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

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Basic information

Entry
Database: PDB / ID: 8fsi
TitleThe structure of a crystallizable variant of E. coli pyruvate formate-lyase activating enzyme bound to SAM
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 / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.46 Å
AuthorsMoody, J.D. / 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
DepositionJan 10, 2023Deposition 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)29,1016
Polymers28,2371
Non-polymers8645
Water4,558253
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization, This protein can pass a radical to pyruvate-formate-lyase
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)46.405, 59.094, 83.545
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

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Protein , 1 types, 1 molecules A

#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 for reproducible crystallization
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: pflA, act, b0902, JW0885 / Plasmid: pET42_SUMO
Details (production host): Kanamycin-resistant, cleavable N-terminal 10xHis-SUMO tag
Production host: Escherichia coli B (bacteria) / Variant (production host): BL21(DE3)RIL
References: UniProt: P0A9N4, [formate-C-acetyltransferase]-activating enzyme

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Non-polymers , 5 types, 258 molecules

#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.: 2 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 253 / 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: 2.03 Å3/Da / Density % sol: 39.44 % / Description: rod-like
Crystal growTemperature: 300 K / Method: vapor diffusion, hanging drop / pH: 9
Details: 4 uL of protein (10 mg/mL PFL-AE-CCR8 in 12.5 mM HEPES, 200 mM KCl, 3.5 mM SAM, 5.0 mM WT 7-mer PFL peptide, and 2.5 mM DTT) with 1 uL of crystallization reservoir solution (28% PEG 3350, ...Details: 4 uL of protein (10 mg/mL PFL-AE-CCR8 in 12.5 mM HEPES, 200 mM KCl, 3.5 mM SAM, 5.0 mM WT 7-mer PFL peptide, and 2.5 mM DTT) with 1 uL of crystallization reservoir solution (28% PEG 3350, 100 mM glycine, pH 9.0) in hanging drop format over 50 uL of crystallization reservoir solution

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9791 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Feb 22, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9791 Å / Relative weight: 1
ReflectionResolution: 1.46→41.77 Å / Num. obs: 39365 / % possible obs: 96.88 % / Redundancy: 7.6 % / Biso Wilson estimate: 16.31 Å2 / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.05246 / Rpim(I) all: 0.01912 / Rrim(I) all: 0.056 / Net I/σ(I): 19.76
Reflection shellResolution: 1.46→1.512 Å / Redundancy: 7.7 % / Rmerge(I) obs: 0.2999 / Mean I/σ(I) obs: 3.66 / Num. unique obs: 3661 / CC1/2: 0.959 / CC star: 0.989 / Rpim(I) all: 0.1104 / Rrim(I) all: 0.3205 / % possible all: 91.96

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
Cootmodel building
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.46→41.77 Å / SU ML: 0.1297 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 14.752
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.173 1890 4.8 %
Rwork0.1511 37523 -
obs0.1522 39365 96.49 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 24.39 Å2
Refinement stepCycle: LAST / Resolution: 1.46→41.77 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1975 0 38 253 2266
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01172109
X-RAY DIFFRACTIONf_angle_d1.19762865
X-RAY DIFFRACTIONf_chiral_restr0.0834301
X-RAY DIFFRACTIONf_plane_restr0.0125366
X-RAY DIFFRACTIONf_dihedral_angle_d16.9913801
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.46-1.490.22381270.2032341X-RAY DIFFRACTION85.69
1.49-1.530.19331380.16922603X-RAY DIFFRACTION95.27
1.53-1.580.15381430.14872624X-RAY DIFFRACTION96.44
1.58-1.630.16771370.14722662X-RAY DIFFRACTION97.36
1.63-1.690.18711150.15392654X-RAY DIFFRACTION96.11
1.69-1.760.18551250.15472702X-RAY DIFFRACTION98.06
1.76-1.840.17091420.14262711X-RAY DIFFRACTION98.01
1.84-1.930.1761440.14692673X-RAY DIFFRACTION97.47
1.93-2.050.17451440.15092649X-RAY DIFFRACTION96.58
2.05-2.210.20271360.14912751X-RAY DIFFRACTION98.67
2.21-2.440.15681260.14322744X-RAY DIFFRACTION98.19
2.44-2.790.19531210.15092712X-RAY DIFFRACTION96.3
2.79-3.510.18241460.15582820X-RAY DIFFRACTION99.33
3.51-41.770.14881480.14962877X-RAY DIFFRACTION97.11
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)
11.319904165670.5821613752420.7447716989911.493783313670.8823412369763.612641764120.01054252275990.0765317460388-0.0147555618036-0.06148077237260.0324888018236-0.104979632495-0.07625531307140.338446178045-0.05807758610520.09237516338640.001533469784430.02057583417270.1296194753290.01873157854280.110190616934.39227759005-6.9818625008922.7235927181
22.237395035580.3469593226840.712405948362.96094994397-0.4483922794393.20516125101-0.0394694178441-0.072685404432-0.04160358835020.2823432571820.070651225846-0.0295186982118-0.04594143474050.10086533059-0.01705184143270.1036898197850.02814602873780.01600195511510.09196826154110.02057076201790.09571658901510.749299928493-7.4647294641833.506671099
31.15650652554-0.2648001549590.9312421595180.94321639945-0.3710900046662.684737900910.0636865105543-0.123570851376-0.168445621353-0.0688979516790.1313073728080.2637841363620.25896222108-0.390344071357-0.1696316408190.134932381694-0.0250178905342-0.01463666362840.1413437774490.05944858123250.188034355806-13.9173848053-12.204637954620.4953450955
44.19453034224-0.3987036889870.2223986748271.65997036611-0.871704726652.95214866592-0.01389167829920.1431141667160.047104398595-0.2432016515720.02182838127890.0556552811898-0.123081466119-0.0718612083001-0.002034758384080.186598069824-0.00551490183228-0.02665077918250.1378291703630.03951039870780.170995491241-12.0174426727-2.2265408021312.5979926221
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 51 )1 - 511 - 51
22chain 'A' and (resid 52 through 114 )52 - 11452 - 114
33chain 'A' and (resid 115 through 187 )115 - 187115 - 187
44chain 'A' and (resid 188 through 245 )188 - 245188 - 245

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