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- PDB-8uke: Crystal Structure of Norbelladine O-methyltransferase variant in ... -

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Basic information

Entry
Database: PDB / ID: 8uke
TitleCrystal Structure of Norbelladine O-methyltransferase variant in complex with SAH
ComponentsNorbelladine 4'-O-methyltransferase
KeywordsTRANSFERASE / Methyltransferase / SAH / ML-guided Protein Engineering
Function / homology
Function and homology information


norbelladine O-methyltransferase / alkaloid metabolic process / S-adenosylmethionine-dependent methyltransferase activity / O-methyltransferase activity / methylation / metal ion binding
Similarity search - Function
: / Class I-like SAM-dependent O-methyltransferase / O-methyltransferase / SAM-dependent O-methyltransferase class I-type profile. / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
S-ADENOSYL-L-HOMOCYSTEINE / Norbelladine 4'-O-methyltransferase
Similarity search - Component
Biological speciesNarcissus aff. pseudonarcissus MK-2014 (plant)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.4 Å
AuthorsKim, W. / Zhang, Y.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM148356 United States
CitationJournal: Nat Commun / Year: 2024
Title: Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme.
Authors: d'Oelsnitz, S. / Diaz, D.J. / Kim, W. / Acosta, D.J. / Dangerfield, T.L. / Schechter, M.W. / Minus, M.B. / Howard, J.R. / Do, H. / Loy, J.M. / Alper, H.S. / Zhang, Y.J. / Ellington, A.D.
History
DepositionOct 12, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 20, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Norbelladine 4'-O-methyltransferase
B: Norbelladine 4'-O-methyltransferase
C: Norbelladine 4'-O-methyltransferase
D: Norbelladine 4'-O-methyltransferase
E: Norbelladine 4'-O-methyltransferase
F: Norbelladine 4'-O-methyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)166,38321
Polymers163,7166
Non-polymers2,66715
Water5,386299
1
A: Norbelladine 4'-O-methyltransferase
B: Norbelladine 4'-O-methyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,5018
Polymers54,5722
Non-polymers9296
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4250 Å2
ΔGint-47 kcal/mol
Surface area18130 Å2
MethodPISA
2
C: Norbelladine 4'-O-methyltransferase
hetero molecules

E: Norbelladine 4'-O-methyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,4617
Polymers54,5722
Non-polymers8895
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y+1/2,-z1
Buried area4210 Å2
ΔGint-48 kcal/mol
Surface area18250 Å2
MethodPISA
3
D: Norbelladine 4'-O-methyltransferase
F: Norbelladine 4'-O-methyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,4216
Polymers54,5722
Non-polymers8494
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4270 Å2
ΔGint-48 kcal/mol
Surface area18410 Å2
MethodPISA
Unit cell
Length a, b, c (Å)95.973, 79.581, 98.379
Angle α, β, γ (deg.)90.00, 105.62, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
Norbelladine 4'-O-methyltransferase


Mass: 27286.047 Da / Num. of mol.: 6 / Mutation: E36P, G40E, A53M
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Narcissus aff. pseudonarcissus MK-2014 (plant)
Gene: N4OMT
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A077EWA5
#2: Chemical
ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE


Type: L-peptide linking / Mass: 384.411 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: C14H20N6O5S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 299 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.21 Å3/Da / Density % sol: 44.34 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 7 / Details: 0.1M Calcium Acetate, 0.1M MES pH 7.0, 26% PEG3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 1.00002 Å
DetectorType: DECTRIS PILATUS3 2M / Detector: PIXEL / Date: Jun 25, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.00002 Å / Relative weight: 1
ReflectionResolution: 2.4→50 Å / Num. obs: 55771 / % possible obs: 99.3 % / Redundancy: 3.8 % / CC1/2: 0.966 / CC star: 0.991 / Rmerge(I) obs: 0.163 / Rpim(I) all: 0.095 / Rrim(I) all: 0.189 / Χ2: 0.441 / Net I/σ(I): 2.5 / Num. measured all: 211904
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2CC starRpim(I) allRrim(I) allΧ2% possible all
2.4-2.443.20.44126060.7780.9350.2780.5240.42893.9
2.44-2.493.50.43827500.7970.9420.2670.5150.4298.3
2.49-2.533.90.4328020.8120.9470.2520.50.43499.9
2.53-2.5940.41227970.8480.9580.2390.4780.41799.8
2.59-2.6440.38227420.8720.9650.2190.4410.44199.9
2.64-2.740.35927820.8760.9660.2070.4160.44499.8
2.7-2.7740.34128000.8830.9680.1970.3950.44299.8
2.77-2.8540.31928050.910.9760.1840.370.43999.9
2.85-2.933.90.2927830.9240.980.1690.3370.44199.9
2.93-3.023.90.26328050.9210.9790.1530.3050.44499.7
3.02-3.133.90.23427670.9470.9860.1360.2710.43799.6
3.13-3.263.80.20128040.9520.9880.1190.2350.45999.5
3.26-3.413.50.16127830.9670.9920.0990.190.46299.4
3.41-3.583.30.1327440.9750.9940.0830.1550.4697.9
3.58-3.813.90.12128250.9820.9960.070.140.47100
3.81-4.140.128010.9880.9970.0570.1150.457100
4.1-4.5240.08428410.9910.9980.0480.0970.468100
4.52-5.173.90.08128080.9920.9980.0470.0940.43399.5
5.17-6.513.60.09628300.9880.9970.0580.1130.41499.4
6.51-503.80.06228960.9960.9990.0360.0710.41199.3

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Processing

Software
NameVersionClassification
PHENIX(1.18rc3_3805: ???)refinement
HKL-2000data scaling
HKL-2000data reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.4→47.69 Å / SU ML: 0.29 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 24.78 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2494 1981 3.55 %
Rwork0.1894 --
obs0.1915 55735 99.16 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.4→47.69 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms11193 0 165 299 11657
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00311609
X-RAY DIFFRACTIONf_angle_d0.61115768
X-RAY DIFFRACTIONf_dihedral_angle_d25.154354
X-RAY DIFFRACTIONf_chiral_restr0.0441769
X-RAY DIFFRACTIONf_plane_restr0.0041995
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.4-2.460.34611270.23793584X-RAY DIFFRACTION94
2.46-2.520.26641390.22313842X-RAY DIFFRACTION100
2.52-2.60.24281470.2173846X-RAY DIFFRACTION100
2.6-2.680.30161410.20873839X-RAY DIFFRACTION100
2.68-2.780.28081380.21453826X-RAY DIFFRACTION100
2.78-2.890.26531480.21213874X-RAY DIFFRACTION100
2.89-3.020.30181390.21283852X-RAY DIFFRACTION100
3.02-3.180.28031460.2163864X-RAY DIFFRACTION100
3.18-3.380.28181350.19683798X-RAY DIFFRACTION99
3.38-3.640.22581490.18573835X-RAY DIFFRACTION98
3.64-4.010.22461370.17253864X-RAY DIFFRACTION100
4.01-4.590.20851450.14713890X-RAY DIFFRACTION100
4.59-5.770.21441430.1693895X-RAY DIFFRACTION99
5.78-47.690.21991470.16653945X-RAY DIFFRACTION99

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