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- PDB-1ih8: NH3-dependent NAD+ Synthetase from Bacillus subtilis Complexed wi... -

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Basic information

Entry
Database: PDB / ID: 1ih8
TitleNH3-dependent NAD+ Synthetase from Bacillus subtilis Complexed with AMP-CPP and Mg2+ ions.
ComponentsNH(3)-DEPENDENT NAD(+) synthetase
KeywordsLIGASE / amidotransferase / ATP pyrophosphatase / active-site loops
Function / homology
Function and homology information


NAD+ synthase / NAD+ synthase activity / NAD+ synthase (glutamine-hydrolyzing) activity / glutaminase activity / NAD+ biosynthetic process / sporulation resulting in formation of a cellular spore / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
NH(3)-dependent NAD(+) synthetase / NAD(+) synthetase / NAD/GMP synthase / NAD synthase / HUPs / Rossmann-like alpha/beta/alpha sandwich fold / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
DIPHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER / NH(3)-dependent NAD(+) synthetase
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodX-RAY DIFFRACTION / FOURIER SYNTHESIS / Resolution: 1.9 Å
AuthorsDevedjiev, Y. / Symersky, J. / Singh, R. / Jedrzejas, M. / Brouillette, C. / Brouillette, W. / Muccio, D. / Chattopadhyay, D. / DeLucas, L.
CitationJournal: Acta Crystallogr.,Sect.D / Year: 2001
Title: Stabilization of active-site loops in NH3-dependent NAD+ synthetase from Bacillus subtilis.
Authors: Devedjiev, Y. / Symersky, J. / Singh, R. / Jedrzejas, M. / Brouillette, C. / Brouillette, W. / Muccio, D. / Chattopadhyay, D. / DeLucas, L.
History
DepositionApr 18, 2001Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 6, 2001Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Aug 16, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / 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_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_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_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_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: NH(3)-DEPENDENT NAD(+) synthetase
B: NH(3)-DEPENDENT NAD(+) synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)61,1866
Polymers60,6082
Non-polymers5784
Water10,629590
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6430 Å2
ΔGint-62 kcal/mol
Surface area21450 Å2
MethodPISA
Unit cell
Length a, b, c (Å)52.599, 85.411, 60.253
Angle α, β, γ (deg.)90.00, 110.88, 90.00
Int Tables number4
Space group name H-MP1211
DetailsThe biological assembly is a homodimer consisting of chains A and B in the same asymmetric unit.

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Components

#1: Protein NH(3)-DEPENDENT NAD(+) synthetase / NAD(+) SYNTHETASE


Mass: 30303.994 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: OutB / Plasmid: pET21 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DH3) plysS
References: UniProt: P08164, NAD+ synthase (glutamine-hydrolysing)
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#3: Chemical ChemComp-APC / DIPHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER / ALPHA,BETA-METHYLENEADENOSINE-5'-TRIPHOSPHATE


Mass: 505.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H18N5O12P3 / Comment: AMP-CPP, energy-carrying molecule analogue*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 590 / 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: 2.1 Å3/Da / Density % sol: 39 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: VAPOR DIFFUSION IN MICROSEEDED HANGING DROPS. 20-26% PEG400, 0.05 M HEPES SODIUM SALT, PH=7.5, 0.1 M MAGNESIUM CHLORIDE, 0.002 M AMP-CPP., VAPOR DIFFUSION, HANGING DROP, temperature 295K
Crystal grow
*PLUS
Details: used microseeding
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formula
110 mMHEPES-Na1drop
220 mg/mlprotein1drop
320-26 %(v/v)PEG4001reservoir
450 mMHEPES-Na1reservoir
50.1 M1reservoirMgCl2
62 mMAMP-CPP1reservoir

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Data collection

DiffractionMean temperature: 120 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU200 / Wavelength: 1.5418 Å
DetectorType: MACSCIENCE DIP100 / Detector: IMAGE PLATE / Date: Jun 27, 2000 / Details: mirrors
RadiationMonochromator: Ni filter / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.9→30 Å / Num. all: 112884 / Num. obs: 38796 / % possible obs: 98.8 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -10 / Redundancy: 2.9 % / Biso Wilson estimate: 10.8 Å2 / Rmerge(I) obs: 0.048 / Net I/σ(I): 20.4
Reflection shellResolution: 1.9→1.97 Å / Redundancy: 2.5 % / Rmerge(I) obs: 0.154 / Mean I/σ(I) obs: 7.6 / % possible all: 98.4
Reflection
*PLUS
Num. measured all: 112884
Reflection shell
*PLUS
% possible obs: 98.8 %

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Processing

Software
NameClassification
CNSrefinement
DENZOdata reduction
SCALEPACKdata scaling
CNSphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: pdb entry 1NSY

1nsy


Resolution: 1.9→29.83 Å / Rfactor Rfree error: 0.005 / Data cutoff high absF: 346000.4 / Data cutoff high rms absF: 10000 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 2 / σ(I): 1.36 / Stereochemistry target values: Engh & Huber
Details: Subunit A has all 271 residues and methyleneadenosine triphosphate. In the subunit B, residues 1083-1086, 1205-1224, and methyleneadenosine triphosphate are disordered and not included in the model.
RfactorNum. reflection% reflectionSelection details
Rfree0.205 1900 5 %RANDOM
Rwork0.167 ---
all0.175 38796 --
obs0.167 37901 96.4 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 48.29 Å2 / ksol: 0.371 e/Å3
Displacement parametersBiso mean: 15.4 Å2
Baniso -1Baniso -2Baniso -3
1--0.72 Å20 Å21.46 Å2
2---0.32 Å20 Å2
3---1.05 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.22 Å0.18 Å
Luzzati d res low-30 Å
Luzzati sigma a0.15 Å0.06 Å
Refinement stepCycle: LAST / Resolution: 1.9→29.83 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4072 0 34 590 4696
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.005
X-RAY DIFFRACTIONc_angle_deg1.1
X-RAY DIFFRACTIONc_dihedral_angle_d20.1
X-RAY DIFFRACTIONc_improper_angle_d0.71
X-RAY DIFFRACTIONc_mcbond_it1.171.5
X-RAY DIFFRACTIONc_mcangle_it1.72
X-RAY DIFFRACTIONc_scbond_it2.032
X-RAY DIFFRACTIONc_scangle_it32.5
LS refinement shellResolution: 1.9→1.97 Å / Rfactor Rfree error: 0.017 / Total num. of bins used: 10
RfactorNum. reflection% reflection
Rfree0.244 202 5.5 %
Rwork0.175 3442 -
obs-2102 92.9 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2AMPCPP.PARAMPROTEIN.LINK
X-RAY DIFFRACTION3ION.PARAMWATER.TOP
X-RAY DIFFRACTION4WATER_REP.PARAMAMPCPP.TOP
X-RAY DIFFRACTION5ION.TOP
Software
*PLUS
Name: CNS / Version: 1 / Classification: refinement
Refinement
*PLUS
σ(F): 2 / % reflection Rfree: 5 %
Solvent computation
*PLUS
Displacement parameters
*PLUS
Biso mean: 15.4 Å2
Refine LS restraints
*PLUS
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg20.1
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.71
X-RAY DIFFRACTIONc_mcbond_it1.5
X-RAY DIFFRACTIONc_scbond_it2
X-RAY DIFFRACTIONc_mcangle_it2
X-RAY DIFFRACTIONc_scangle_it2.5
LS refinement shell
*PLUS
Rfactor Rfree: 0.244 / % reflection Rfree: 5.5 % / Rfactor Rwork: 0.175

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