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- PDB-5h81: HETEROYOHIMBINE SYNTHASE THAS2 FROM CATHARANTHUS ROSEUS - COMPLEX... -

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Basic information

Entry
Database: PDB / ID: 5h81
TitleHETEROYOHIMBINE SYNTHASE THAS2 FROM CATHARANTHUS ROSEUS - COMPLEX WITH NADP+
Componentsheteroyohimbine synthase THAS2
KeywordsOXIDOREDUCTASE / heteroyohimbine synthase / medium chain dehydrogenase/reductase / NADP+ dependent enzyme / zinc binding site
Function / homology
Function and homology information


alkaloid metabolic process / oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor / nucleotide binding / metal ion binding
Similarity search - Function
: / Quinone Oxidoreductase; Chain A, domain 1 / Medium-chain alcohol dehydrogenases, catalytic domain / Alcohol dehydrogenase-like, C-terminal / Zinc-binding dehydrogenase / Alcohol dehydrogenase, N-terminal / Alcohol dehydrogenase GroES-like domain / GroES-like superfamily / NAD(P)-binding Rossmann-like Domain / NAD(P)-binding domain superfamily ...: / Quinone Oxidoreductase; Chain A, domain 1 / Medium-chain alcohol dehydrogenases, catalytic domain / Alcohol dehydrogenase-like, C-terminal / Zinc-binding dehydrogenase / Alcohol dehydrogenase, N-terminal / Alcohol dehydrogenase GroES-like domain / GroES-like superfamily / NAD(P)-binding Rossmann-like Domain / NAD(P)-binding domain superfamily / Alpha-Beta Complex / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Heteroyohimbine synthase THAS2
Similarity search - Component
Biological speciesCatharanthus roseus (Madagascar periwinkle)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsStavrinides, A. / Tatsis, E.C. / Caputi, L. / Foureau, E. / Stevenson, C.E.M. / Lawson, D.M. / Courdavault, V. / O'Connor, S.E.
Funding support United Kingdom, France, 4items
OrganizationGrant numberCountry
European Research Council311363 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/J004561/1 United Kingdom
Region CentreABISAL grant France
Biotechnology and Biological Sciences Research CouncilDoctoral Training Partnership United Kingdom
CitationJournal: Nat Commun / Year: 2016
Title: Structural investigation of heteroyohimbine alkaloid synthesis reveals active site elements that control stereoselectivity.
Authors: Stavrinides, A. / Tatsis, E.C. / Caputi, L. / Foureau, E. / Stevenson, C.E. / Lawson, D.M. / Courdavault, V. / O'Connor, S.E.
History
DepositionDec 23, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Jul 27, 2016Provider: repository / Type: Initial release
Revision 1.1Jan 10, 2024Group: Author supporting evidence / Data collection ...Author supporting evidence / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_audit_support / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: heteroyohimbine synthase THAS2
B: heteroyohimbine synthase THAS2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)86,9868
Polymers85,2382
Non-polymers1,7486
Water5,242291
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4900 Å2
ΔGint-24 kcal/mol
Surface area27930 Å2
MethodPISA
Unit cell
Length a, b, c (Å)70.960, 83.790, 117.160
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Component-ID: _ / Ens-ID: 1 / Beg auth comp-ID: PRO / Beg label comp-ID: PRO / End auth comp-ID: SER / End label comp-ID: SER / Refine code: _ / Auth seq-ID: 8 - 370 / Label seq-ID: 26 - 388

Dom-IDAuth asym-IDLabel asym-ID
1AA
2BB

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Components

#1: Protein heteroyohimbine synthase THAS2


Mass: 42618.859 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Native N-terminal MET is replaced by an affinity tag with the sequence MAHHHHHHSSGLEVLFQGP.
Source: (gene. exp.) Catharanthus roseus (Madagascar periwinkle)
Gene: Cr_021691 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): SOLUBL21 / References: UniProt: A0A1C7D193*PLUS
#2: Chemical ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE


Mass: 743.405 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H28N7O17P3
#3: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 291 / Source method: isolated from a natural source / Formula: H2O

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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: 2.04 Å3/Da / Density % sol: 39.8 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / Details: NULL

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04-1 / Wavelength: 0.92 Å
DetectorType: DECTRIS PILATUS 2M / Detector: PIXEL / Date: Aug 1, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.92 Å / Relative weight: 1
ReflectionResolution: 2.1→35.48 Å / Num. obs: 41492 / % possible obs: 99.9 % / Redundancy: 7.4 % / Biso Wilson estimate: 26.8 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.106 / Rpim(I) all: 0.041 / Net I/σ(I): 16.5 / Num. measured all: 307785
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allCC1/2Rpim(I) all% possible all
2.1-2.157.60.9052.42330930540.7250.34999.8
9.39-35.486.70.02665.5362454110.0198.5

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Processing

Software
NameVersionClassification
Aimless0.3.6data scaling
REFMAC5.8.0135refinement
PDB_EXTRACT3.15data extraction
PHASERphasing
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5FI3

5fi3


Resolution: 2.1→35.48 Å / Cor.coef. Fo:Fc: 0.954 / Cor.coef. Fo:Fc free: 0.927 / WRfactor Rfree: 0.2016 / WRfactor Rwork: 0.1622 / FOM work R set: 0.836 / SU B: 10.137 / SU ML: 0.141 / SU R Cruickshank DPI: 0.2306 / SU Rfree: 0.1876 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.231 / ESU R Free: 0.188 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2319 2110 5.1 %RANDOM
Rwork0.1863 ---
obs0.1885 39326 99.82 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 101.44 Å2 / Biso mean: 35.5 Å2 / Biso min: 14.94 Å2
Baniso -1Baniso -2Baniso -3
1-1.2 Å20 Å20 Å2
2---0.73 Å20 Å2
3----0.47 Å2
Refinement stepCycle: final / Resolution: 2.1→35.48 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5242 0 100 291 5633
Biso mean--45.03 37.11 -
Num. residues----690
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0195481
X-RAY DIFFRACTIONr_bond_other_d0.0050.025159
X-RAY DIFFRACTIONr_angle_refined_deg1.4691.9657438
X-RAY DIFFRACTIONr_angle_other_deg1.2233.00211868
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.3875685
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.68924210
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.65915875
X-RAY DIFFRACTIONr_dihedral_angle_4_deg13.2041522
X-RAY DIFFRACTIONr_chiral_restr0.0980.2834
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.0216282
X-RAY DIFFRACTIONr_gen_planes_other0.0060.021206
X-RAY DIFFRACTIONr_mcbond_it1.3681.632755
X-RAY DIFFRACTIONr_mcbond_other1.3681.632754
X-RAY DIFFRACTIONr_mcangle_it2.0712.4373435
Refine LS restraints NCS

Ens-ID: 1 / Number: 39776 / Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Rms dev position: 0.07 Å / Weight position: 0.05

Dom-IDAuth asym-ID
1A
2B
LS refinement shellResolution: 2.1→2.154 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.32 169 -
Rwork0.262 2871 -
all-3040 -
obs--99.87 %
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)
13.5975-0.05480.08182.76091.37652.7330.0622-0.21660.18980.1866-0.0152-0.015-0.233-0.2456-0.0470.10580.0618-0.01860.0815-0.00950.048-13.368133.7854-12.3673
20.45930.17910.25874.0662-1.84094.7401-0.0093-0.12250.1390.39920.2669-0.7727-0.37150.3974-0.25760.13990.0238-0.04650.2144-0.17170.42382.671535.292-6.5716
31.7259-1.24860.29092.7179-0.10921.1744-0.0023-0.1490.06870.210.1322-0.50450.06970.1795-0.12990.0970.0428-0.08460.1108-0.05510.20870.867421.0605-11.7673
42.5042-0.37461.53143.9426-0.7014.7730.1095-0.0021-0.138-0.23480.08530.24730.1811-0.3435-0.19480.18170.0542-0.09830.1163-0.03850.243-3.020.0211-15.8694
50.2250.0701-0.63676.3417-3.31483.9318-0.0578-0.106-0.01840.34060.1036-0.36080.05080.0607-0.04570.08740.0892-0.13420.233-0.06710.30754.100111.4953-6.647
66.3245-1.1950.3521.90770.50123.52560.12960.094-0.2982-0.2538-0.1131-0.23380.0711-0.1661-0.01650.12210.0498-0.00050.1008-0.01070.0847-11.291325.3452-24.5076
74.7299-0.04620.97811.9455-0.75962.1581-0.04070.0861-0.2233-0.3635-0.0457-0.19640.22150.11490.08640.10050.01330.04750.0420.01970.0411-3.8228-31.733414.301
84.15572.9713-3.80014.2538-2.23953.6083-0.21930.27830.1809-0.60940.2880.19680.163-0.2265-0.06870.24580.00440.04730.13490.02790.1490.2047-19.86280.3079
91.35590.7029-0.18524.4903-0.0130.89010.014-0.15040.16480.0598-0.16410.2072-0.07580.04160.15020.0107-0.0246-0.00940.1469-0.02770.0587-2.0953-11.821719.3467
106.73482.1922-1.44466.5276-0.9783.4010.03040.30020.5485-0.2833-0.04691.2473-0.3952-0.74670.01660.19540.0784-0.10090.2873-0.08640.4017-10.24685.411813.4434
110.9762-0.3277-0.17756.5114-2.65312.81090.06730.05440.1512-0.2123-0.10670.2189-0.207-0.16090.03940.0806-0.0159-0.00780.1577-0.04420.10971.4453-2.662711.1396
125.97862.37520.43673.27570.35783.94590.0113-0.20530.19680.0855-0.06050.1641-0.1273-0.20570.04910.04760.00170.00110.11740.00730.012-12.7488-23.475524.5751
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A8 - 72
2X-RAY DIFFRACTION2A73 - 108
3X-RAY DIFFRACTION3A109 - 220
4X-RAY DIFFRACTION4A221 - 300
5X-RAY DIFFRACTION5A301 - 330
6X-RAY DIFFRACTION6A331 - 370
7X-RAY DIFFRACTION7B8 - 97
8X-RAY DIFFRACTION8B98 - 133
9X-RAY DIFFRACTION9B134 - 246
10X-RAY DIFFRACTION10B247 - 278
11X-RAY DIFFRACTION11B279 - 330
12X-RAY DIFFRACTION12B331 - 370

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