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- PDB-8a8o: PAPS reductase from Methanothermococcus thermolithotrophicus refi... -

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Basic information

Entry
Database: PDB / ID: 8a8o
TitlePAPS reductase from Methanothermococcus thermolithotrophicus refined to 1.45 A
Components
  • Alpha-subunit of the PAPS reductase from Methanothermococcus thermolithotrophicus
  • Beta-subunit of the PAPS reductase from Methanothermococcus thermolithotrophicus
KeywordsOXIDOREDUCTASE / sulfate assimilation / methanogens / archaea / PAPS / PAP / sulfite / thermophile / methane / activation / marine / FAD / [4Fe-4S]-cluster / ferredoxin / electron transfer
Function / homologyFLAVIN-ADENINE DINUCLEOTIDE / IRON/SULFUR CLUSTER
Function and homology information
Biological speciesMethanothermococcus thermolithotrophicus DSM 2095 (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / AB INITIO PHASING / Resolution: 1.45 Å
AuthorsJespersen, M. / Wagner, T.
Funding support Germany, 2items
OrganizationGrant numberCountry
Max Planck Society Germany
German Research Foundation (DFG)WA 4053/1-1 Germany
CitationJournal: Nat Microbiol / Year: 2023
Title: Assimilatory sulfate reduction in the marine methanogen Methanothermococcus thermolithotrophicus.
Authors: Jespersen, M. / Wagner, T.
History
DepositionJun 23, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 26, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Alpha-subunit of the PAPS reductase from Methanothermococcus thermolithotrophicus
B: Alpha-subunit of the PAPS reductase from Methanothermococcus thermolithotrophicus
C: Beta-subunit of the PAPS reductase from Methanothermococcus thermolithotrophicus
D: Beta-subunit of the PAPS reductase from Methanothermococcus thermolithotrophicus
hetero molecules


Theoretical massNumber of molelcules
Total (without water)156,65027
Polymers152,3184
Non-polymers4,33223
Water15,835879
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Confirmed by gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)63.714, 123.650, 88.842
Angle α, β, γ (deg.)90.000, 104.440, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 2 types, 4 molecules ABCD

#1: Protein Alpha-subunit of the PAPS reductase from Methanothermococcus thermolithotrophicus


Mass: 64438.516 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: The alpha and beta subunits of the PAPS reductase were co-expressed. The alpha subunit contained a His-tag at the C-terminal position (additional sequence SSGHHHHHH).
Source: (gene. exp.) Methanothermococcus thermolithotrophicus DSM 2095 (archaea)
Strain: DSM 2095 / Tissue: / / Cell: / / Cell line: / / Organ: / / Variant: / / Plasmid: pET-28a(+)
Details (production host): The DNA sequence of the PAPS reductase alpha and beta subunits were synthesized in tandem and cloned in the pET-28a(+) by using the restriction sites NcoI and BamHI.
Cell (production host): / / Cell line (production host): / / Organ (production host): / / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Tissue (production host): / / Variant (production host): /
#2: Protein Beta-subunit of the PAPS reductase from Methanothermococcus thermolithotrophicus


Mass: 11720.575 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methanothermococcus thermolithotrophicus DSM 2095 (archaea)
Strain: DSM 2095 / Tissue: / / Cell: / / Cell line: / / Organ: / / Variant: / / Plasmid: pET-28a(+)
Details (production host): The DNA sequence of the PAPS reductase alpha and beta subunits were synthesized in tandem and cloned in the pET-28a(+) by using the restriction sites NcoI and BamHI.
Cell (production host): / / Cell line (production host): / / Organ (production host): / / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Tissue (production host): / / Variant (production host): /

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Non-polymers , 8 types, 902 molecules

#3: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#5: Chemical
ChemComp-MPD / (4S)-2-METHYL-2,4-PENTANEDIOL


Mass: 118.174 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C6H14O2 / Comment: precipitant*YM
#6: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C3H8O3
#7: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#8: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Na
#9: Chemical
ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 879 / 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: 2.23 Å3/Da / Density % sol: 44.84 %
Description: The crystals were brown, elongated plate-shaped.
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7
Details: Purified PAPS reductase from Methanothermococcus thermolithotrophicus at a concentration of 20 mg.ml-1 was anaerobically crystallized inside a Coy tent (N2/H2 at a 96:4 ratio) under yellow ...Details: Purified PAPS reductase from Methanothermococcus thermolithotrophicus at a concentration of 20 mg.ml-1 was anaerobically crystallized inside a Coy tent (N2/H2 at a 96:4 ratio) under yellow light condition. 0.7 ul of PAPSR in 25 mM Tris/HCl pH 7.6, 2 mM dithiothreitol, 0.5 mM FAD, and 10 % v/v glycerol was mixed with 0.7 ul reservoir solution. The drops were spotted in a 96-Well MRC 2-drop crystallization plates in polystyrene (SWISSCI) containing 90 ul of crystallization solution in the reservoir. Crystals appeared after a few days in the following crystallization condition: 35 % v/v 2-Methyl-2,4-pentanediol, 100 mM Tris pH 7.0 and 200 mM NaCl. Prior to transfer to liquid nitrogen, the crystal was soaked in 10 mM disodium 3'-Phosphoadenosine 5'-phosphate for 7 min.
PH range: / / Temp details: 293 +/- 1 K

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 0.97625 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Apr 21, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97625 Å / Relative weight: 1
ReflectionResolution: 1.45→86.04 Å / Num. obs: 146296 / % possible obs: 95 % / Redundancy: 6.8 % / Biso Wilson estimate: 19.2 Å2 / CC1/2: 0.997 / Rmerge(I) obs: 0.079 / Rpim(I) all: 0.033 / Rrim(I) all: 0.086 / Net I/σ(I): 11.9
Reflection shellResolution: 1.45→1.64 Å / Redundancy: 7.1 % / Rmerge(I) obs: 1.056 / Mean I/σ(I) obs: 1.7 / Num. unique obs: 7316 / CC1/2: 0.567 / Rpim(I) all: 0.425 / Rrim(I) all: 1.139 / % possible all: 73.8

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
PDB_EXTRACT3.27data extraction
autoPROCdata reduction
autoPROCdata scaling
CRANK2phasing
RefinementMethod to determine structure: AB INITIO PHASING / Resolution: 1.45→52.04 Å / SU ML: 0.11 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 23.52 / Stereochemistry target values: ML
Details: Model was refined with hydrogens in riding position. The final cycles of refinement were performed without applying non-crystallography symmetry and without translation-liberation screw.
RfactorNum. reflection% reflection
Rfree0.178 7256 4.96 %
Rwork0.1514 139027 -
obs0.1527 146283 62.29 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 140.41 Å2 / Biso mean: 32.1826 Å2 / Biso min: 9.18 Å2
Refinement stepCycle: final / Resolution: 1.45→52.04 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10278 0 223 879 11380
Biso mean--29.75 33.83 -
Num. residues----1303
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.45-1.470.143410.34232728
1.47-1.480.440860.32741051111
1.48-1.50.332790.28842542633
1.5-1.520.3242200.2764334536
1.52-1.540.3054340.27996096438
1.54-1.560.2472460.279487191712
1.56-1.580.261670.25131163123016
1.58-1.610.2759660.24831405147119
1.61-1.630.2756910.25211677176823
1.63-1.660.23061280.24092036216428
1.66-1.690.25711270.23992517264434
1.69-1.720.26121610.22852958311940
1.72-1.750.24332010.22943623382449
1.75-1.790.2442240.22094473469760
1.79-1.830.23553230.2265707603077
1.83-1.870.22973680.21666852722092
1.87-1.920.2463990.20787334773399
1.92-1.970.2283880.189674317819100
1.97-2.030.20063820.169674567838100
2.03-2.090.18363520.16374647816100
2.09-2.170.19923720.154275027874100
2.17-2.250.17753950.147973837778100
2.25-2.360.16993970.149274357832100
2.36-2.480.17593780.146774667844100
2.48-2.630.18043620.151774557817100
2.63-2.840.17963690.148374997868100
2.84-3.120.18253950.1574117806100
3.12-3.580.15554000.132574647864100
3.58-4.50.13784060.111374577863100
4.5-52.040.15963890.135675607949100

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