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- PDB-9dg8: Tetrahydroprotoberberine N-methyltransferase E204A mutant in comp... -

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Basic information

Entry
Database: PDB / ID: 9dg8
TitleTetrahydroprotoberberine N-methyltransferase E204A mutant in complex with (S)-reticuline and SAM
ComponentsTetrahydroprotoberberine N-methyltransferase
KeywordsTRANSFERASE / methyltransferase / benzylisoquinoline alkaloid
Function / homology(S)-tetrahydroprotoberberine N-methyltransferase / (S)-tetrahydroprotoberberine N-methyltransferase activity / Mycolic acid cyclopropane synthetase / methylation / S-adenosyl-L-methionine-dependent methyltransferase superfamily / (S)-reticuline / S-ADENOSYLMETHIONINE / Tetrahydroprotoberberine N-methyltransferase
Function and homology information
Biological speciesGlaucium flavum (plant)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.2 Å
AuthorsLang, D.E. / Ng, K.K.S.
Funding support Canada, 1items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
CitationJournal: To Be Published
Title: The stereospecific activities of the tetrahydroprotoberberine N-methyltransferase with alternative substrates provide insight into the catalytic mechanisms of benzylisoquinoline alkaloid N-methylation.
Authors: Lang, D.E. / Ng, K.K.S. / Rehman, F. / Morris, J.S. / Facchini, P.J.
History
DepositionSep 2, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 17, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tetrahydroprotoberberine N-methyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)44,4334
Polymers43,6131
Non-polymers8203
Water3,909217
1
A: Tetrahydroprotoberberine N-methyltransferase
hetero molecules

A: Tetrahydroprotoberberine N-methyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)88,8658
Polymers87,2252
Non-polymers1,6406
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation6_554-x,-x+y,-z-1/31
Buried area2570 Å2
ΔGint-12 kcal/mol
Surface area28340 Å2
MethodPISA
Unit cell
Length a, b, c (Å)104.098, 104.098, 82.196
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221
Symmetry operation#1: x,y,z
#2: -y,x-y,z+2/3
#3: -x+y,-x,z+1/3
#4: x-y,-y,-z+1/3
#5: -x,-x+y,-z+2/3
#6: y,x,-z

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Components

#1: Protein Tetrahydroprotoberberine N-methyltransferase


Mass: 43612.695 Da / Num. of mol.: 1 / Mutation: E204A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Glaucium flavum (plant) / Production host: Escherichia coli (E. coli)
References: UniProt: A0A5S8WF76, (S)-tetrahydroprotoberberine N-methyltransferase
#2: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE


Mass: 398.437 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H22N6O5S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-REN / (S)-reticuline / (1S)-1-(3-hydroxy-4-methoxybenzyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-ol


Mass: 329.390 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H23NO4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 217 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: 2.95 Å3/Da / Density % sol: 58.27 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 5% glycerol, 0.1 M Tris-Cl, 0.5 mM SAM, 0.5 mM (S)-reticuline, 15 mM ammonium sulfate, 29% pentaerythritol ethoxylate (15/4 EO/OH)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-2 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jul 3, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 2.2→39.523 Å / Num. obs: 26423 / % possible obs: 99.77 % / Redundancy: 6.7 % / Biso Wilson estimate: 35.83 Å2 / CC1/2: 0.999 / Rsym value: 0.025 / Net I/σ(I): 13.31
Reflection shellResolution: 2.2→2.28 Å / Redundancy: 6.5 % / Mean I/σ(I) obs: 4.23 / Num. unique obs: 2598 / CC1/2: 0.949 / Rsym value: 0.127 / % possible all: 99.65

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Processing

Software
NameVersionClassification
REFMAC5.8.0430refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.2→39.523 Å / Cor.coef. Fo:Fc: 0.962 / Cor.coef. Fo:Fc free: 0.953 / SU B: 3.732 / SU ML: 0.095 / Cross valid method: FREE R-VALUE / ESU R: 0.18 / ESU R Free: 0.153 / Details: Hydrogens have not been used
RfactorNum. reflection% reflection
Rfree0.1963 1362 5.155 %
Rwork0.166 25059 -
all0.167 --
obs-26421 99.773 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: BABINET MODEL PLUS MASK
Displacement parametersBiso mean: 51.674 Å2
Baniso -1Baniso -2Baniso -3
1--0.053 Å2-0.026 Å2-0 Å2
2---0.053 Å20 Å2
3---0.171 Å2
Refinement stepCycle: LAST / Resolution: 2.2→39.523 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2693 0 57 217 2967
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0050.0122871
X-RAY DIFFRACTIONr_angle_refined_deg1.41.8363873
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.1175347
X-RAY DIFFRACTIONr_dihedral_angle_2_deg5.4358
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.62810537
X-RAY DIFFRACTIONr_dihedral_angle_6_deg12.44610132
X-RAY DIFFRACTIONr_chiral_restr0.10.2418
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.022160
X-RAY DIFFRACTIONr_nbd_refined0.2020.21380
X-RAY DIFFRACTIONr_nbtor_refined0.3050.21975
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1770.2224
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1980.279
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.1730.229
X-RAY DIFFRACTIONr_mcbond_it8.1546.3711376
X-RAY DIFFRACTIONr_mcangle_it9.5510.6151727
X-RAY DIFFRACTIONr_scbond_it12.3457.2211495
X-RAY DIFFRACTIONr_scangle_it14.7711.922146
X-RAY DIFFRACTIONr_lrange_it15.07355.2864632
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
2.2-2.2570.275990.19918330.20219380.950.97599.69040.199
2.257-2.3190.267930.19817650.20218640.9560.97499.67810.198
2.319-2.3860.2321060.17517070.17818150.9640.98199.88980.175
2.386-2.4590.229990.17716830.1817840.9660.9899.88790.177
2.459-2.5390.187890.15916400.1617330.9790.98499.76920.159
2.539-2.6280.198690.15715930.15916620.980.9851000.157
2.628-2.7270.21960.15515250.15816230.9760.98599.87680.155
2.727-2.8370.226660.17314800.17515530.9730.98299.54930.173
2.837-2.9630.214730.16914260.17115010.9740.98299.86680.169
2.963-3.1070.195720.17813480.17914220.9760.9899.85940.178
3.107-3.2740.219640.1712980.17313640.9760.98299.85340.17
3.274-3.4710.191660.15812310.1612990.9760.98599.8460.158
3.471-3.7080.162590.15311460.15312110.9840.98599.50450.153
3.708-4.0020.203650.15510860.15811510.9760.9851000.155
4.002-4.380.139630.1439840.14310490.9880.98799.80930.143
4.38-4.8890.153490.1399150.1399660.9860.98899.7930.139
4.889-5.6310.184430.1578040.1598520.9760.98499.41310.157
5.631-6.8620.271440.2136950.2167390.9620.9731000.213
6.862-9.5620.19240.1675610.1685860.9780.98299.82940.167
9.562-39.5230.184230.223390.2183630.9750.97199.72450.22

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