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- PDB-8ai5: Crystal structure of radical SAM epimerase EpeE C223A mutant from... -

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Basic information

Entry
Database: PDB / ID: 8ai5
TitleCrystal structure of radical SAM epimerase EpeE C223A mutant from Bacillus subtilis with [4Fe-4S] clusters, S-adenosyl-L-homocysteine and RiPP peptide 6 bound
Components
  • Putative exported peptide YydF
  • Putative peptide biosynthesis protein YydG
KeywordsMETAL BINDING PROTEIN / radical SAM / metalloenzyme / iron-sulfur / RiPP
Function / homology
Function and homology information


catalytic activity / 4 iron, 4 sulfur cluster binding / metal ion binding
Similarity search - Function
YydF/SAG 2028 / Peptide radical SAM maturase , YydG / 4Fe-4S single cluster domain / Radical SAM superfamily / Radical SAM core domain profile. / Radical SAM / Aldolase-type TIM barrel
Similarity search - Domain/homology
DI(HYDROXYETHYL)ETHER / S-ADENOSYL-L-HOMOCYSTEINE / IRON/SULFUR CLUSTER / Putative peptide biosynthesis protein YydG / Putative exported peptide YydF
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.15 Å
AuthorsPolsinelli, I. / Fyfe, C.D. / Legrand, P. / Kubiak, X. / Chavas, L.M.G. / Berteau, O. / Benjdia, A.
Funding support France, European Union, 2items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-17-CE11-0014 France
European Research Council (ERC)617053European Union
Citation
Journal: Nat.Chem.Biol. / Year: 2024
Title: Structural and mechanistic basis for RiPP epimerization by a radical SAM enzyme.
Authors: Kubiak, X. / Polsinelli, I. / Chavas, L.M.G. / Fyfe, C.D. / Guillot, A. / Fradale, L. / Brewee, C. / Grimaldi, S. / Gerbaud, G. / Thureau, A. / Legrand, P. / Berteau, O. / Benjdia, A.
#1: Journal: Nat Chem / Year: 2017
Title: Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis
Authors: Benjdia, A. / Guillot, A. / Ruffie, P. / Leprince, J. / Berteau, O.
History
DepositionJul 25, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2024Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 13, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Putative peptide biosynthesis protein YydG
B: Putative peptide biosynthesis protein YydG
C: Putative exported peptide YydF
D: Putative exported peptide YydF
hetero molecules


Theoretical massNumber of molelcules
Total (without water)85,51023
Polymers82,4404
Non-polymers3,07019
Water3,513195
1
A: Putative peptide biosynthesis protein YydG
C: Putative exported peptide YydF
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,66210
Polymers41,2202
Non-polymers1,4428
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3470 Å2
ΔGint-42 kcal/mol
Surface area14570 Å2
MethodPISA
2
B: Putative peptide biosynthesis protein YydG
D: Putative exported peptide YydF
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,84813
Polymers41,2202
Non-polymers1,62811
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3690 Å2
ΔGint-40 kcal/mol
Surface area15600 Å2
MethodPISA
Unit cell
Length a, b, c (Å)78.98, 92.52, 127.52
Angle α, β, γ (deg.)90, 90, 90
Int Tables number19
Space group name H-MP212121

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Components

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Protein / Protein/peptide , 2 types, 4 molecules ABCD

#1: Protein Putative peptide biosynthesis protein YydG


Mass: 39920.379 Da / Num. of mol.: 2 / Mutation: C223A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Strain: 168 / Gene: yydG, BSU40170 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q45595
#2: Protein/peptide Putative exported peptide YydF


Mass: 1299.459 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Bacillus subtilis (bacteria) / References: UniProt: Q45596

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Non-polymers , 5 types, 214 molecules

#3: Chemical
ChemComp-SF4 / IRON/SULFUR CLUSTER / Iron–sulfur cluster


Mass: 351.640 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE / S-Adenosyl-L-homocysteine


Type: L-peptide linking / Mass: 384.411 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C14H20N6O5S / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C2H6O2
#6: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H10O3
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 195 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.83 Å3/Da / Density % sol: 56.47 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / Details: 15% (w/v) PEG 1000, 100 mM Tris

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.97857 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Sep 10, 2021 / Details: KB Mirrors
RadiationMonochromator: channel-cut Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97857 Å / Relative weight: 1
ReflectionResolution: 2.15→49.61 Å / Num. obs: 775084 / % possible obs: 100 % / Redundancy: 15 % / CC1/2: 1 / Rmerge(I) obs: 0.071 / Rpim(I) all: 0.019 / Rrim(I) all: 0.074 / Net I/σ(I): 18.9
Reflection shellResolution: 2.15→2.21 Å / Rmerge(I) obs: 2.909 / Mean I/σ(I) obs: 0.9 / Num. unique obs: 51591 / CC1/2: 0.525 / Rpim(I) all: 0.815 / Rrim(I) all: 3.022

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Processing

Software
NameVersionClassification
BUSTER2.10.4refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
MxCuBEdata collection
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Fe-SAD experimental model

Resolution: 2.15→46.26 Å / Cor.coef. Fo:Fc: 0.958 / Cor.coef. Fo:Fc free: 0.947 / SU R Cruickshank DPI: 0.195 / Cross valid method: THROUGHOUT / SU R Blow DPI: 0.197 / SU Rfree Blow DPI: 0.156 / SU Rfree Cruickshank DPI: 0.157
RfactorNum. reflection% reflectionSelection details
Rfree0.2182 2577 -RANDOM
Rwork0.2007 ---
obs0.2016 51522 100 %-
Displacement parametersBiso mean: 70.69 Å2
Baniso -1Baniso -2Baniso -3
1--7.7374 Å20 Å20 Å2
2--6.8529 Å20 Å2
3---0.8845 Å2
Refine analyzeLuzzati coordinate error obs: 0.28 Å
Refinement stepCycle: LAST / Resolution: 2.15→46.26 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5411 0 142 195 5748
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0085787HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.917821HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d2062SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes987HARMONIC5
X-RAY DIFFRACTIONt_it5787HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion745SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact4569SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion3.06
X-RAY DIFFRACTIONt_other_torsion16.49
LS refinement shellResolution: 2.15→2.17 Å
RfactorNum. reflection% reflection
Rfree0.3562 52 -
Rwork0.3261 --
obs0.3276 1031 100 %
Refinement TLS params.

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)
11.88130.3667-0.55781.3691-0.07161.109-0.0181-0.01420.0554-0.0142-0.0626-0.05530.0554-0.05530.0807-0.14950.04030.0011-0.30450.0296-0.28391.0632-14.1573-23.3491
22.3353-0.09281.13071.6964-0.02322.31970.0866-0.0782-0.006-0.0782-0.08890.2193-0.0060.21930.0023-0.25160.03240.0647-0.30760.0025-0.28231.6147-28.225-22.5369
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|* }A2 - 317
2X-RAY DIFFRACTION1{ A|* }A401 - 505
3X-RAY DIFFRACTION1{ A|* }A601 - 724
4X-RAY DIFFRACTION2{ B|* }B-6 - 316
5X-RAY DIFFRACTION2{ B|* }B401 - 509
6X-RAY DIFFRACTION2{ B|* }B601 - 671

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