
25 mMimidazole1drop
35-8 mMligand1drop
40.1 MMES1reservoirpH5.6-6.0
5100-200 mMammonium acetate1reservoir
62.0 mMGSH1reservoir
735000 nmSDS1reservoir
818-22 %(w/v)PEG33501reservoir

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 1.2398 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Jun 20, 2003 / Details: mirrors
RadiationMonochromator: yes / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.2398 Å / Relative weight: 1
ReflectionResolution: 1.74→50 Å / Num. all: 100785 / Num. obs: 97827 / % possible obs: 97.1 % / Observed criterion σ(F): -3 / Redundancy: 4.1 % / Biso Wilson estimate: 22.6 Å2 / Rmerge(I) obs: 0.077 / Rsym value: 0.095 / Net I/σ(I): 28.5
Reflection shellResolution: 1.74→1.8 Å / Redundancy: 3.8 % / Rmerge(I) obs: 0.631 / Mean I/σ(I) obs: 2.85 / Num. unique all: 9417 / Rsym value: 0.538 / % possible all: 95
Reflection
*PLUS
Num. obs: 97889 / % possible obs: 97.4 % / Num. measured all: 412431 / Rmerge(I) obs: 0.095
Reflection shell
*PLUS
% possible obs: 95 % / Rmerge(I) obs: 0.538 / Mean I/σ(I) obs: 2.9

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Processing

Software
NameVersionClassification
CNS1.1refinement
HKL-2000data reduction
SCALEPACKdata scaling
CNSphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: wild-type bovine enos heme domain

Resolution: 1.74→48.44 Å / Rfactor Rfree error: 0.003 / Data cutoff high absF: 2019469.95 / Data cutoff high rms absF: 2019469.95 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.22 4908 5 %RANDOM
Rwork0.193 ---
obs0.193 97827 97.5 %-
all-100785 --
Solvent computationSolvent model: FLAT MODEL / Bsol: 43.2618 Å2 / ksol: 0.381694 e/Å3
Displacement parametersBiso mean: 25.5 Å2
Baniso -1Baniso -2Baniso -3
1-4.35 Å20 Å20 Å2
2---2.63 Å20 Å2
3----1.73 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.21 Å0.19 Å
Luzzati d res low-5 Å
Luzzati sigma a0.11 Å0.09 Å
Refinement stepCycle: LAST / Resolution: 1.74→48.44 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6439 0 191 604 7234
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_bond_d_na
X-RAY DIFFRACTIONc_bond_d_prot
X-RAY DIFFRACTIONc_angle_d
X-RAY DIFFRACTIONc_angle_d_na
X-RAY DIFFRACTIONc_angle_d_prot
X-RAY DIFFRACTIONc_angle_deg1.4
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d22.6
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.99
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it
X-RAY DIFFRACTIONc_mcangle_it
X-RAY DIFFRACTIONc_scbond_it
X-RAY DIFFRACTIONc_scangle_it
LS refinement shellResolution: 1.74→1.8 Å / Rfactor Rfree error: 0.014 / Total num. of bins used: 10
RfactorNum. reflection% reflection
Rfree0.286 443 4.9 %
Rwork0.259 8596 -
obs-8596 90.8 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2WATER_REP.PARAMWATER.TOP
X-RAY DIFFRACTION3HETERO.PARAMHETERO.TOP
X-RAY DIFFRACTION4DP1.PARAMDP1.TOP
Refinement
*PLUS
Num. reflection obs: 92919 / % reflection Rfree: 5 % / Rfactor Rfree: 0.221 / Rfactor Rwork: 0.198
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg22.6
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.99

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