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- PDB-8dq7: The structure of NicA2 variant F104L/A107T/S146I/G317D/H368R/L449... -

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Basic information

Entry
Database: PDB / ID: 8dq7
TitleThe structure of NicA2 variant F104L/A107T/S146I/G317D/H368R/L449V/N462S from Pseudomonas putida
ComponentsAmine oxidase
KeywordsFLAVOPROTEIN / nicotine-degrading enzyme
Function / homology
Function and homology information


nicotine dehydrogenase / nicotine catabolic process / alkaloid metabolic process / oxidoreductase activity / periplasmic space / 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 / Nicotine dehydrogenase
Similarity search - Component
Biological speciesPseudomonas putida S16 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 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

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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)96,9274
Polymers95,3522
Non-polymers1,5752
Water8,035446
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5980 Å2
ΔGint-27 kcal/mol
Surface area27890 Å2
MethodPISA
Unit cell
Length a, b, c (Å)85.510, 85.510, 122.390
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

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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-FDA / DIHYDROFLAVINE-ADENINE DINUCLEOTIDE


Mass: 787.566 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H35N9O15P2 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 446 / 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: 2.12 Å3/Da / Density % sol: 47.58 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop
Details: 100 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: 2.1→42.76 Å / Num. obs: 51280 / % possible obs: 99.9 % / Redundancy: 7.6 % / Biso Wilson estimate: 26.8 Å2 / CC1/2: 0.999 / Net I/σ(I): 17.6
Reflection shellResolution: 2.1→2.16 Å / Mean I/σ(I) obs: 4.3 / Num. unique obs: 4187 / 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: 2.1→42.76 Å / SU ML: 0.1931 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 20.0747
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2001 2550 4.98 %
Rwork0.151 48670 -
obs0.1534 51220 99.96 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 29.18 Å2
Refinement stepCycle: LAST / Resolution: 2.1→42.76 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6629 0 106 446 7181
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01236919
X-RAY DIFFRACTIONf_angle_d1.05099416
X-RAY DIFFRACTIONf_chiral_restr0.06341040
X-RAY DIFFRACTIONf_plane_restr0.00841211
X-RAY DIFFRACTIONf_dihedral_angle_d10.6809950
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1-2.140.26941480.18012694X-RAY DIFFRACTION100
2.14-2.180.23581320.17332700X-RAY DIFFRACTION100
2.18-2.230.2241620.16412670X-RAY DIFFRACTION100
2.23-2.280.23351090.15532709X-RAY DIFFRACTION100
2.28-2.340.22871450.15692684X-RAY DIFFRACTION100
2.34-2.40.21961260.15072721X-RAY DIFFRACTION99.96
2.4-2.470.20821350.14872707X-RAY DIFFRACTION100
2.47-2.550.19071370.15342708X-RAY DIFFRACTION100
2.55-2.650.21011220.1512730X-RAY DIFFRACTION100
2.65-2.750.21531490.15712698X-RAY DIFFRACTION100
2.75-2.880.2221640.15672681X-RAY DIFFRACTION100
2.88-3.030.18211360.15112692X-RAY DIFFRACTION100
3.03-3.220.19811250.14612715X-RAY DIFFRACTION100
3.22-3.470.21371530.14532709X-RAY DIFFRACTION100
3.47-3.810.18241460.14312693X-RAY DIFFRACTION100
3.82-4.370.19031480.1372721X-RAY DIFFRACTION99.97
4.37-5.50.171500.14422706X-RAY DIFFRACTION99.9
5.5-42.760.19251630.16512732X-RAY DIFFRACTION99.52
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)
10.262863913736-0.1587500021990.4032833373820.399323097011-0.06664900002380.4784725361020.03262537590940.09783573303230.1235179532220.00161486999315-0.05657766334230.0639477298316-0.1711537183450.04319362474272.8121493919E-50.207523087648-0.00860169342609-0.02480783382940.194852893670.01543885579520.16894840711342.575417989-2.62522956-5.00649825498
20.1170438950690.3671522642730.3315313375480.1933749905830.6419671527470.6741352351930.0753187356838-0.0428490547864-0.03295427618450.0802065576655-0.1348303871480.02533710305510.147711046963-0.141479466509-0.0001005058424680.220116486991-0.0248916695154-0.02465006974280.175886197807-0.008644291418130.21415175109324.1728698069-19.1761639919-1.01429584454
30.163983638867-0.238368150111-0.001988230575030.281952073880.1067508901680.1416733327540.1150284755710.061360349987-0.2911200267560.0749671576252-0.04575672569550.08989048413360.00607268651116-0.09725132013010.0001412681570710.3105588760920.00136587132791-0.1025682384870.241690526878-0.04152183598310.32591185800227.943945576-31.7710126111-5.54746454219
40.81480314417-0.137986881489-0.05617702506920.37303629636-0.2200077031210.6318808278660.08990973498570.155138695592-0.00447681562804-0.0505300318146-0.05264384305570.0569507457651-0.003663443275230.01771848735572.46483741556E-50.2277228845687.16561475497E-5-0.05595515214640.1742845504150.01402069337790.17749055623739.8395648271-11.298737326-3.5342779791
50.3310172655820.5551982271860.2382808866110.620431409535-0.20706639350.519566466305-0.05940138689240.0662524047830.135306721242-0.0318339755904-0.01300068814260.138354998174-0.02811099275640.105031546464-0.02860933384990.1906950794420.0294618739497-0.01961490228650.183447238879-0.04265787903950.18529798543339.0215330695-14.3910300496-12.5508503785
60.09550167159390.05399797050480.146784181210.3052146567640.2611561575481.1046260048-0.1348117976110.3441410173150.45096682338-1.058451692910.5116773692770.265049463758-0.1434436592230.3279390039450.09562591345650.387111085015-0.093871642523-0.1084673250440.2938573492810.02697941920040.41076366579124.2469967676-14.4209203411-23.4743437365
70.3551373726740.01897955043610.04480930744841.23456752675-0.4167420586810.06431022089310.1390437557540.108929284044-0.0989667868536-0.0386072330664-0.02220522958830.0450160134040.129476150770.09513988731590.03318005535240.1454925151380.0239035837644-0.02684947111740.183586679802-0.01505646879940.16813963068842.672096997-14.5785590643-4.86974113822
80.539582753034-0.1633514880010.1251683646840.231996407931-0.2777351798710.424859965102-0.0378771412023-0.0205333613332-0.05985248352080.1001057796250.0277012579628-0.10605567946-0.06725102059720.1249818549974.54890561247E-50.187538908842-0.012246355189-0.01874385446440.1925792368010.02574072934290.16459382027440.02372920170.011514230565324.5892352221
90.3260909440770.345046075777-0.6462792963910.163749041949-0.178768165570.493530010188-0.1375447631370.104830414851-0.0215904684599-0.02901362949560.06320203816570.02756599111980.134014031979-0.131714626426-4.06936517696E-60.189117719681-0.02650905687440.00703970914870.2366564207070.01891808667130.22080888467923.5171589218-18.748011563220.7018951597
100.394445993718-0.201770164923-0.07743020292310.2464120216380.1705921291320.246370342137-0.0121898766670.1681510899270.02425929696350.0004528441256580.07992428832020.3005487416650.0575783529632-0.0512015674731-5.72121048915E-50.244212902433-0.01022119078370.0314321891470.3235270100940.1138215059730.32776788379810.7512928628-14.082267421324.4382558829
110.3854787354140.05956299105850.2649476178480.640559603083-0.05427373507690.617432920134-0.0451874720594-0.096192351939-0.0382184740090.1588503765710.07415209079330.00611445568978-0.0324803696043-0.009873516928273.00501932594E-50.1856798631620.00993402713757-0.01033173969790.2400066281580.04339169461710.18745075669831.4590667208-2.9221594624823.2438376776
120.8990529088820.8893258120690.2828172544820.776014001762-0.4094621165010.5137948998380.107009348862-0.124325525978-0.2028800664130.16104610097-0.170783944753-0.162626629628-0.08869665364580.0243057942399-0.02513232438950.2244295545710.01043474153580.04971474787710.2277572117970.02948985727240.20861008884128.4391836261-8.054975186335.3595573364
131.312034817530.06025010186620.4658323968320.404305179023-0.1128573467590.111320814475-0.0220327236336-0.0362653766148-0.06910161988820.1048148656680.1084184238490.0949587245058-0.096336291156-0.146160993550.02264183015790.1809539998390.02867895818170.01633641032490.150669223390.02107048466240.17349343052228.2446695613-0.29836065172524.7687107834
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 371 )AA294 - 371244 - 321
66chain 'A' and (resid 372 through 403 )AA372 - 403322 - 353
77chain 'A' and (resid 404 through 482 )AA404 - 482354 - 432
88chain 'B' and (resid 51 through 113 )BC51 - 1131 - 63
99chain 'B' and (resid 114 through 183 )BC114 - 18364 - 133
1010chain 'B' and (resid 184 through 219 )BC184 - 219134 - 169
1111chain 'B' and (resid 220 through 293 )BC220 - 293170 - 243
1212chain 'B' and (resid 294 through 403 )BC294 - 403244 - 353
1313chain 'B' and (resid 404 through 482 )BC404 - 482354 - 432

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