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- PDB-8dq8: The structure of NicA2 variant F104L/A107T/S146I/G317D/H368R/L449... -

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Basic information

Entry
Database: PDB / ID: 8dq8
TitleThe structure of NicA2 variant F104L/A107T/S146I/G317D/H368R/L449V/N462S in complex with N-methylmyosmine
ComponentsAmine oxidase
KeywordsFLAVOPROTEIN / nicotine-degrading enzyme
Function / homology
Function and homology information


nicotine dehydrogenase / nicotine catabolic process / alkaloid metabolic process / periplasmic space / oxidoreductase activity / nucleotide binding
Similarity search - Function
Flavin amine oxidase / Amine oxidase / Flavin containing amine oxidoreductase / Twin arginine translocation (Tat) signal profile. / Twin-arginine translocation pathway, signal sequence / FAD/NAD(P)-binding domain superfamily
Similarity search - Domain/homology
DIHYDROFLAVINE-ADENINE DINUCLEOTIDE / (S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE / Nicotine dehydrogenase
Similarity search - Component
Biological speciesPseudomonas putida S16 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.9 Å
AuthorsWu, K. / Dulchavsky, M. / Li, J. / Bardwell, J.C.A.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat.Chem.Biol. / Year: 2023
Title: Directed evolution unlocks oxygen reactivity for a nicotine-degrading flavoenzyme.
Authors: Dulchavsky, M. / Mitra, R. / Wu, K. / Li, J. / Boer, K. / Liu, X. / Zhang, Z. / Vasquez, C. / Clark, C.T. / Funckes, K. / Shankar, K. / Bonnet-Zahedi, S. / Siddiq, M. / Sepulveda, Y. / ...Authors: Dulchavsky, M. / Mitra, R. / Wu, K. / Li, J. / Boer, K. / Liu, X. / Zhang, Z. / Vasquez, C. / Clark, C.T. / Funckes, K. / Shankar, K. / Bonnet-Zahedi, S. / Siddiq, M. / Sepulveda, Y. / Suhandynata, R.T. / Momper, J.D. / Calabrese, A.N. / George, O. / Stull, F. / Bardwell, J.C.A.
History
DepositionJul 18, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 26, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Nov 8, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Amine oxidase
B: Amine oxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,3758
Polymers95,3522
Non-polymers2,0246
Water10,863603
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7270 Å2
ΔGint-9 kcal/mol
Surface area27930 Å2
MethodPISA
Unit cell
Length a, b, c (Å)86.270, 86.270, 122.470
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number78
Space group name H-MP43
Space group name HallP4cw
Symmetry operation#1: x,y,z
#2: -y,x,z+3/4
#3: y,-x,z+1/4
#4: -x,-y,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 51 through 212 or resid 214...
d_2ens_1(chain "B" and (resid 51 through 136 or (resid 137...

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1GLYASNA1 - 162
d_12ens_1TYRTRPA164 - 190
d_13ens_1TYRGLYA192 - 238
d_14ens_1LYSVALA240 - 253
d_15ens_1VALLYSA255 - 352
d_16ens_1PHESERA354 - 432
d_17ens_1NMMNMMB1
d_18ens_1FDAFDAC
d_19ens_1EDOEDOD
d_21ens_1GLYASNE1 - 162
d_22ens_1TYRTRPE164 - 190
d_23ens_1TYRGLYE192 - 238
d_24ens_1LYSVALE240 - 253
d_25ens_1VALLYSE255 - 352
d_26ens_1PHESERE354 - 432
d_27ens_1NMMNMMF1
d_28ens_1FDAFDAG
d_29ens_1EDOEDOH

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Components

#1: Protein Amine oxidase


Mass: 47675.852 Da / Num. of mol.: 2 / Mutation: F104L, A107T, S146I,G317D, H368R, L449V, N462S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas putida S16 (bacteria) / Strain: S16 / Gene: PPS_4081 / Production host: Escherichia coli (E. coli) / References: UniProt: F8G0P2
#2: Chemical ChemComp-NCT / (S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE / (S)-(-)-NICOTINE / 3-[(2S)-1-METHYL-2-PYRROLIDINYL] PYRIDINE


Mass: 162.232 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H14N2 / Feature type: SUBJECT OF INVESTIGATION / Comment: alkaloid*YM
#3: Chemical ChemComp-FDA / DIHYDROFLAVINE-ADENINE DINUCLEOTIDE


Mass: 787.566 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: C27H35N9O15P2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 603 / 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: 2.39 Å3/Da / Density % sol: 48.53 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop
Details: 80 mM sodium citrate tribasic dihydrate pH 5.0, 18% PEG 6000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-G / Wavelength: 0.9787 Å
DetectorType: RAYONIX MX300-HS / Detector: CCD / Date: Feb 4, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9787 Å / Relative weight: 1
ReflectionResolution: 1.9→32.64 Å / Num. obs: 70427 / % possible obs: 100 % / Redundancy: 7.6 % / Biso Wilson estimate: 20.07 Å2 / CC1/2: 0.999 / Net I/σ(I): 15.9
Reflection shellResolution: 1.9→1.94 Å / Mean I/σ(I) obs: 4.2 / Num. unique obs: 4531 / CC1/2: 0.917

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
MOSFLMdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7c4a

7c4a


Resolution: 1.9→32.64 Å / SU ML: 0.1664 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 17.2178
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1767 3521 5 %
Rwork0.14 66846 -
obs0.1418 70367 99.98 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 21.77 Å2
Refinement stepCycle: LAST / Resolution: 1.9→32.64 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6640 0 138 603 7381
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01616974
X-RAY DIFFRACTIONf_angle_d1.18679491
X-RAY DIFFRACTIONf_chiral_restr0.08831047
X-RAY DIFFRACTIONf_plane_restr0.01081218
X-RAY DIFFRACTIONf_dihedral_angle_d10.8858972
Refine LS restraints NCSType: Torsion NCS / Rms dev position: 0.606563498219 Å
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.9-1.930.22491460.17262649X-RAY DIFFRACTION100
1.93-1.950.22431640.17062657X-RAY DIFFRACTION100
1.95-1.980.16811280.14542658X-RAY DIFFRACTION100
1.98-2.010.17511640.14142659X-RAY DIFFRACTION100
2.01-2.050.17171560.13572663X-RAY DIFFRACTION100
2.05-2.080.22881470.13792644X-RAY DIFFRACTION100
2.08-2.120.18461430.14122680X-RAY DIFFRACTION100
2.12-2.160.19821180.13352676X-RAY DIFFRACTION100
2.16-2.20.2291190.13992688X-RAY DIFFRACTION100
2.2-2.250.17641120.13682688X-RAY DIFFRACTION100
2.25-2.310.15271600.13892684X-RAY DIFFRACTION100
2.31-2.360.19621330.1432623X-RAY DIFFRACTION100
2.36-2.430.21141240.14612721X-RAY DIFFRACTION100
2.43-2.50.18171360.14242675X-RAY DIFFRACTION99.96
2.5-2.580.17671840.1452635X-RAY DIFFRACTION100
2.58-2.670.17611050.14392684X-RAY DIFFRACTION100
2.67-2.780.16691190.14212716X-RAY DIFFRACTION100
2.78-2.90.21721570.14752638X-RAY DIFFRACTION100
2.9-3.060.19961500.14632672X-RAY DIFFRACTION100
3.06-3.250.16191350.13992690X-RAY DIFFRACTION100
3.25-3.50.16861370.13082689X-RAY DIFFRACTION100
3.5-3.850.16351260.13122688X-RAY DIFFRACTION100
3.85-4.410.15671790.12552659X-RAY DIFFRACTION99.96
4.41-5.550.15351440.13072687X-RAY DIFFRACTION99.93
5.55-32.640.15991350.15722723X-RAY DIFFRACTION99.62
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.520665162730.3103776493960.256458936830.6862600701380.05462442901671.05999201192-0.05079625414690.1909461862370.144426969865-0.020856672656-0.0122945551155-0.00429059195584-0.1888887430720.1151308572390.08317041745440.136518641688-0.00702130189327-0.01817741518620.1077673440070.03795180406510.11202133718743.0242121385-2.88700106854-4.93240617822
20.5145099996790.3182166110480.4896961619560.4829850448610.2580909050330.7724125153990.040696276028-0.00778715151736-0.03191441673880.0289970676684-0.04596857519950.06809885605360.0809193624707-0.03384943462220.01593326870260.1381657295590.00478393035708-0.00100844342390.151738530019-0.007476254352480.17448177322824.5874554603-19.3685570338-0.799054649539
33.43181036762-0.7957611529761.601094241281.16116509749-0.383453277092.132870879160.1383661035730.0468936665949-0.531938534557-0.04487622339550.04072244828930.1309536069150.119339653697-0.0711615072713-0.1791089974120.1813392542990.00969101172506-0.05341707009470.120665311403-0.03907534323320.22304662722728.1319358105-31.9429654102-5.39872545873
41.15785913905-0.0272576808918-0.07661504360080.585706773178-0.08193002996770.6352677969210.05089628410990.122597436150.0472993505281-0.0395284766179-0.06136792873260.00637280315448-0.03909980506870.08828211304330.007196918181540.1322461661290.00600171977887-0.03534178408410.1327843106650.007714496109070.092213874590340.3062445267-11.440067814-3.51038810684
51.579457986050.3283546877880.3176981462490.443346839711-0.02360414297350.428750820263-0.06097752179870.163923122144-0.0210074867808-0.0964447877919-0.0339712594404-0.0279607655382-0.07749782540850.1598430527220.08280003421540.1411167117610.0051476667197-0.01060066507640.204754014115-0.007388117015360.1042193529150.5654753131-14.7347717407-10.7371988039
62.38429590246-0.7098067064560.1186126440073.503428817220.1449944388612.32636116073-0.1469786207410.2463239674270.188868325937-0.4139416496710.1774639861810.0883882592689-0.0603921519370.107274896696-0.03355927318750.171537954596-0.0517016015943-0.05085663110130.1541972014010.01472015584560.14990174497422.1896926516-14.5491483078-19.5531314777
71.057671768740.01539655397060.4970948997671.05533283941-0.3347931095730.7566115548370.04654624686740.171225280953-0.0687415389462-0.0802124139634-0.00158641878322-0.01390840932610.05246566069330.119130911732-0.03410975995960.09637868685270.0158205924332-0.001431181756330.12783884756-0.002981812343230.097856090606442.8950069148-14.8700394497-4.81786576642
80.9291889970670.36382684084-0.035067327861.32559883108-0.3205585670741.04901525013-0.00343917029703-0.0260103087577-0.0003395127068190.165567652889-0.0541493298128-0.1120586004-0.1107562566930.1972381601720.07139571018650.109637336715-0.00720677902086-0.03445844478390.1292975360550.01918093601480.11134730386140.2361942615-0.061759589897524.715832131
90.5730007029030.261233517128-0.3204136427480.597978193603-0.6575333278031.04261375271-0.0647289288640.00891777439121-0.0834758045358-0.008581257662990.06798660110970.04341839963750.0501417648915-0.1078678856520.006086694008140.15489185256-0.002447249561480.007350381936910.130008022439-0.009126232409050.16792715514223.7475450057-18.867492618520.7048046184
102.41880769539-0.5944538418870.2611185988474.11988860983-1.183024770071.961700806880.00278006646206-0.0274413699581-0.0848692575227-9.87631114756E-50.2032313151950.4969459275830.0206308454692-0.19006684505-0.2147121048280.1130382656440.007833699528550.03686782561550.1860595481760.04465031789080.19616958411710.9564341735-14.281804720424.3868098955
110.4620136574230.003454431380990.09843821906211.237407498060.06940539404530.664228306916-0.0569955110606-0.0394759416125-0.001246689922910.1259037430580.0497519612633-0.0626266453915-0.0930707177170.04893196797390.007772513067390.133675009982-0.00101592966795-0.01053514506880.1391905658370.0321269291410.10918936521631.7448027291-2.5484655758423.2944137888
120.72697843730.3853177379350.03653896506050.872180575821-0.267411239110.4589254450130.0324952470272-0.127589851384-0.02609678125030.229185823465-0.06574271003540.00337772279747-0.093458100260.01013961557830.02717114774530.1583528169590.01052923662750.00957276849270.1363472203010.005818459650960.1075918008828.4625993991-4.7617233795530.9516345892
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 51 through 113 )AA51 - 1131 - 63
22chain 'A' and (resid 114 through 183 )AA114 - 18364 - 133
33chain 'A' and (resid 184 through 219 )AA184 - 219134 - 169
44chain 'A' and (resid 220 through 293 )AA220 - 293170 - 243
55chain 'A' and (resid 294 through 345 )AA294 - 345244 - 295
66chain 'A' and (resid 346 through 403 )AA346 - 403296 - 353
77chain 'A' and (resid 404 through 482 )AA404 - 482354 - 432
88chain 'B' and (resid 51 through 113 )BE51 - 1131 - 63
99chain 'B' and (resid 114 through 183 )BE114 - 18364 - 133
1010chain 'B' and (resid 184 through 219 )BE184 - 219134 - 169
1111chain 'B' and (resid 220 through 293 )BE220 - 293170 - 243
1212chain 'B' and (resid 294 through 482 )BE294 - 482244 - 432

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