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- PDB-6rt8: Structure of catharanthine synthase - an alpha-beta hydrolase fro... -

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Basic information

Entry
Database: PDB / ID: 6rt8
TitleStructure of catharanthine synthase - an alpha-beta hydrolase from Catharanthus roseus with a cleaviminium intermediate bound
ComponentsCatharanthine synthase
KeywordsHYDROLASE / alkaloid / catharanthine / natural product / biosynthesis / alpha/beta hydrolase fold
Function / homology
Function and homology information


Lyases / alkaloid metabolic process / hydrolase activity / lyase activity / nucleus / cytosol
Similarity search - Function
Alpha/beta hydrolase fold-3 / alpha/beta hydrolase fold / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
18-carboxymethoxy-cleaviminium / Catharanthine synthase
Similarity search - Component
Biological speciesCatharanthus roseus (Madagascar periwinkle)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.19 Å
AuthorsCaputi, L. / Franke, J. / Bussey, K. / Farrow, S.C. / Curcino Vieira, I.J. / Stevenson, C.E.M. / Lawson, D.M. / O'Connor, S.E.
Funding support Belgium, United Kingdom, 2items
OrganizationGrant numberCountry
European Research Council788301 Belgium
Biotechnology and Biological Sciences Research CouncilBB/P012523/1 United Kingdom
CitationJournal: Nat.Chem.Biol. / Year: 2020
Title: Structural basis of cycloaddition in biosynthesis of iboga and aspidosperma alkaloids.
Authors: Caputi, L. / Franke, J. / Bussey, K. / Farrow, S.C. / Vieira, I.J.C. / Stevenson, C.E.M. / Lawson, D.M. / O'Connor, S.E.
History
DepositionMay 22, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 4, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 1, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / 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 / _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: Catharanthine synthase
B: Catharanthine synthase
C: Catharanthine synthase
D: Catharanthine synthase
E: Catharanthine synthase
F: Catharanthine synthase
G: Catharanthine synthase
H: Catharanthine synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)300,44420
Polymers296,6168
Non-polymers3,82912
Water6,990388
1
A: Catharanthine synthase
B: Catharanthine synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,1115
Polymers74,1542
Non-polymers9573
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2720 Å2
ΔGint-15 kcal/mol
Surface area23910 Å2
MethodPISA
2
C: Catharanthine synthase
D: Catharanthine synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,1115
Polymers74,1542
Non-polymers9573
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2730 Å2
ΔGint-14 kcal/mol
Surface area23860 Å2
MethodPISA
3
E: Catharanthine synthase
F: Catharanthine synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,1115
Polymers74,1542
Non-polymers9573
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2690 Å2
ΔGint-15 kcal/mol
Surface area23700 Å2
MethodPISA
4
G: Catharanthine synthase
H: Catharanthine synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,1115
Polymers74,1542
Non-polymers9573
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2700 Å2
ΔGint-15 kcal/mol
Surface area23460 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.540, 121.030, 157.980
Angle α, β, γ (deg.)90.000, 99.020, 90.000
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14A
24E
15A
25F
16A
26G
17A
27H
18B
28C
19B
29D
110B
210E
111B
211F
112B
212G
113B
213H
114C
214D
115C
215E
116C
216F
117C
217G
118C
218H
119D
219E
120D
220F
121D
221G
122D
222H
123E
223F
124E
224G
125E
225H
126F
226G
127F
227H
128G
228H

NCS domain segments:

Component-ID: 0 / Refine code: 0

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ASPASPLYSLYSAA9 - 32710 - 328
21ASPASPLYSLYSBB9 - 32710 - 328
12ASPASPLYSLYSAA9 - 32710 - 328
22ASPASPLYSLYSCC9 - 32710 - 328
13ASPASPLYSLYSAA9 - 32710 - 328
23ASPASPLYSLYSDD9 - 32710 - 328
14ASPASPILEILEAA9 - 32610 - 327
24ASPASPILEILEEE9 - 32610 - 327
15ASPASPLYSLYSAA9 - 32710 - 328
25ASPASPLYSLYSFF9 - 32710 - 328
16ASPASPLYSLYSAA9 - 32710 - 328
26ASPASPLYSLYSGG9 - 32710 - 328
17GLUGLUILEILEAA10 - 32611 - 327
27GLUGLUILEILEHH10 - 32611 - 327
18ASPASPLYSLYSBB9 - 32710 - 328
28ASPASPLYSLYSCC9 - 32710 - 328
19ASPASPLYSLYSBB9 - 32710 - 328
29ASPASPLYSLYSDD9 - 32710 - 328
110ASPASPILEILEBB9 - 32610 - 327
210ASPASPILEILEEE9 - 32610 - 327
111ASPASPLYSLYSBB9 - 32710 - 328
211ASPASPLYSLYSFF9 - 32710 - 328
112ASPASPLYSLYSBB9 - 32710 - 328
212ASPASPLYSLYSGG9 - 32710 - 328
113GLUGLUILEILEBB10 - 32611 - 327
213GLUGLUILEILEHH10 - 32611 - 327
114ASPASPLYSLYSCC9 - 32710 - 328
214ASPASPLYSLYSDD9 - 32710 - 328
115ASPASPILEILECC9 - 32610 - 327
215ASPASPILEILEEE9 - 32610 - 327
116ASPASPLYSLYSCC9 - 32710 - 328
216ASPASPLYSLYSFF9 - 32710 - 328
117ASPASPLYSLYSCC9 - 32710 - 328
217ASPASPLYSLYSGG9 - 32710 - 328
118GLUGLUILEILECC10 - 32611 - 327
218GLUGLUILEILEHH10 - 32611 - 327
119ASPASPILEILEDD9 - 32610 - 327
219ASPASPILEILEEE9 - 32610 - 327
120ASPASPLYSLYSDD9 - 32710 - 328
220ASPASPLYSLYSFF9 - 32710 - 328
121ASPASPLYSLYSDD9 - 32710 - 328
221ASPASPLYSLYSGG9 - 32710 - 328
122GLUGLUILEILEDD10 - 32611 - 327
222GLUGLUILEILEHH10 - 32611 - 327
123ASPASPILEILEEE9 - 32610 - 327
223ASPASPILEILEFF9 - 32610 - 327
124ASPASPILEILEEE9 - 32610 - 327
224ASPASPILEILEGG9 - 32610 - 327
125GLUGLUILEILEEE10 - 32611 - 327
225GLUGLUILEILEHH10 - 32611 - 327
126ASPASPLYSLYSFF9 - 32710 - 328
226ASPASPLYSLYSGG9 - 32710 - 328
127GLUGLUILEILEFF10 - 32611 - 327
227GLUGLUILEILEHH10 - 32611 - 327
128GLUGLUILEILEGG10 - 32611 - 327
228GLUGLUILEILEHH10 - 32611 - 327

NCS ensembles :
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28

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Components

#1: Protein
Catharanthine synthase / Hydrolase 1 / CrHL1


Mass: 37076.957 Da / Num. of mol.: 8
Mutation: Native N-terminal MET is replaced by a GLY-PRO dipeptide left over from cleavage of the affinity tag
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Catharanthus roseus (Madagascar periwinkle)
Gene: CS, HL1, Caros025416 / Plasmid: pOPINF / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): SOLUBL21 / References: UniProt: A0A2P1GIW2, Lyases
#2: Chemical
ChemComp-KJE / 18-carboxymethoxy-cleaviminium


Mass: 337.435 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C21H25N2O2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-P6G / HEXAETHYLENE GLYCOL / POLYETHYLENE GLYCOL PEG400 / Polyethylene glycol


Mass: 282.331 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C12H26O7 / Comment: precipitant*YM
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 388 / 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: 2.6 Å3/Da / Density % sol: 52.61 % / Description: NULL
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.5 / Details: NULL

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9763 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Dec 14, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 2.19→52.01 Å / Num. obs: 152727 / % possible obs: 98.3 % / Redundancy: 7 % / Biso Wilson estimate: 41.2 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.093 / Rpim(I) all: 0.038 / Rrim(I) all: 0.1 / Net I/σ(I): 14.1
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
2.19-2.2371.1295259474780.5590.4571.219297.7
12-52.016.60.02262609450.9990.0090.02471.195.8

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Processing

Software
NameVersionClassification
XDS20161101data reduction
Aimless0.7.4data scaling
REFMAC5.8.0238refinement
PDB_EXTRACT3.25data extraction
PHASER2.7.17phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6RS4
Resolution: 2.19→52.01 Å / Cor.coef. Fo:Fc: 0.965 / Cor.coef. Fo:Fc free: 0.952 / SU B: 21.651 / SU ML: 0.228 / SU R Cruickshank DPI: 0.2427 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.243 / ESU R Free: 0.196
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2342 7594 5 %RANDOM
Rwork0.1939 ---
obs0.1959 145106 98.1 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.1 Å
Displacement parametersBiso max: 202.59 Å2 / Biso mean: 62.531 Å2 / Biso min: 27.34 Å2
Baniso -1Baniso -2Baniso -3
1--0.78 Å20 Å22.8 Å2
2--0.88 Å20 Å2
3----0.94 Å2
Refinement stepCycle: final / Resolution: 2.19→52.01 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms19468 0 276 388 20132
Biso mean--67.91 54.54 -
Num. residues----2501
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.01320341
X-RAY DIFFRACTIONr_bond_other_d0.0010.01718236
X-RAY DIFFRACTIONr_angle_refined_deg1.5221.64227773
X-RAY DIFFRACTIONr_angle_other_deg1.2691.57542165
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.21552483
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.65522.582945
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.221152945
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.9431576
X-RAY DIFFRACTIONr_chiral_restr0.0720.22653
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.0222813
X-RAY DIFFRACTIONr_gen_planes_other0.0010.024411
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A104070.05
12B104070.05
21A104930.04
22C104930.04
31A104790.04
32D104790.04
41A97550.06
42E97550.06
51A103570.04
52F103570.04
61A105030.04
62G105030.04
71A102350.05
72H102350.05
81B104220.06
82C104220.06
91B104260.05
92D104260.05
101B98150.04
102E98150.04
111B102650.05
112F102650.05
121B104340.04
122G104340.04
131B102340.05
132H102340.05
141C104680.05
142D104680.05
151C97580.06
152E97580.06
161C103590.05
162F103590.05
171C104990.05
172G104990.05
181C102550.05
182H102550.05
191D97440.06
192E97440.06
201D103490.04
202F103490.04
211D105120.03
212G105120.03
221D102940.04
222H102940.04
231E97320.05
232F97320.05
241E97610.05
242G97610.05
251E96320.05
252H96320.05
261F103880.04
262G103880.04
271F101020.04
272H101020.04
281G102710.04
282H102710.04
LS refinement shellResolution: 2.19→2.247 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.39 588 -
Rwork0.408 10578 -
all-11166 -
obs--97.46 %
Refinement TLS params.

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

IDL112)L122)L132)L222)L232)L332)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T112)T122)T132)T222)T232)T332)Origin x (Å)Origin y (Å)Origin z (Å)
11.6503-0.3113-0.02831.1178-0.11712.37280.123-0.05240.12050.0770.04190.0185-0.2314-0.132-0.16490.09620.04730.09580.10660.03080.106881.9742-9.756618.9403
22.29940.31090.33251.49330.14162.6992-0.02130.0566-0.1098-0.03960.2020.1087-0.0606-0.3454-0.18070.04040.0860.0590.28250.16390.113974.186-13.0321-18.0666
32.33680.5694-0.69022.0218-0.06912.088-0.0670.0238-0.1711-0.15110.07370.1170.0605-0.2026-0.00670.08150.01670.03730.17010.030.058264.7312-4.9775104.1061
41.74960.2459-0.68013.1653-0.34321.89420.0502-0.03560.1197-0.1542-0.0115-0.0758-0.1771-0.0017-0.03870.1190.00080.06970.1571-0.0410.061988.990720.291488.8966
54.97090.2462-0.74291.59540.37372.8568-0.0426-1.3348-0.75150.1816-0.1953-0.15610.20470.05490.2380.03940.0110.04950.46920.32860.38838.107311.759610.9934
64.00971.4138-0.91542.13-0.49551.7328-0.34540.8395-0.4088-0.22960.24160.03760.0213-0.24380.10380.0437-0.05920.03510.2445-0.1240.170445.889221.543-24.2025
72.107-0.3883-0.59111.81180.19061.98280.22140.02180.3384-0.0197-0.12860.0755-0.3057-0.0402-0.09280.24280.00510.17830.17160.00680.172236.191721.95164.3078
82.9767-0.7752-1.4783.1705-0.10042.7280.1209-0.33550.09950.1758-0.017-0.406-0.15920.431-0.1040.1909-0.00350.06320.24-0.00740.098964.012-0.362250.8484
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A9 - 401
2X-RAY DIFFRACTION2B9 - 401
3X-RAY DIFFRACTION3C9 - 401
4X-RAY DIFFRACTION4D9 - 401
5X-RAY DIFFRACTION5E9 - 401
6X-RAY DIFFRACTION6F9 - 401
7X-RAY DIFFRACTION7G9 - 401
8X-RAY DIFFRACTION8H9 - 401

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