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- PDB-8ewa: Crystal Structure of UDP-N-acetylmuramate-L-alanine ligase (UDP-N... -

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Basic information

Entry
Database: PDB / ID: 8ewa
TitleCrystal Structure of UDP-N-acetylmuramate-L-alanine ligase (UDP-N-acetylmuramoyl-L-alanine synthetase, MurC) Pseudomonas aeruginosa in complex with ligand AZ-13644923
ComponentsUDP-N-acetylmuramate--L-alanine ligase
KeywordsLIGASE / SSGCID / murC / Cell wall biogenesis / peptidoglycan biosynthesis / Structural Genomics / Seattle Structural Genomics Center for Infectious Disease
Function / homology
Function and homology information


UDP-N-acetylmuramate-L-alanine ligase / UDP-N-acetylmuramate-L-alanine ligase activity / peptidoglycan biosynthetic process / cell wall organization / regulation of cell shape / cell cycle / cell division / ATP binding / cytoplasm
Similarity search - Function
UDP-N-acetylmuramate--L-alanine ligase / Mur ligase, N-terminal catalytic domain / Mur ligase family, catalytic domain / Mur ligase, C-terminal / Mur ligase family, glutamate ligase domain / Mur ligase, C-terminal domain superfamily / Mur ligase, central / Mur-like, catalytic domain superfamily / Mur ligase middle domain
Similarity search - Domain/homology
Chem-WYI / UDP-N-acetylmuramate--L-alanine ligase
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.85 Å
AuthorsSeattle Structural Genomics Center for Infectious Disease (SSGCID)
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)75N93022C00036 United States
CitationJournal: to be published
Title: Crystal Structure of UDP-N-acetylmuramate-L-alanine ligase (UDP-N-acetylmuramoyl-L-alanine synthetase, MurC) Pseudomonas aeruginosa in complex with ligand AZ-13644923
Authors: Abendroth, J. / Hill, P. / Horanyi, P.S. / Lorimer, D.D. / Edwards, T.E.
History
DepositionOct 21, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 9, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: UDP-N-acetylmuramate--L-alanine ligase
B: UDP-N-acetylmuramate--L-alanine ligase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)69,2787
Polymers67,9532
Non-polymers1,3255
Water8,593477
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3370 Å2
ΔGint-51 kcal/mol
Surface area24230 Å2
MethodPISA
Unit cell
Length a, b, c (Å)71.270, 76.950, 110.230
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 UDP-N-acetylmuramate--L-alanine ligase / UDP-N-acetylmuramoyl-L-alanine synthetase


Mass: 33976.645 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Gene: murC, PA4411 / Plasmid: PsaeA.00137.b.B5 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: Q9HW02, UDP-N-acetylmuramate-L-alanine ligase
#2: Chemical ChemComp-WYI / (2R)-2-({4-[(5-tert-butyl-1-methyl-1H-pyrazol-3-yl)amino]-1H-pyrazolo[3,4-d]pyrimidin-6-yl}amino)-2-phenylethyl (2R)-2-(aminomethyl)morpholine-4-carboxylate


Mass: 548.640 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H36N10O3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-CL / CHLORIDE ION / Chloride


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


Mass: 18.015 Da / Num. of mol.: 477 / 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: 2.23 Å3/Da / Density % sol: 44.9 %
Crystal growTemperature: 287 K / Method: vapor diffusion, sitting drop / pH: 5.9
Details: Molecular Dimensions Morpheus II, optimization screen around condition A1 and A10: 100mM Tris base/HCl pH 8.6, 11% (w/V) PEG 4000, 18% (V/V) 1,2,4-butanetriol, 30mM lithum sulfate, 30mM ...Details: Molecular Dimensions Morpheus II, optimization screen around condition A1 and A10: 100mM Tris base/HCl pH 8.6, 11% (w/V) PEG 4000, 18% (V/V) 1,2,4-butanetriol, 30mM lithum sulfate, 30mM potassium sulfate: PseaA.00137.b.B5.PW39129 at 12mg/ml + 1mM BSI111803 / AZ13644923: tray: 325495b11: cryo: direct: puck tui7-6

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-F / Wavelength: 0.97872 Å
DetectorType: RAYONIX MX-300 / Detector: CCD / Date: Oct 6, 2022
RadiationMonochromator: Diamond [111] / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97872 Å / Relative weight: 1
ReflectionResolution: 1.85→50 Å / Num. obs: 52456 / % possible obs: 100 % / Redundancy: 7.314 % / Biso Wilson estimate: 32.163 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.074 / Rrim(I) all: 0.079 / Χ2: 0.931 / Net I/σ(I): 16.44
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
1.85-1.97.3870.6343.3338360.9090.682100
1.9-1.957.3940.4744.3737300.9410.51100
1.95-2.017.390.3735.5236290.9560.401100
2.01-2.077.4020.2847.0835460.9740.306100
2.07-2.147.3930.2328.5834090.9820.24999.9
2.14-2.217.3980.18610.3933070.9880.2100
2.21-2.297.3940.15812.1632170.990.17100
2.29-2.397.3940.13313.8930950.9930.143100
2.39-2.497.3810.11415.5729720.9940.123100
2.49-2.627.3880.10217.5428240.9950.109100
2.62-2.767.3530.08520.2927140.9960.09199.9
2.76-2.937.3350.07523.2525740.9960.08100
2.93-3.137.2850.06625.8124060.9970.071100
3.13-3.387.2820.05529.1122710.9980.06100
3.38-3.77.2140.0531.7320710.9980.054100
3.7-4.147.150.04633.919080.9980.05100
4.14-4.787.0880.04435.516990.9970.048100
4.78-5.857.0060.04335.0114440.9980.04799.9
5.85-8.276.8490.04434.3511310.9970.048100
8.27-506.1830.04235.046730.9950.04699

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Processing

Software
NameVersionClassification
XDSdata reduction
XSCALEdata scaling
PHENIX1.20.1 4742refinement
PDB_EXTRACT3.27data extraction
PHASERphasing
PHENIXmodel building
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 8EGN, same crystal form

8egn


Resolution: 1.85→47.24 Å / SU ML: 0.2126 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 20.6822
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflectionSelection details
Rfree0.2145 2037 3.88 %0
Rwork0.1672 50416 --
obs0.169 52453 99.97 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 29.73 Å2
Refinement stepCycle: LAST / Resolution: 1.85→47.24 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4482 0 91 477 5050
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00834752
X-RAY DIFFRACTIONf_angle_d0.95676494
X-RAY DIFFRACTIONf_chiral_restr0.0647758
X-RAY DIFFRACTIONf_plane_restr0.0083852
X-RAY DIFFRACTIONf_dihedral_angle_d14.47821724
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.85-1.890.32371440.26663304X-RAY DIFFRACTION100
1.89-1.940.26871480.2133307X-RAY DIFFRACTION99.94
1.94-1.990.20841300.18413313X-RAY DIFFRACTION100
1.99-2.050.24911500.17443305X-RAY DIFFRACTION100
2.05-2.120.23591160.1793341X-RAY DIFFRACTION100
2.12-2.190.23241610.19163299X-RAY DIFFRACTION99.91
2.19-2.280.21381270.17443354X-RAY DIFFRACTION100
2.28-2.390.25971470.17273318X-RAY DIFFRACTION100
2.39-2.510.28151370.17543322X-RAY DIFFRACTION99.97
2.51-2.670.21411440.18463349X-RAY DIFFRACTION100
2.67-2.870.22021420.17423361X-RAY DIFFRACTION99.94
2.87-3.160.20241240.16873388X-RAY DIFFRACTION100
3.16-3.620.20221040.15333429X-RAY DIFFRACTION100
3.62-4.560.15211250.1393444X-RAY DIFFRACTION99.97
4.56-47.240.21571380.15913582X-RAY DIFFRACTION99.87
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)
11.896867348740.00129138550895-0.06786464764362.63444570179-0.6157673165212.121860639240.01840186780160.0502116657307-0.082976905691-0.2150637315660.05699892126610.02992329471960.1560549477570.02913462504-0.04373916370720.145259354199-0.0274379018371-0.0314788134360.143467455565-0.03498050589230.131270803478-24.05322491784.0961536963131.0865568403
24.09195165237-0.00615697314214-1.108130109474.755122434557.51691535158E-53.08036937143-0.0897276339818-0.08238059008430.317534769085-0.0915265460470.1175419503970.454649586733-0.316756104829-0.210004665636-0.02669773855940.1518393385770.000194049752421-0.01130329817630.139055600175-0.002983111625390.177047526556-33.846065132712.299170586538.8700787797
34.717771576581.769440553641.00285660092.004593675030.1843744582393.681382263240.07312522724-0.03918311651990.01839062525510.0282174066078-0.02968129730310.0802867105122-0.0399108981912-0.137529155829-0.04757233911960.1757560715470.0587888906305-0.006446652550770.177663373616-0.02778826782070.1411497034-2.20577493182-1.9003113133148.33862069
42.62375261816-0.017760711814-0.2890382511071.627795534860.1105924513662.725868565990.0495203897756-0.166672726314-0.08285585582890.0356350991962-0.101276848127-0.002845510086810.03803404855030.003972932551010.03468066904520.119223980453-0.002816312052180.000362687592560.09544066976690.03422689165380.13538258124212.2412059503-1.0782242735420.5916387091
52.9389389024-0.474920525993-1.311549896473.978975858320.4087412148513.913226581080.005567871362050.09348951791250.198982784417-0.2620996221260.1075364060770.116208889304-0.3027212829490.131938474716-0.09675621423150.203518497714-0.0429179355611-0.02314600177520.1313327980960.002383242098750.167472755718-10.648525275213.87743947067.34637793997
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'B' and (resid 115 through 264 )BD115 - 264103 - 245
22chain 'B' and (resid 265 through 322 )BD265 - 322246 - 303
33chain 'A' and (resid 13 through 114 )AA13 - 1141 - 102
44chain 'A' and (resid 115 through 322 )AA115 - 322103 - 302
55chain 'B' and (resid 13 through 114 )BD13 - 1141 - 102

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