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

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Basic information

Entry
Database: PDB / ID: 8fol
TitleThe structure of a crystallizable variant of E. coli pyruvate formate-lyase activating enzyme bound to SAM, alternate crystal form
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: 2.65 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 31, 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)29,1337
Polymers28,2371
Non-polymers8966
Water18010
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: assay for oligomerization, This enzyme has been observed passing a radical to pyruvate formate-lyase
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)49.653, 59.011, 96.792
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
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

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Non-polymers , 5 types, 16 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.: 1 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 10 / 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: 2.51 Å3/Da / Density % sol: 51.08 % / Description: rod-like
Crystal growTemperature: 300 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 4 uL of protein (10 mg/mL PFL-AE-CCR8 in 12.5 mM HEPES, 200 mM KCl, with 3.65 mM SAM, 1.2 mM WT 7-mer PFL peptide, 0.13% glycerol, and 1 mM DTT) were combined with 1 uL of crystallization ...Details: 4 uL of protein (10 mg/mL PFL-AE-CCR8 in 12.5 mM HEPES, 200 mM KCl, with 3.65 mM SAM, 1.2 mM WT 7-mer PFL peptide, 0.13% glycerol, and 1 mM DTT) were combined with 1 uL of crystallization reservoir solution (18% PEG 3350, 100 mM HEPES, pH 7.5) 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: ROTATING ANODE / Type: RIGAKU MICROMAX-007 / Wavelength: 1.542 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: Aug 14, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.542 Å / Relative weight: 1
ReflectionResolution: 2.65→35.37 Å / Num. obs: 8650 / % possible obs: 97.67 % / Redundancy: 4.3 % / Biso Wilson estimate: 55.81 Å2 / CC1/2: 0.996 / CC star: 0.999 / Rmerge(I) obs: 0.1248 / Rpim(I) all: 0.06559 / Rrim(I) all: 0.1417 / Net I/σ(I): 10.26
Reflection shellResolution: 2.65→2.745 Å / Redundancy: 4.2 % / Rmerge(I) obs: 0.8407 / Mean I/σ(I) obs: 1.29 / Num. unique obs: 752 / CC1/2: 0.71 / CC star: 0.911 / Rpim(I) all: 0.4408 / Rrim(I) all: 0.9537 / % possible all: 88.01

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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: 2.65→35.37 Å / SU ML: 0.3407 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 23.0446
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2142 426 5 %
Rwork0.1947 8090 -
obs0.1957 8516 97.67 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 54.7 Å2
Refinement stepCycle: LAST / Resolution: 2.65→35.37 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1975 0 39 10 2024
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00612069
X-RAY DIFFRACTIONf_angle_d0.78592804
X-RAY DIFFRACTIONf_chiral_restr0.0941296
X-RAY DIFFRACTIONf_plane_restr0.0031358
X-RAY DIFFRACTIONf_dihedral_angle_d16.5804755
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.65-2.7450.30471360.27742572X-RAY DIFFRACTION95.59
3.03-3.820.23911430.21032721X-RAY DIFFRACTION99.34
3.82-35.370.17671470.16492797X-RAY DIFFRACTION98.04
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)
15.84872926578-2.12194379443-1.126757019488.04274928122-1.185299389442.985653515170.301453535050.0309984409314-0.1240373162120.310616106614-0.174185401093-0.118073836580.270515363658-0.0630507781904-0.1289863765760.4169910654420.00866364147365-0.06520744795950.4667307444710.03149823164940.32317921908253.655175738659.289883743232.1008085876
23.04720392495-2.873264289464.540513091525.20805274315-3.991452989756.91791482868-0.239170256468-0.352154273520.02854141058840.04908400384560.3079715680690.202569145185-0.113722315669-0.486122438365-0.0350766639740.322103261096-0.03191197665240.02282556156150.4362071663890.005683070855740.39154811414549.932162426563.751040327231.5476275332
36.35319805091-1.264858455290.3287379615394.68038897520.4140212089985.483782655120.196408643125-0.280019367819-0.03288744554430.321543734770.0160436437898-0.600500930972-0.05829804700450.418383170349-0.2980640258630.4202389093370.00749137329032-0.06800365392680.40971961499-0.02150248694490.38593302373660.329654666865.100489901531.9850643516
46.25192375895-2.396095267883.437224746766.73147869586-1.311122827135.90081635314-0.0795019712222-0.07843247096410.2975204126090.4239776194370.111788529055-0.0441424414056-0.6931755723340.2811862729350.001242187770230.325726746979-0.1050433147230.04974581186170.4641229943660.009382490869830.35639750196661.817388073671.038057597726.0997617092
54.77597825166-0.8750872025511.358574043052.084238629421.139745307191.70583966929-0.09866761228350.2561846203740.0104096923961-0.26957233563-0.07119474373690.135459510334-0.42237701101-0.01024303882560.1159954015880.431841274649-0.01289659115290.002608418411330.330789340305-0.02956418722110.33176661538452.443934406670.256954748412.0977014499
64.54133035582-0.9044003015741.326722176193.735189226511.634133917284.9944261046-0.1479127112810.2495312036520.204977314084-0.403725540987-0.0211239354607-0.176292864886-0.2892598724880.377211384720.1684043381480.442490339251-0.05735701210380.05354375214170.4815125964890.09993778238590.34161685780359.086119329666.35827882919.88925480503
75.30563184347-2.67448534071-6.748274488164.629410815163.071262025898.59033546653-0.2153271052320.401799013518-0.7149920030830.402520092149-0.7175063705030.5893963883680.290245276338-0.8794280162291.000558426520.600421672142-0.0623993948923-0.06301989829820.727163280119-0.07884784685170.8066524062636.907119587460.568083747615.0860180664
85.898758214641.980155813773.389297616366.03998044191-0.06651895277885.72616430970.1874811943690.367494291836-0.524949403467-0.0802461062802-0.02663063026360.1763599643070.3891210056880.568982579084-0.2431887520560.6369709656030.02662192713040.1074556298380.425162031972-0.1115319162840.43155676068757.523367753455.85783502312.58434073199
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 66 )27 - 6627 - 66
33chain 'A' and (resid 67 through 96 )67 - 9667 - 96
44chain 'A' and (resid 97 through 122 )97 - 12297 - 122
55chain 'A' and (resid 123 through 146 )123 - 146123 - 146
66chain 'A' and (resid 147 through 200 )147 - 200147 - 200
77chain 'A' and (resid 201 through 225 )201 - 225201 - 225
88chain 'A' and (resid 226 through 245 )226 - 245226 - 245

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