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- PDB-8kbw: The crystal structure of syn-copalyl diphosphate synthase from Or... -

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Basic information

Entry
Database: PDB / ID: 8kbw
TitleThe crystal structure of syn-copalyl diphosphate synthase from Oryza sativa
ComponentsSyn-copalyl diphosphate synthase, chloroplastic
KeywordsISOMERASE / syn-copalyl diphosphate synthase / labdane-related diterpenoids / apo-state crystal structure / plant defense / PLANT PROTEIN
Function / homology
Function and homology information


syn-copalyl-diphosphate synthase / syn-copalyl diphosphate synthase activity / gibberellin biosynthetic process / terpene synthase activity / chloroplast / defense response / magnesium ion binding
Similarity search - Function
Terpene synthase, N-terminal domain / Terpene synthase, N-terminal domain superfamily / Terpene synthase, N-terminal domain / Terpenoid cyclases/protein prenyltransferase alpha-alpha toroid / Isoprenoid synthase domain superfamily
Similarity search - Domain/homology
Syn-copalyl diphosphate synthase, chloroplastic
Similarity search - Component
Biological speciesOryza sativa Japonica Group (Japanese rice)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.49 Å
AuthorsMa, X.L. / Xu, H.F. / Jiang, T.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Commun Chem / Year: 2023
Title: Structural and functional investigations of syn-copalyl diphosphate synthase from Oryza sativa.
Authors: Xiaoli Ma / Haifeng Xu / Yuru Tong / Yunfeng Luo / Qinghua Dong / Tao Jiang /
Abstract: The large superfamily of labdane-related diterpenoids is defined by the cyclization of linear geranylgeranyl pyrophosphate (GGPP), catalyzed by copalyl diphosphate synthases (CPSs) to form the basic ...The large superfamily of labdane-related diterpenoids is defined by the cyclization of linear geranylgeranyl pyrophosphate (GGPP), catalyzed by copalyl diphosphate synthases (CPSs) to form the basic decalin core, the copalyl diphosphates (CPPs). Three stereochemically distinct CPPs have been found in plants, namely (+)-CPP, ent-CPP and syn-CPP. Here, we used X-ray crystallography and cryo-EM methods to describe different oligomeric structures of a syn-copalyl diphosphate synthase from Oryza sativa (OsCyc1), and provided a cryo-EM structure of OsCyc1 mutant in complex with the substrate GGPP. Further analysis showed that tetramers are the dominant form of OsCyc1 in solution and are not necessary for enzyme activity in vitro. Through rational design, we identified an OsCyc1 mutant that can generate ent-CPP in addition to syn-CPP. Our work provides a structural and mechanistic basis for comparing different CPSs and paves the way for further enzyme design to obtain diterpene derivatives with specific chirality.
History
DepositionAug 4, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 6, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Syn-copalyl diphosphate synthase, chloroplastic
C: Syn-copalyl diphosphate synthase, chloroplastic
E: Syn-copalyl diphosphate synthase, chloroplastic
D: Syn-copalyl diphosphate synthase, chloroplastic
F: Syn-copalyl diphosphate synthase, chloroplastic
A: Syn-copalyl diphosphate synthase, chloroplastic


Theoretical massNumber of molelcules
Total (without water)530,5656
Polymers530,5656
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)129.702, 174.342, 296.550
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21
31
41
51
61

NCS domain segments:

Ens-ID: 1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11THRTHRLYSLYS(chain 'A' and (resid 81 through 113 or resid 124...AF81 - 11381 - 113
12PROPROSERSER(chain 'A' and (resid 81 through 113 or resid 124...AF124 - 139124 - 139
13ASPASPTRPTRP(chain 'A' and (resid 81 through 113 or resid 124...AF142 - 184142 - 184
14LEULEUMETMET(chain 'A' and (resid 81 through 113 or resid 124...AF195 - 208195 - 208
15PROPROILEILE(chain 'A' and (resid 81 through 113 or resid 124...AF221 - 226221 - 226
16ILEILEHISHIS(chain 'A' and (resid 81 through 113 or resid 124...AF237 - 401237 - 401
17SERSERASPASP(chain 'A' and (resid 81 through 113 or resid 124...AF407 - 452407 - 452
18ALAALALEULEU(chain 'A' and (resid 81 through 113 or resid 124...AF457 - 538457 - 538
19SERSERLYSLYS(chain 'A' and (resid 81 through 113 or resid 124...AF540 - 544540 - 544
110GLNGLNGLNGLN(chain 'A' and (resid 81 through 113 or resid 124...AF551 - 694551 - 694
111ASNASNILEILE(chain 'A' and (resid 81 through 113 or resid 124...AF696 - 767696 - 767
21THRTHRLYSLYS(chain 'B' and (resid 81 through 113 or resid 124...BA81 - 11381 - 113
22PROPROSERSER(chain 'B' and (resid 81 through 113 or resid 124...BA124 - 139124 - 139
23ASPASPTRPTRP(chain 'B' and (resid 81 through 113 or resid 124...BA142 - 184142 - 184
24LEULEUMETMET(chain 'B' and (resid 81 through 113 or resid 124...BA195 - 208195 - 208
25PROPROILEILE(chain 'B' and (resid 81 through 113 or resid 124...BA221 - 226221 - 226
26ILEILEHISHIS(chain 'B' and (resid 81 through 113 or resid 124...BA237 - 401237 - 401
27SERSERASPASP(chain 'B' and (resid 81 through 113 or resid 124...BA407 - 452407 - 452
28ALAALALEULEU(chain 'B' and (resid 81 through 113 or resid 124...BA457 - 538457 - 538
29SERSERLYSLYS(chain 'B' and (resid 81 through 113 or resid 124...BA540 - 544540 - 544
210GLNGLNGLNGLN(chain 'B' and (resid 81 through 113 or resid 124...BA551 - 694551 - 694
211ASNASNILEILE(chain 'B' and (resid 81 through 113 or resid 124...BA696 - 767696 - 767
31THRTHRLYSLYS(chain 'C' and (resid 81 through 184 or resid 195...CB81 - 11381 - 113
32PROPROSERSER(chain 'C' and (resid 81 through 184 or resid 195...CB124 - 139124 - 139
33ASPASPTRPTRP(chain 'C' and (resid 81 through 184 or resid 195...CB142 - 184142 - 184
34LEULEUMETMET(chain 'C' and (resid 81 through 184 or resid 195...CB195 - 208195 - 208
35PROPROILEILE(chain 'C' and (resid 81 through 184 or resid 195...CB221 - 226221 - 226
36ILEILEHISHIS(chain 'C' and (resid 81 through 184 or resid 195...CB237 - 401237 - 401
37SERSERASPASP(chain 'C' and (resid 81 through 184 or resid 195...CB407 - 452407 - 452
38ALAALALEULEU(chain 'C' and (resid 81 through 184 or resid 195...CB457 - 538457 - 538
39SERSERLYSLYS(chain 'C' and (resid 81 through 184 or resid 195...CB540 - 544540 - 544
310GLNGLNGLNGLN(chain 'C' and (resid 81 through 184 or resid 195...CB551 - 694551 - 694
311ASNASNILEILE(chain 'C' and (resid 81 through 184 or resid 195...CB696 - 767696 - 767
41THRTHRLYSLYS(chain 'D' and (resid 81 through 113 or resid 124...DD81 - 11381 - 113
42PROPROSERSER(chain 'D' and (resid 81 through 113 or resid 124...DD124 - 139124 - 139
43ASPASPTRPTRP(chain 'D' and (resid 81 through 113 or resid 124...DD142 - 184142 - 184
44LEULEUMETMET(chain 'D' and (resid 81 through 113 or resid 124...DD195 - 208195 - 208
45PROPROILEILE(chain 'D' and (resid 81 through 113 or resid 124...DD221 - 226221 - 226
46ILEILEHISHIS(chain 'D' and (resid 81 through 113 or resid 124...DD237 - 401237 - 401
47SERSERASPASP(chain 'D' and (resid 81 through 113 or resid 124...DD407 - 452407 - 452
48ALAALALEULEU(chain 'D' and (resid 81 through 113 or resid 124...DD457 - 538457 - 538
49SERSERLYSLYS(chain 'D' and (resid 81 through 113 or resid 124...DD540 - 544540 - 544
410GLNGLNGLNGLN(chain 'D' and (resid 81 through 113 or resid 124...DD551 - 694551 - 694
411ASNASNILEILE(chain 'D' and (resid 81 through 113 or resid 124...DD696 - 767696 - 767
51THRTHRLYSLYS(chain 'E' and (resid 81 through 113 or resid 124...EC81 - 11381 - 113
52PROPROSERSER(chain 'E' and (resid 81 through 113 or resid 124...EC124 - 139124 - 139
53ASPASPTRPTRP(chain 'E' and (resid 81 through 113 or resid 124...EC142 - 184142 - 184
54LEULEUMETMET(chain 'E' and (resid 81 through 113 or resid 124...EC195 - 208195 - 208
55PROPROILEILE(chain 'E' and (resid 81 through 113 or resid 124...EC221 - 226221 - 226
56ILEILEHISHIS(chain 'E' and (resid 81 through 113 or resid 124...EC237 - 401237 - 401
57SERSERASPASP(chain 'E' and (resid 81 through 113 or resid 124...EC407 - 452407 - 452
58ALAALALEULEU(chain 'E' and (resid 81 through 113 or resid 124...EC457 - 538457 - 538
59SERSERLYSLYS(chain 'E' and (resid 81 through 113 or resid 124...EC540 - 544540 - 544
510GLNGLNGLNGLN(chain 'E' and (resid 81 through 113 or resid 124...EC551 - 694551 - 694
511ASNASNILEILE(chain 'E' and (resid 81 through 113 or resid 124...EC696 - 767696 - 767
61THRTHRLYSLYS(chain 'F' and (resid 81 through 113 or resid 124...FE81 - 11381 - 113
62PROPROSERSER(chain 'F' and (resid 81 through 113 or resid 124...FE124 - 139124 - 139
63ASPASPTRPTRP(chain 'F' and (resid 81 through 113 or resid 124...FE142 - 184142 - 184
64LEULEUMETMET(chain 'F' and (resid 81 through 113 or resid 124...FE195 - 208195 - 208
65PROPROILEILE(chain 'F' and (resid 81 through 113 or resid 124...FE221 - 226221 - 226
66ILEILEHISHIS(chain 'F' and (resid 81 through 113 or resid 124...FE237 - 401237 - 401
67SERSERASPASP(chain 'F' and (resid 81 through 113 or resid 124...FE407 - 452407 - 452
68ALAALALEULEU(chain 'F' and (resid 81 through 113 or resid 124...FE457 - 538457 - 538
69SERSERLYSLYS(chain 'F' and (resid 81 through 113 or resid 124...FE540 - 544540 - 544
610GLNGLNGLNGLN(chain 'F' and (resid 81 through 113 or resid 124...FE551 - 694551 - 694
611ASNASNILEILE(chain 'F' and (resid 81 through 113 or resid 124...FE696 - 767696 - 767

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Components

#1: Protein
Syn-copalyl diphosphate synthase, chloroplastic / OsCPSsyn / Syn-CPP synthase / OsCPS4 / OsCyc1


Mass: 88427.445 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryza sativa Japonica Group (Japanese rice)
Gene: CPS4, CYC1, Os04g0178300, LOC_Os04g09900, OSJNBa0096F01.12
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q0JF02, syn-copalyl-diphosphate synthase

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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: 3.16 Å3/Da / Density % sol: 61.07 %
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop
Details: 2% v/v 1,4-dioxane, 0.1 M Tris pH 8.0, 15% w/v polyethylene glycol 3,350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL18U1 / Wavelength: 0.97853 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: May 6, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97853 Å / Relative weight: 1
ReflectionResolution: 3.49→97.62 Å / Num. obs: 85822 / % possible obs: 96.51 % / Redundancy: 13.5 % / Biso Wilson estimate: 86.54 Å2 / CC1/2: 0.995 / Rpim(I) all: 0.1111 / Net I/σ(I): 10.14
Reflection shellResolution: 3.49→3.61 Å / Num. unique obs: 8453 / CC1/2: 0.511

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
XDSdata reduction
PHENIXphasing
XDSdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.49→97.62 Å / SU ML: 0.4821 / Cross valid method: FREE R-VALUE / σ(F): 0 / Phase error: 27.3109
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2627 1947 2.35 %
Rwork0.2012 80922 -
obs0.2027 82869 96.52 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 81.33 Å2
Refinement stepCycle: LAST / Resolution: 3.49→97.62 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms32459 0 0 0 32459
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.011333178
X-RAY DIFFRACTIONf_angle_d1.399944880
X-RAY DIFFRACTIONf_chiral_restr0.0714906
X-RAY DIFFRACTIONf_plane_restr0.00855757
X-RAY DIFFRACTIONf_dihedral_angle_d21.005712273
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.49-3.580.39251370.31475332X-RAY DIFFRACTION90.68
3.58-3.680.35831290.28745463X-RAY DIFFRACTION92.14
3.68-3.790.34571350.26825569X-RAY DIFFRACTION94.37
3.79-3.910.33361360.2395617X-RAY DIFFRACTION94.65
3.91-4.050.26021360.22445700X-RAY DIFFRACTION95.83
4.05-4.210.26411380.20535738X-RAY DIFFRACTION97.09
4.21-4.40.27341390.19755804X-RAY DIFFRACTION97.54
4.4-4.630.25421400.18575833X-RAY DIFFRACTION97.69
4.63-4.920.2271380.18145855X-RAY DIFFRACTION98.36
4.92-5.30.26451410.19155882X-RAY DIFFRACTION98.03
5.3-5.840.27491440.19935918X-RAY DIFFRACTION98.59
5.84-6.680.29631420.21245964X-RAY DIFFRACTION98.91
6.68-8.420.22041450.17556070X-RAY DIFFRACTION99.58
8.42-97.620.19581470.15486177X-RAY DIFFRACTION97.5

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