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- PDB-8fo0: The structure of a crystallizable variant of E. coli pyruvate for... -

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Basic information

Entry
Database: PDB / ID: 8fo0
TitleThe structure of a crystallizable variant of E. coli pyruvate formate-lyase activating enzyme bound to a partially cleaved SAM molecule
ComponentsPyruvate formate-lyase 1-activating enzyme
KeywordsOXIDOREDUCTASE / radical SAM / activase / PFL
Function / homology
Function and homology information


[formate-C-acetyltransferase]-activating enzyme / [formate-C-acetyltransferase]-activating enzyme activity / potassium ion binding / protein maturation / glucose metabolic process / 4 iron, 4 sulfur cluster binding / oxidoreductase activity / DNA damage response / cytosol
Similarity search - Function
Pyruvate formate-lyase 1 activating enzyme / Radical-activating enzyme, conserved site / Organic radical enzyme activase / Organic radical-activating enzymes / Pyruvate formate-lyase activase / Radical activating enzymes signature. / 4Fe-4S single cluster domain / Radical SAM superfamily / Radical SAM core domain profile. / Radical SAM / Aldolase-type TIM barrel
Similarity search - Domain/homology
: / S-ADENOSYLMETHIONINE / IRON/SULFUR CLUSTER / Pyruvate formate-lyase 1-activating enzyme
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.69 Å
AuthorsMoody, J.D. / Saxton, A.J. / Galambas, A. / Lawrence, C.M. / Broderick, J.B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH GM 131889, NIH GM 054608 United States
CitationJournal: J.Biol.Chem. / Year: 2023
Title: Computational engineering of previously crystallized pyruvate formate-lyase activating enzyme reveals insights into SAM binding and reductive cleavage.
Authors: Moody, J.D. / Hill, S. / Lundahl, M.N. / Saxton, A.J. / Galambas, A. / Broderick, W.E. / Lawrence, C.M. / Broderick, J.B.
History
DepositionDec 29, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 24, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 21, 2023Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2May 22, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pyruvate formate-lyase 1-activating enzyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,0264
Polymers28,2371
Non-polymers7893
Water2,468137
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: assay for oligomerization, Enzyme activity assay for transfer of a radical from PFL-AE-CCR8 to PFL
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)46.580, 58.551, 84.484
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein Pyruvate formate-lyase 1-activating enzyme / Formate-C-acetyltransferase-activating enzyme 1 / PFL-activating enzyme 1


Mass: 28237.273 Da / Num. of mol.: 1
Mutation: S1E, E53K, A93E, R111H, Q139K, E151R, K154Q, N158E, K222E, K225R, K226A, E230R
Source method: isolated from a genetically manipulated source
Details: Computationally redesigned protein / Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: pflA, act, b0902, JW0885 / Plasmid: pET42_SUMO / Details (production host): Kanamycin-resistant / Production host: Escherichia coli B (bacteria) / Variant (production host): BL21(DE3)RIL
References: UniProt: P0A9N4, [formate-C-acetyltransferase]-activating enzyme
#2: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE


Mass: 398.437 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H22N6O5S / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 137 / Source method: isolated from a natural source / Formula: H2O
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: 2.04 Å3/Da / Density % sol: 39.79 % / Description: Rod-like
Crystal growTemperature: 300 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 4 uL of protein (10 mg/mL PFL-AE-CCR8 in 25 mM Tris, 500 200 mM KCl, with 3.5 mM SAM, 5.0 mM 7-mer RVSAYAV peptide, 0.13% glycerol, and 2.5 mM DTT) added to 1 uL of crystallization reservoir ...Details: 4 uL of protein (10 mg/mL PFL-AE-CCR8 in 25 mM Tris, 500 200 mM KCl, with 3.5 mM SAM, 5.0 mM 7-mer RVSAYAV peptide, 0.13% glycerol, and 2.5 mM DTT) added to 1 uL of crystallization reservoir solution (30% PEG 3350, 100 mM Tris, pH 8.5) in hanging drop format over a reservoir containing of 50 uL of crystallization reservoir solution.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 / Wavelength: 1.542 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: Dec 30, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.542 Å / Relative weight: 1
ReflectionResolution: 1.69→34.26 Å / Num. obs: 26396 / % possible obs: 99.66 % / Redundancy: 11.7 % / Biso Wilson estimate: 26.46 Å2 / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.06465 / Rpim(I) all: 0.01912 / Rrim(I) all: 0.06748 / Net I/σ(I): 23.76
Reflection shellResolution: 1.69→1.753 Å / Redundancy: 8.5 % / Rmerge(I) obs: 0.683 / Mean I/σ(I) obs: 1.8 / Num. unique obs: 2527 / CC1/2: 0.898 / CC star: 0.973 / Rpim(I) all: 0.2236 / Rrim(I) all: 0.721 / % possible all: 97.15

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Processing

Software
NameVersionClassification
CrystalCleardata collection
PHENIX1.20.1-4487refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.69→34.26 Å / SU ML: 0.1926 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 18.4475
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Details: The cleaved and un-cleaved SAM molecule were refined separately and later combined as alternative conformations of a single molecule.
RfactorNum. reflection% reflectionSelection details
Rfree0.188 1294 4.9 %Random selection within each resolution shell
Rwork0.1642 25089 --
obs0.1653 26383 99.68 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 36.31 Å2
Refinement stepCycle: LAST / Resolution: 1.69→34.26 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1972 0 36 137 2145
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.03262171
X-RAY DIFFRACTIONf_angle_d0.86132951
X-RAY DIFFRACTIONf_chiral_restr0.2923312
X-RAY DIFFRACTIONf_plane_restr0.0045375
X-RAY DIFFRACTIONf_dihedral_angle_d16.8313793
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.69-1.760.30291270.26332686X-RAY DIFFRACTION97.4
1.76-1.840.23131550.2222723X-RAY DIFFRACTION99.86
1.84-1.940.27051510.20552760X-RAY DIFFRACTION100
1.94-2.060.18131340.17432755X-RAY DIFFRACTION99.97
2.06-2.220.20421340.16912794X-RAY DIFFRACTION100
2.22-2.440.19151500.15612782X-RAY DIFFRACTION99.97
2.44-2.790.19921310.16952807X-RAY DIFFRACTION100
2.79-3.520.18411750.16622797X-RAY DIFFRACTION99.93
3.52-34.260.15911370.14362985X-RAY DIFFRACTION99.94
Refinement TLS params.

Method: refined / 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)
12.00899091761-0.5092145741591.630206631337.1842375096-2.011156873066.465944835130.107750849763-0.0430123320879-0.0413564529612-0.0195196699386-0.1256882805640.1296474048940.416234582895-0.1402385224790.05217512716120.142221155589-0.03444109592730.01369276395170.2057691932360.001484535125670.158639782562-3.9503978167160.365691450628.5511329354
29.415694843511.34480497647-6.546278847321.61684149761-1.105209344164.61223628675-0.000463598324520.3090809672060.236210208045-0.1969508251610.1110304798570.264620289694-0.0215312398738-0.38298796457-0.1017647137170.2428292871950.0256515813826-0.05096984787670.2553722109450.02090520874350.228925194993-4.9283108665770.411174269917.7888759749
35.01987593903-4.97528496042-0.5799355984095.001244422761.012207307693.09948723967-0.517954127314-0.431441656989-0.2346421800370.8012149402760.4869987705030.4242505712060.397743717642-0.348645204890.005570545514360.340637951532-0.03994861089490.07882532243090.2737786748110.007066653788690.297052719972-8.2026230429557.679086207537.6589866276
44.28332858137-2.1312516041.26114864246.01710527933-1.277376904836.28121359810.0106230632533-0.147099793667-0.06654197572970.318028078360.0668536474974-0.09161975433970.255860737612-0.0207540506802-0.04259584841470.111894524631-0.0287486112741-0.02001124819410.132856299842-0.02180854946340.1607432055520.2206393998764.02349413234.9337313148
52.68318596930.681896282115-1.194230169084.50551110073-0.3992066809667.423074633840.0767830505246-0.0432803148560.3147405893260.05998916086590.005733837155610.0616016120908-0.593816572437-0.129827635936-0.1350998236290.1600545197420.047239009626-0.04221477465730.175067008259-0.03144716636780.2351826883435.0738301225672.526412657432.2431035778
61.92239808097-0.120922635991-1.046548656841.096464270790.4447390377266.31308951608-0.00896597087486-0.03581942630590.155179685752-0.1196089991940.156388132227-0.267083714879-0.2653011535670.437090588435-0.1395916212220.171663440311-0.01114482183450.02002885965150.206347841174-0.05218381343290.26910239311414.672419105269.957096479319.0528453823
78.50255379284-1.600406962763.754506614752.944704788090.3111017880176.407309543210.1263791800320.0149082646318-0.23220126144-0.3901571976850.0783555870837-0.0676930137910.373159453746-0.00684240240553-0.2432666900770.289545566972-0.04210594850390.07295218712080.191333037390.007901959059410.1812173493478.5546020289362.188568432916.1693644543
83.06634128483-4.55848951727-1.07710097937.394527726431.12401723434.973204199570.7411504503471.41810577013-0.560086884225-0.946408207747-1.020996808320.676771129701-0.551164811761-0.3457053915220.2337166544630.4908849758220.035919637044-0.05592145755640.56182229605-0.1306818234270.4289442559292.5638374712563.15001805343.13242555078
95.162437175252.925308550724.393905114486.030508112142.924350162413.83358728689-0.211784123251-0.369990033249-0.3555253335640.1851084209490.304271487226-0.4134358087890.7321709153010.657197793898-0.1038543712780.2810524375710.1141872304860.04717855573740.456159486293-0.09112863136020.37438149899221.984732846857.343072962315.4236965358
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION / Auth asym-ID: A / Label asym-ID: A

IDRefine TLS-IDSelection detailsAuth seq-IDLabel seq-ID
11chain 'A' and (resid 1 through 26 )1 - 261 - 26
22chain 'A' and (resid 27 through 51 )27 - 5127 - 51
33chain 'A' and (resid 52 through 65 )52 - 6552 - 65
44chain 'A' and (resid 66 through 96 )66 - 9666 - 96
55chain 'A' and (resid 97 through 122 )97 - 12297 - 122
66chain 'A' and (resid 123 through 187 )123 - 187123 - 187
77chain 'A' and (resid 188 through 211 )188 - 211188 - 211
88chain 'A' and (resid 212 through 225 )212 - 225212 - 225
99chain 'A' and (resid 226 through 245 )226 - 245226 - 245

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