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- PDB-7ux7: Crystal structure of MfnG, an L- and D-tyrosine O-methyltransfera... -

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Basic information

Entry
Database: PDB / ID: 7ux7
TitleCrystal structure of MfnG, an L- and D-tyrosine O-methyltransferase from the marformycin biosynthesis pathway of Streptomyces drozdowiczii, with SAH bound at 1.2 A resolution (P212121 - form II)
ComponentsMfnG
KeywordsTRANSFERASE / O-methyltransferase / O-methyl-tyrosine / Marformycin synthesis / SAM-dependent methyltransferase
Function / homology
Function and homology information


O-methyltransferase activity / methylation / protein dimerization activity
Similarity search - Function
: / Plant methyltransferase dimerisation / O-methyltransferase dimerisation domain / O-methyltransferase domain / O-methyltransferase COMT-type / O-methyltransferase domain / SAM-dependent O-methyltransferase class II-type profile. / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
S-ADENOSYL-L-HOMOCYSTEINE / Unknown ligand / MfnG
Similarity search - Component
Biological speciesStreptomyces drozdowiczii (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.14 Å
AuthorsMiller, M.D. / Wu, K.-L. / Xu, W. / Xiao, H. / Philips Jr., G.N.
Funding support United States, 9items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM115261 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01-CA217255 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM133706 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R21-CA255894 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01-AI165079 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR170014 United States
Robert A. Welch FoundationC-1970 United States
Department of Defense (DOD, United States)W81XWH-21-1-0789 United States
National Science Foundation (NSF, United States)STC 1231306 United States
CitationJournal: Protein Sci. / Year: 2022
Title: Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid.
Authors: Wu, K.L. / Moore, J.A. / Miller, M.D. / Chen, Y. / Lee, C. / Xu, W. / Peng, Z. / Duan, Q. / Phillips Jr., G.N. / Uribe, R.A. / Xiao, H.
History
DepositionMay 5, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 12, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: MfnG
B: MfnG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)84,8536
Polymers84,0842
Non-polymers7694
Water15,511861
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Size Exclusion Chromatography (SEC) supports the assignment of a dimer as the assembly in solution
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10270 Å2
ΔGint-58 kcal/mol
Surface area27650 Å2
MethodPISA
Unit cell
Length a, b, c (Å)60.694, 86.334, 134.504
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 MfnG


Mass: 42042.246 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces drozdowiczii (bacteria) / Plasmid: pET22b-T5-MfnG-TEV-His / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0D4WTP2
#2: Chemical ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE


Type: L-peptide linking / Mass: 384.411 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C14H20N6O5S
#3: Chemical ChemComp-UNL / UNKNOWN LIGAND


Num. of mol.: 2 / Source method: obtained synthetically
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 861 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Sequence detailsThe construct was cloned into the EcoRI and HindIII sites of pET22b with an N-terminal Met (0) ...The construct was cloned into the EcoRI and HindIII sites of pET22b with an N-terminal Met (0) added and the native GUG-start codon being expressed as V. A C-terminal expression and 6-His tag ASENLYFQ/GGGHHHHHHG leaving a C-terminal ASENLYFQ after removal of the 6-His tag with TEV protease.

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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: 41.3 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8
Details: 0.1 M Tris pH 8, 30% Polyethylene glycol monomethyl ether (PEG MME) 2000, Additive: 0.002 M S-Adenosyl methionine (SAM/AdoMet)

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Data collection

DiffractionMean temperature: 100 K
Crystal treatment: flash cooled by immersion in liquid nitrogen
Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-D / Wavelength: 0.9762 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Feb 10, 2021
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9762 Å / Relative weight: 1
ReflectionResolution: 1.139→72.655 Å / Num. obs: 214260 / % possible obs: 89.6 % / Redundancy: 32.75 % / Biso Wilson estimate: 11.85 Å2
Details: merged and scaled data post-processed by STARANISO for conversion from intensities to structure factor amplitudes
CC1/2: 0.996 / Rmerge(I) obs: 0.143 / Rpim(I) all: 0.0248 / Rrim(I) all: 0.1452 / Net I/σ(I): 15.13 / Num. measured all: 7017400
Reflection shellResolution: 1.139→1.204 Å / Redundancy: 23.24 % / Rmerge(I) obs: 1.5681 / Mean I/σ(I) obs: 2.17 / Num. measured all: 247895 / Num. measured obs: 247895 / Num. unique all: 10668 / Num. unique obs: 10668 / CC1/2: 0.727 / Rpim(I) all: 0.3222 / Rrim(I) all: 1.6026 / % possible all: 42.5

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX1.20.1refinement
autoPROC1.0.5 20201211data processing
XDSJan 31, 2020data reduction
Aimless0.7.4data scaling
STARANISO2.3.54data scaling
PHASER2.8.3phasing
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7UX6

7ux6


Resolution: 1.14→35.18 Å / SU ML: 0.08 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 13.04 / Stereochemistry target values: ML
Details: 1. HYDROGENS HAVE BEEN INCLUDED AT THEIR RIDING POSITIONS USING THE ELECTRON CLOUD DISTANCES. 2. THE STRUCTURE CONTAINS AND UNKNOWN LIGAND UNL BOUND IN THE ACTIVE SITE. THE COMPOUND IS A ...Details: 1. HYDROGENS HAVE BEEN INCLUDED AT THEIR RIDING POSITIONS USING THE ELECTRON CLOUD DISTANCES. 2. THE STRUCTURE CONTAINS AND UNKNOWN LIGAND UNL BOUND IN THE ACTIVE SITE. THE COMPOUND IS A METABOLITE THAT CO-PURIFIED WITH THE PROTEIN. THE STRUCTURE LOOKS SIMILAR TO NIACIN, NICOTINAMIDE, BENZOATE, NITORBENZENE ETC. SINCE THE PRECIENSE SPECIES IS NOT KNOW, IT WAS MODELED AS AN UNL. 3. THE C-TERMINUS OF CHAIN A PACKS AGAINST CHAIN B FROM A SYMMETRY MATE IN THE UNIT CELL. THIS SHIFTS THE POSITION OF SEVERAL RESIDUES IN CHAIN B TO ACCOMMODATE THE C-TERMINAL TAG REGION. HOWEVER, THE INTERACTION IS NOT COMPLETE. AFTER MODELING THIS MAJOR SHIFTED PORTION, THERE IS RESIDUAL DIFFERENCE DENSITY FOR A CONFORMATION THAT IS SIMILAR TO THE WHAT IS SEEN IN CHAIN A AND OTHER CRYSTAL FORMS. OCCUPANCY REFINEMENT SUGGESTED ABOUT 60:40 SPLIT. SINCE ONLY THE MAJOR PORTION OF THE CHAIN A C-TERMINUS (367-383)COULD BE MODELED, THIS WAS FIXED AT 0.6 OCCUPANCY TO MATCH THE CHAIN B PORTION THAT IT INTERACTS WITH, WHILE THE OTHER 0.4 OCCUPANCY PORTION COULD NOT BE MODELED DUE TO DISORDER. 4. EVEN THOUGH THE CRYSTALLIZATION DROPS WERE SETUP WITH S-ADENOSYLMETHIONINE (SAM/ ADOMET), ELECTRON DENSITY CLEARLY SHOWS THAT THE COFACTOR HAS BROKEN DOWN TO S-ADENOSYL-L-HOMOCYSTEINE (SAH/ADOHCY). 5. THE DATA WERE PROCESSED WITH STARTANISO DUE TO THE ANISOTROPIC RESOLUTION EXTENT.
RfactorNum. reflection% reflection
Rfree0.142 10679 4.98 %
Rwork0.119 --
obs0.121 214231 83.4 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 18.98 Å2
Refinement stepCycle: LAST / Resolution: 1.14→35.18 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5690 0 70 861 6621
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0136505
X-RAY DIFFRACTIONf_angle_d1.358927
X-RAY DIFFRACTIONf_dihedral_angle_d13.9682387
X-RAY DIFFRACTIONf_chiral_restr0.099964
X-RAY DIFFRACTIONf_plane_restr0.0151232
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.14-1.150.2688640.22341198X-RAY DIFFRACTION15
1.15-1.170.2286790.22191606X-RAY DIFFRACTION20
1.17-1.180.21681160.21222269X-RAY DIFFRACTION28
1.18-1.20.19361760.20133029X-RAY DIFFRACTION38
1.2-1.210.20922060.19783855X-RAY DIFFRACTION48
1.21-1.230.19132540.18284522X-RAY DIFFRACTION56
1.23-1.240.19882630.17265273X-RAY DIFFRACTION65
1.24-1.260.19383630.16716457X-RAY DIFFRACTION80
1.26-1.280.18683880.1537374X-RAY DIFFRACTION91
1.28-1.30.1633880.13657687X-RAY DIFFRACTION95
1.3-1.330.17044060.1297769X-RAY DIFFRACTION96
1.33-1.350.16884260.12367780X-RAY DIFFRACTION96
1.35-1.380.1654050.11867818X-RAY DIFFRACTION97
1.38-1.410.13984000.11077829X-RAY DIFFRACTION97
1.41-1.440.13184100.10697825X-RAY DIFFRACTION97
1.44-1.470.12814260.10447868X-RAY DIFFRACTION97
1.47-1.510.12794160.09687914X-RAY DIFFRACTION97
1.51-1.550.12914220.09447883X-RAY DIFFRACTION97
1.55-1.590.12014290.08857929X-RAY DIFFRACTION98
1.59-1.640.13454240.09187970X-RAY DIFFRACTION98
1.64-1.70.11824330.09227968X-RAY DIFFRACTION98
1.7-1.770.11893950.09277984X-RAY DIFFRACTION98
1.77-1.850.13364220.09688067X-RAY DIFFRACTION99
1.85-1.950.13254400.09958056X-RAY DIFFRACTION99
1.95-2.070.12753970.10238109X-RAY DIFFRACTION99
2.07-2.230.12084350.1018124X-RAY DIFFRACTION99
2.23-2.450.13154410.10458172X-RAY DIFFRACTION99
2.45-2.810.13274210.11928229X-RAY DIFFRACTION100
2.81-3.540.15454300.13358343X-RAY DIFFRACTION100
3.54-35.180.15174040.14558645X-RAY DIFFRACTION100

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