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- PDB-4j9t: Crystal structure of a putative, de novo designed unnatural amino... -

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Basic information

Entry
Database: PDB / ID: 4j9t
TitleCrystal structure of a putative, de novo designed unnatural amino acid dependent metalloprotein, northeast structural genomics consortium target OR61
Componentsdesigned unnatural amino acid dependent metalloprotein
KeywordsStructural Genomics / Unknown Function / PSI-Biology / Northeast Structural Genomics Consortium / NESG / a beta-propeller / novel metalloenzyme
Function / homology
Function and homology information


ganglioside catabolic process / oligosaccharide catabolic process / exo-alpha-sialidase / exo-alpha-sialidase activity / intracellular membrane-bounded organelle / extracellular region / membrane / cytoplasm
Similarity search - Function
Alpha-galactosidase, NEW3 domain / NPCBM-associated, NEW3 domain of alpha-galactosidase / BNR repeat-like domain / Sialidase family / Sialidase / Coagulation factor 5/8 C-terminal domain, discoidin domain / Coagulation factors 5/8 type C domain (FA58C) profile. / F5/8 type C domain / Coagulation factor 5/8 C-terminal domain / Neuraminidase - #10 ...Alpha-galactosidase, NEW3 domain / NPCBM-associated, NEW3 domain of alpha-galactosidase / BNR repeat-like domain / Sialidase family / Sialidase / Coagulation factor 5/8 C-terminal domain, discoidin domain / Coagulation factors 5/8 type C domain (FA58C) profile. / F5/8 type C domain / Coagulation factor 5/8 C-terminal domain / Neuraminidase - #10 / Sialidase superfamily / 6 Propeller / Neuraminidase / Twin arginine translocation (Tat) signal profile. / Twin-arginine translocation pathway, signal sequence / Galactose-binding-like domain superfamily / Immunoglobulin E-set / Immunoglobulin-like fold / Mainly Beta
Similarity search - Domain/homology
Biological speciesMicromonospora viridifaciens (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.4 Å
AuthorsForouhar, F. / Lew, S. / Seetharaman, J. / Mills, J.H. / Khare, S.D. / Everett, J.K. / Baker, D. / Montelione, G.T. / Hunt, J.F. / Tong, L. / Northeast Structural Genomics Consortium (NESG)
CitationJournal: J.Am.Chem.Soc. / Year: 2013
Title: Computational design of an unnatural amino Acid dependent metalloprotein with atomic level accuracy.
Authors: Mills, J.H. / Khare, S.D. / Bolduc, J.M. / Forouhar, F. / Mulligan, V.K. / Lew, S. / Seetharaman, J. / Tong, L. / Stoddard, B.L. / Baker, D.
History
DepositionFeb 17, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 27, 2013Provider: repository / Type: Initial release
Revision 1.1Sep 25, 2013Group: Database references
Revision 1.2Nov 15, 2017Group: Refinement description / Category: software / Item: _software.name
Revision 1.3Jul 17, 2019Group: Data collection / Refinement description / Category: software
Item: _software.classification / _software.name / _software.version
Revision 1.4Sep 20, 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 / 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 / _struct_ref_seq_dif.details / _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: designed unnatural amino acid dependent metalloprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,7163
Polymers38,5491
Non-polymers1672
Water9,494527
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)46.163, 79.677, 83.777
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein designed unnatural amino acid dependent metalloprotein / Neuraminidase / Sialidase


Mass: 38549.270 Da / Num. of mol.: 1
Mutation: R68S, I69H, D92A, D131H, F155W, A156E, T226A, F234I, D259S, E260H, R276A, N311D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Micromonospora viridifaciens (bacteria)
Gene: nedA / Plasmid: pET29b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q02834
#2: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical ChemComp-ARS / ARSENIC


Mass: 74.922 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: As
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 527 / Source method: isolated from a natural source / Formula: H2O
Compound detailsresidue 92, which is modeled as Ala in the current structure, is modified by unnatural amino acid ...residue 92, which is modeled as Ala in the current structure, is modified by unnatural amino acid biprimidine Ala.

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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 Å3/Da / Density % sol: 38.45 %
Crystal growTemperature: 277 K / Method: microbatch under oil / pH: 6.5
Details: protein buffer: 10 mM Tris HCl (pH 8). Precipitation cocktail: 100 MM NA CACODYLATE (PH 6.5), 5 mM FeCl2, 15% PEG 3350 Microbatch under oil, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X4C / Wavelength: 0.97012 Å
DetectorType: MAR CCD 165 mm / Detector: CCD / Date: Oct 12, 2012 / Details: mirrors
RadiationMonochromator: Si 111 CHANNEL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97012 Å / Relative weight: 1
ReflectionResolution: 1.4→31.021 Å / Num. obs: 61532 / % possible obs: 99.9 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 6.1 % / Biso Wilson estimate: 11.2 Å2 / Rmerge(I) obs: 0.059 / Rsym value: 0.041 / Net I/σ(I): 29.2
Reflection shellResolution: 1.4→1.45 Å / Redundancy: 4.9 % / Rmerge(I) obs: 0.42 / Mean I/σ(I) obs: 4.9 / Num. unique all: 6060 / Rsym value: 0.405 / % possible all: 99.7

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Processing

Software
NameVersionClassification
PHENIX(phenix.refine: 1.7_650)refinement
COMOphasing
XTALVIEWrefinement
CNSrefinement
ADSCQuantumdata collection
HKL-2000data reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1EUU

1euu


Resolution: 1.4→31.021 Å / SU ML: 0.18 / Cross valid method: THROUGHOUT / σ(F): 1.34 / σ(I): 1.3 / Phase error: 13.94 / Stereochemistry target values: ML
Details: the electron density near Cys-351 was modeled as As ion, however, Fe2+ could be another possibility
RfactorNum. reflection% reflectionSelection details
Rfree0.1662 6183 10.06 %RANDOM
Rwork0.1349 ---
all0.1394 61546 --
obs0.1382 61460 99.86 %-
Solvent computationShrinkage radii: 0.83 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 40.338 Å2 / ksol: 0.396 e/Å3
Displacement parameters
Baniso -1Baniso -2Baniso -3
1--1.2254 Å20 Å20 Å2
2--0.8007 Å20 Å2
3---0.4246 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.13 Å0.13 Å
Luzzati d res low-5 Å
Luzzati sigma a0.09 Å0.09 Å
Refinement stepCycle: LAST / Resolution: 1.4→31.021 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2675 0 7 527 3209
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0072752
X-RAY DIFFRACTIONf_angle_d1.1483756
X-RAY DIFFRACTIONf_dihedral_angle_d12.698981
X-RAY DIFFRACTIONf_chiral_restr0.069403
X-RAY DIFFRACTIONf_plane_restr0.005504
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.4003-1.41620.37821980.29951769X-RAY DIFFRACTION98
1.4162-1.43280.26841860.2471808X-RAY DIFFRACTION100
1.4328-1.45030.25782230.21491820X-RAY DIFFRACTION100
1.4503-1.46870.19271930.17381813X-RAY DIFFRACTION100
1.4687-1.4880.222060.16621827X-RAY DIFFRACTION100
1.488-1.50840.20062150.15091799X-RAY DIFFRACTION100
1.5084-1.52990.19492210.14511800X-RAY DIFFRACTION100
1.5299-1.55280.19651830.1271852X-RAY DIFFRACTION100
1.5528-1.5770.18491950.11961831X-RAY DIFFRACTION100
1.577-1.60290.17612010.11581832X-RAY DIFFRACTION100
1.6029-1.63050.1761940.11191831X-RAY DIFFRACTION100
1.6305-1.66020.15942100.10531841X-RAY DIFFRACTION100
1.6602-1.69210.14851910.10641820X-RAY DIFFRACTION100
1.6921-1.72660.16672190.10871824X-RAY DIFFRACTION100
1.7266-1.76420.15292150.10091815X-RAY DIFFRACTION100
1.7642-1.80520.15662120.10241861X-RAY DIFFRACTION100
1.8052-1.85030.15292010.10571825X-RAY DIFFRACTION100
1.8503-1.90040.14582080.11631824X-RAY DIFFRACTION100
1.9004-1.95630.1662140.11851831X-RAY DIFFRACTION100
1.9563-2.01940.16182090.1231824X-RAY DIFFRACTION100
2.0194-2.09160.15721950.12381884X-RAY DIFFRACTION100
2.0916-2.17530.15472060.12631850X-RAY DIFFRACTION100
2.1753-2.27430.1551840.12311874X-RAY DIFFRACTION100
2.2743-2.39410.15992160.131839X-RAY DIFFRACTION100
2.3941-2.54410.16512020.13311873X-RAY DIFFRACTION100
2.5441-2.74040.16422140.13431849X-RAY DIFFRACTION100
2.7404-3.01590.16222040.14011873X-RAY DIFFRACTION100
3.0159-3.45190.15012210.13721890X-RAY DIFFRACTION100
3.4519-4.34710.14472120.1311913X-RAY DIFFRACTION100
4.3471-31.02860.17192350.1711985X-RAY DIFFRACTION99

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