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- PDB-1kyq: Met8p: A bifunctional NAD-dependent dehydrogenase and ferrochelat... -

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Basic information

Entry
Database: PDB / ID: 1kyq
TitleMet8p: A bifunctional NAD-dependent dehydrogenase and ferrochelatase involved in siroheme synthesis.
ComponentsSiroheme biosynthesis protein MET8
KeywordsOXIDOREDUCTASE / LYASE / homodimer
Function / homology
Function and homology information


precorrin-2 dehydrogenase / precorrin-2 dehydrogenase activity / sirohydrochlorin ferrochelatase / sirohydrochlorin ferrochelatase activity / siroheme biosynthetic process / ferrochelatase activity / sulfate assimilation / methionine biosynthetic process
Similarity search - Function
Siro heme synthase C-terminal domain-like / Siroheme synthase; domain 3 / Siroheme biosynthesis protein Met8, C-terminal / Sirohaem biosynthesis protein C-terminal / Siroheme biosynthesis protein Met8 / : / Sirohaem synthase, central domain / Siroheme synthase, central domain / Putative NAD(P)-binding / Sirohaem biosynthesis protein central ...Siro heme synthase C-terminal domain-like / Siroheme synthase; domain 3 / Siroheme biosynthesis protein Met8, C-terminal / Sirohaem biosynthesis protein C-terminal / Siroheme biosynthesis protein Met8 / : / Sirohaem synthase, central domain / Siroheme synthase, central domain / Putative NAD(P)-binding / Sirohaem biosynthesis protein central / Double Stranded RNA Binding Domain / NAD(P)-binding Rossmann-like Domain / NAD(P)-binding domain superfamily / Rossmann fold / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Siroheme biosynthesis protein MET8
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.2 Å
AuthorsSchubert, H.L. / Raux, E. / Brindley, A.A. / Wilson, K.S. / Hill, C.P. / Warren, M.J.
Citation
Journal: EMBO J. / Year: 2002
Title: The structure of Saccharomyces cerevisiae Met8p, a bifunctional dehydrogenase and ferrochelatase.
Authors: Schubert, H.L. / Raux, E. / Brindley, A.A. / Leech, H.K. / Wilson, K.S. / Hill, C.P. / Warren, M.J.
#1: Journal: Biophys.J. / Year: 1999
Title: The Role of Saccharomyces cerevisiae Met1p and Met8p in Sirohaem and Cobalamin Biosynthesis
Authors: Raux, E. / McVeigh, T. / Peters, S.E. / Leustek, T. / Warren, M.J.
History
DepositionFeb 5, 2002Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 8, 2002Provider: repository / Type: Initial release
Revision 1.1Apr 28, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Remark 999SEQUENCE During several rounds of independent PCR and sequencing from S. cervisiae, the authors ...SEQUENCE During several rounds of independent PCR and sequencing from S. cervisiae, the authors discovered several discrepancies in the primary sequence: K15R, I33(MSE) (a helpful mutation for solving the structure), E61K, D102N.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Siroheme biosynthesis protein MET8
B: Siroheme biosynthesis protein MET8
C: Siroheme biosynthesis protein MET8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)98,8586
Polymers96,8683
Non-polymers1,9903
Water11,061614
1
A: Siroheme biosynthesis protein MET8
B: Siroheme biosynthesis protein MET8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,9054
Polymers64,5792
Non-polymers1,3272
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8390 Å2
ΔGint-50 kcal/mol
Surface area26090 Å2
MethodPISA
2
C: Siroheme biosynthesis protein MET8
hetero molecules

C: Siroheme biosynthesis protein MET8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,9054
Polymers64,5792
Non-polymers1,3272
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area8760 Å2
ΔGint-55 kcal/mol
Surface area24770 Å2
MethodPISA
Unit cell
Length a, b, c (Å)156.988, 80.856, 103.975
Angle α, β, γ (deg.)90.00, 121.88, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: Protein Siroheme biosynthesis protein MET8 / MET8P


Mass: 32289.268 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: Met8 / Plasmid: pET14b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) Codon Plus
References: UniProt: P15807, Oxidoreductases, protoporphyrin ferrochelatase
#2: Chemical ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Nicotinamide adenine dinucleotide


Mass: 663.425 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Comment: NAD*YM
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 614 / 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: 2.89 Å3/Da / Density % sol: 57.46 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: PEG 4000, CaCl2, Tris, pH 8.5, VAPOR DIFFUSION, HANGING DROP, temperature 298K
Crystal
*PLUS
Density % sol: 60 %
Crystal grow
*PLUS
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formulaDetails
17 mg/mlprotein1drop
22.5 mMNAD1drop
316-18 %PEG40001reservoir
40.1 M1reservoirCaCl2
50.1 MTris-HCl1reservoirpH8.5
62 mMdithiothreitol1reservoir

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-1 / Wavelength: 0.97 Å
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: Mar 30, 2001 / Details: Flat mirror (vertical focusing)
RadiationMonochromator: single crystal Si(311) bent monochromator (horizontal focusing)
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97 Å / Relative weight: 1
ReflectionResolution: 1.9→30 Å / Num. all: 64205 / Num. obs: 64205 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 6.1 % / Biso Wilson estimate: 28 Å2 / Rmerge(I) obs: 0.067 / Rsym value: 0.067 / Net I/σ(I): 15.7
Reflection shellResolution: 2.2→2.28 Å / Redundancy: 2.1 % / Rmerge(I) obs: 0.304 / Mean I/σ(I) obs: 2.9 / Num. unique all: 9417 / Rsym value: 0.304 / % possible all: 100
Reflection
*PLUS
Highest resolution: 2.2 Å / Lowest resolution: 20 Å / Num. obs: 56148 / % possible obs: 98 % / Num. measured all: 117894 / Rmerge(I) obs: 0.07
Reflection shell
*PLUS
% possible obs: 85.6 % / Rmerge(I) obs: 0.304 / Mean I/σ(I) obs: 3

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Processing

Software
NameClassification
SOLVEphasing
REFMACrefinement
DENZOdata reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MAD / Resolution: 2.2→30 Å / σ(F): 0 / σ(I): 0
Details: REFMAC MKLF. PORTIONS OF THE NAD LIGANDS WERE MISSING IN THE DENSITY.
RfactorNum. reflection% reflection
Rfree0.283 2818 5 %
Rwork0.219 --
all0.22 56251 -
obs0.22 56251 -
Refinement stepCycle: LAST / Resolution: 2.2→30 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6579 0 96 614 7289
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONp_bond_d0.007
X-RAY DIFFRACTIONp_angle_d2.2
Refinement
*PLUS
% reflection Rfree: 5 % / Rfactor all: 0.22 / Rfactor obs: 0.221 / Rfactor Rfree: 0.287 / Rfactor Rwork: 0.221
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONp_angle_d
X-RAY DIFFRACTIONp_angle_deg2.1

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