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- PDB-8qwc: Cryo-EM structure of Apo coproheme decarboxylase from Corynebacte... -

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Basic information

Entry
Database: PDB / ID: 8qwc
TitleCryo-EM structure of Apo coproheme decarboxylase from Corynebacterium diphtheria.
ComponentsCoproheme decarboxylase
KeywordsOXIDOREDUCTASE / Heme Binding protein / Heme Biosynthesis / Actinobacteria
Function / homologyoxidoreductase activity, acting on the CH-CH group of donors, oxygen as acceptor / hydrogen peroxide-dependent heme synthase / Heme-dependent peroxidase ChdC/CLD / Chlorite dismutase / heme B biosynthetic process / Dimeric alpha-beta barrel / heme binding / metal ion binding / Coproheme decarboxylase
Function and homology information
Biological speciesCorynebacterium diphtheriae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.27 Å
AuthorsPatil, G. / Guo, Y. / Borek, D. / Hofbauer, S.
Funding support Austria, 3items
OrganizationGrant numberCountry
Austrian Science FundP34934 Austria
Austrian Science FundP36967 Austria
Austrian Science FundW1224 Austria
CitationJournal: Protein Sci / Year: 2025
Title: Insights into the flexibility of the domain-linking loop in actinobacterial coproheme decarboxylase through structures and molecular dynamics simulations.
Authors: Gaurav Patil / Diego Javier Alonso de Armiño / Yirui Guo / Paul G Furtmüller / Dominika Borek / Dario A Estrin / Stefan Hofbauer /
Abstract: Prokaryotic heme biosynthesis in Gram-positive bacteria follows the coproporphyrin-dependent heme biosynthesis pathway. The last step in this pathway is catalyzed by the enzyme coproheme ...Prokaryotic heme biosynthesis in Gram-positive bacteria follows the coproporphyrin-dependent heme biosynthesis pathway. The last step in this pathway is catalyzed by the enzyme coproheme decarboxylase, which oxidatively transforms two propionate groups into vinyl groups yielding heme b. The catalytic reaction cycle of coproheme decarboxylases exhibits four different states: the apo-form, the substrate (coproheme)-bound form, a transient three-propionate intermediate form (monovinyl, monopropionate deuteroheme; MMD), and the product (heme b)-bound form. In this study, we used cryogenic electron microscopy single-particle reconstruction (cryo-EM SPR) to characterize structurally the apo and heme b-bound forms of actinobacterial coproheme decarboxylase from Corynebacterium diphtheriae. The flexible loop that connects the N-terminal and the C-terminal ferredoxin domains of coproheme decarboxylases plays an important role in interactions between the enzyme and porphyrin molecule. To understand the role of this flexible loop, we performed molecular dynamics simulations on the apo and heme b coproheme decarboxylase from Corynebacterium diphtheriae. Our results are discussed in the context of the published structural information on coproheme-bound and MMD-bound coproheme decarboxylase and with respect to the reaction mechanism. Having structural information of all four enzymatically relevant states helps in understanding structural restraints with a functional impact.
History
DepositionOct 19, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 5, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Coproheme decarboxylase
B: Coproheme decarboxylase
C: Coproheme decarboxylase
D: Coproheme decarboxylase
E: Coproheme decarboxylase


Theoretical massNumber of molelcules
Total (without water)136,4455
Polymers136,4455
Non-polymers00
Water5,513306
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14280 Å2
ΔGint-62 kcal/mol
Surface area41790 Å2
MethodPISA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21A
32A
42A
53A
63A
74A
84A
95A
105A
116A
126A
137A
147A
158A
168A
179A
189A
1910A
2010A

NCS domain segments:

Beg auth comp-ID: ASN / Beg label comp-ID: ASN / End auth comp-ID: GLY / End label comp-ID: GLY / Auth asym-ID: A / Label asym-ID: A / Auth seq-ID: 11 - 235 / Label seq-ID: 13 - 237

Dom-IDComponent-IDEns-ID
111
211
322
422
533
633
744
844
955
1055
1166
1266
1377
1477
1588
1688
1799
1899
191010
201010

NCS ensembles :
IDDetails
1Local NCS retraints between domains: 1 2
2Local NCS retraints between domains: 3 4
3Local NCS retraints between domains: 5 6
4Local NCS retraints between domains: 7 8
5Local NCS retraints between domains: 9 10
6Local NCS retraints between domains: 11 12
7Local NCS retraints between domains: 13 14
8Local NCS retraints between domains: 15 16
9Local NCS retraints between domains: 17 18
10Local NCS retraints between domains: 19 20

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Components

#1: Protein
Coproheme decarboxylase


Mass: 27288.961 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Corynebacterium diphtheriae (bacteria) / Gene: chdC / Plasmid: pD441-NH Vector / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q6NGV6
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 306 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Structure of Apo coproheme decarboxylase from Corynebacterium diphtheria
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 136.3 kDa/nm / Experimental value: NO
Source (natural)Organism: Corynebacterium diphtheriae (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pD441-NH Vector
Buffer solutionpH: 7.5 / Details: 100mM Phosphate Buffer
Buffer componentName: Sodium Chloride / Formula: NaCl
SpecimenConc.: 18 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Blotting time; 5.0-5.5 sec. Blotting force; 18 or 19.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: HELIUM / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 72 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 1035
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 25 eV / Phase plate: VOLTA PHASE PLATE

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7MOLREPmodel fitting
11cryoSPARCclassification
12cryoSPARC3D reconstruction
19REFMACmodel refinement
20PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.27 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 638556 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 6XUC

6xuc


Accession code: 6XUC / Source name: PDB / Type: experimental model
RefinementResolution: 2.27→101.748 Å / Cor.coef. Fo:Fc: 0.788 / WRfactor Rwork: 0.394 / SU B: 5.515 / SU ML: 0.118 / Average fsc free: 0 / Average fsc overall: 0.6967 / Average fsc work: 0.6967 / ESU R: 0.252
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.3935 151996 -
all0.394 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: NONE
Displacement parametersBiso mean: 69.78 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.0129283
ELECTRON MICROSCOPYr_bond_other_d00.0168627
ELECTRON MICROSCOPYr_angle_refined_deg1.2541.64612583
ELECTRON MICROSCOPYr_angle_other_deg0.4061.56719706
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.851108
ELECTRON MICROSCOPYr_dihedral_angle_2_deg5.5035114
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.811101492
ELECTRON MICROSCOPYr_dihedral_angle_6_deg17.48310494
ELECTRON MICROSCOPYr_chiral_restr0.0580.21294
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.0211320
ELECTRON MICROSCOPYr_gen_planes_other0.0010.022488
ELECTRON MICROSCOPYr_nbd_refined0.1960.21600
ELECTRON MICROSCOPYr_symmetry_nbd_other0.2080.28272
ELECTRON MICROSCOPYr_nbtor_refined0.1940.24627
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.080.25269
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.2280.2215
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_other0.1040.212
ELECTRON MICROSCOPYr_mcbond_it7.8966.5974435
ELECTRON MICROSCOPYr_mcbond_other7.8876.5954434
ELECTRON MICROSCOPYr_mcangle_it10.79411.8875542
ELECTRON MICROSCOPYr_mcangle_other10.79411.8875543
ELECTRON MICROSCOPYr_scbond_it9.2837.3944848
ELECTRON MICROSCOPYr_scbond_other9.2827.3944848
ELECTRON MICROSCOPYr_scangle_it13.92413.1157041
ELECTRON MICROSCOPYr_scangle_other13.92313.1157042
ELECTRON MICROSCOPYr_lrange_it18.33881.58437959
ELECTRON MICROSCOPYr_lrange_other18.33581.5937920
ELECTRON MICROSCOPYr_ncsr_local_group_10.0960.057195
ELECTRON MICROSCOPYr_ncsr_local_group_20.0890.057225
ELECTRON MICROSCOPYr_ncsr_local_group_30.0830.057299
ELECTRON MICROSCOPYr_ncsr_local_group_40.0930.057259
ELECTRON MICROSCOPYr_ncsr_local_group_50.0870.057265
ELECTRON MICROSCOPYr_ncsr_local_group_60.0950.057233
ELECTRON MICROSCOPYr_ncsr_local_group_70.0860.057270
ELECTRON MICROSCOPYr_ncsr_local_group_80.0780.057330
ELECTRON MICROSCOPYr_ncsr_local_group_90.0890.057272
ELECTRON MICROSCOPYr_ncsr_local_group_100.0910.057261
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)Weight position
11AELECTRON MICROSCOPYLocal ncs0.096210.05011
12AELECTRON MICROSCOPYLocal ncs0.096210.05011
23AELECTRON MICROSCOPYLocal ncs0.088740.05011
24AELECTRON MICROSCOPYLocal ncs0.088740.05011
35AELECTRON MICROSCOPYLocal ncs0.082550.05011
36AELECTRON MICROSCOPYLocal ncs0.082550.05011
47AELECTRON MICROSCOPYLocal ncs0.092620.05011
48AELECTRON MICROSCOPYLocal ncs0.092620.05011
59AELECTRON MICROSCOPYLocal ncs0.08690.05011
510AELECTRON MICROSCOPYLocal ncs0.08690.05011
611AELECTRON MICROSCOPYLocal ncs0.095480.05011
612AELECTRON MICROSCOPYLocal ncs0.095480.05011
713AELECTRON MICROSCOPYLocal ncs0.08590.05011
714AELECTRON MICROSCOPYLocal ncs0.08590.05011
815AELECTRON MICROSCOPYLocal ncs0.078330.05011
816AELECTRON MICROSCOPYLocal ncs0.078330.05011
917AELECTRON MICROSCOPYLocal ncs0.088630.05011
918AELECTRON MICROSCOPYLocal ncs0.088630.05011
1019AELECTRON MICROSCOPYLocal ncs0.090730.05011
1020AELECTRON MICROSCOPYLocal ncs0.090730.05011
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: _ / Total num. of bins used: 20 / % reflection obs: 100 %

Resolution (Å)Rfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc workWRfactor Rwork
2.27-2.3291.712112281.712112280.1111.712
2.329-2.3931.78109311.78109310.2131.78
2.393-2.4621.673107171.673107170.3191.673
2.462-2.5381.541103071.541103070.4461.541
2.538-2.6211.234100581.234100580.621.234
2.621-2.7130.9797140.9797140.7190.97
2.713-2.8150.6793170.6793170.7810.67
2.815-2.930.3490180.3490180.8740.34
2.93-3.060.26686580.26686580.910.266
3.06-3.2090.26782210.26782210.9270.267
3.209-3.3830.28779140.28779140.930.287
3.383-3.5880.28773870.28773870.9490.287
3.588-3.8350.28770190.28770190.9510.287
3.835-4.1420.32464930.32464930.9560.324
4.142-4.5370.31659580.31659580.9570.316
4.537-5.0710.27453790.27453790.9580.274
5.071-5.8530.26247810.26247810.930.262
5.853-7.1620.40140660.40140660.8780.401
7.162-10.1040.41331070.41331070.8770.413
10.104-101.7480.90517220.90517220.9160.905

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