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- PDB-8b8h: Structure of DCS-resistant variant D322N of alanine racemase from... -

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Basic information

Entry
Database: PDB / ID: 8b8h
TitleStructure of DCS-resistant variant D322N of alanine racemase from M. tuberculosis in complex with DCS
ComponentsAlanine racemase
KeywordsISOMERASE / Enzyme / alanine racemase / peptidoglycan biosynthesis
Function / homology
Function and homology information


alanine racemase / D-alanine biosynthetic process / alanine racemase activity / peptidoglycan biosynthetic process / pyridoxal phosphate binding / cytosol
Similarity search - Function
Alanine racemase, pyridoxal-phosphate attachment site / Alanine racemase pyridoxal-phosphate attachment site. / Alanine racemase / Alanine racemase, C-terminal / Alanine racemase, C-terminal domain / Alanine racemase, C-terminal domain / Alanine racemase, N-terminal / Alanine racemase, N-terminal domain / Alanine racemase/group IV decarboxylase, C-terminal / PLP-binding barrel
Similarity search - Domain/homology
Chem-L7N / Chem-OJQ / Alanine racemase
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.78 Å
Authorsde Chiara, C. / Prosser, G. / Ogrodowicz, R.W. / de Carvalho, L.P.S.
Funding support United Kingdom, 5items
OrganizationGrant numberCountry
The Francis Crick InstituteSee list below United Kingdom
Cancer Research UKFC001060 United Kingdom
Medical Research Council (MRC, United Kingdom)FC001060 United Kingdom
Wellcome TrustFC001060 United Kingdom
Wellcome Trust104785/B/14/Z United Kingdom
Citation
Journal: Acs Bio Med Chem Au / Year: 2023
Title: Structure of the d-Cycloserine-Resistant Variant D322N of Alanine Racemase from Mycobacterium tuberculosis .
Authors: de Chiara, C. / Prosser, G.A. / Ogrodowicz, R. / de Carvalho, L.P.S.
#1: Journal: Nat Commun / Year: 2019
Title: Comparative fitness analysis of D-cycloserine resistant mutants reveals both fitness-neutral and high-fitness cost genotypes.
Authors: Evangelopoulos, D. / Prosser, G.A. / Rodgers, A. / Dagg, B.M. / Khatri, B. / Ho, M.M. / Gutierrez, M.G. / Cortes, T. / de Carvalho, L.P.S.
#2: Journal: Nat Chem Biol / Year: 2020
Title: D-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition.
Authors: de Chiara, C. / Homsak, M. / Prosser, G.A. / Douglas, H.L. / Garza-Garcia, A. / Kelly, G. / Purkiss, A.G. / Tate, E.W. / de Carvalho, L.P.S.
#3: Journal: Acta Crystallogr D Biol Crystallogr / Year: 2012
Title: Towards automated crystallographic structure refinement with phenix.refine.
Authors: Afonine, P.V. / Grosse-Kunstleve, R.W. / Echols, N. / Headd, J.J. / Moriarty, N.W. / Mustyakimov, M. / Terwilliger, T.C. / Urzhumtsev, A. / Zwart, P.H. / Adams, P.D.
#4: Journal: Acta Crystallogr D Struct Biol / Year: 2019
Title: Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.
Authors: Liebschner, D. / Afonine, P.V. / Baker, M.L. / Bunkoczi, G. / Chen, V.B. / Croll, T.I. / Hintze, B. / Hung, L.W. / Jain, S. / McCoy, A.J. / Moriarty, N.W. / Oeffner, R.D. / Poon, B.K. / ...Authors: Liebschner, D. / Afonine, P.V. / Baker, M.L. / Bunkoczi, G. / Chen, V.B. / Croll, T.I. / Hintze, B. / Hung, L.W. / Jain, S. / McCoy, A.J. / Moriarty, N.W. / Oeffner, R.D. / Poon, B.K. / Prisant, M.G. / Read, R.J. / Richardson, J.S. / Richardson, D.C. / Sammito, M.D. / Sobolev, O.V. / Stockwell, D.H. / Terwilliger, T.C. / Urzhumtsev, A.G. / Videau, L.L. / Williams, C.J. / Adams, P.D.
History
DepositionOct 4, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 5, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 7, 2024Group: 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: Alanine racemase
B: Alanine racemase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)83,48612
Polymers82,1002
Non-polymers1,38610
Water6,179343
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: homology
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5720 Å2
ΔGint-34 kcal/mol
Surface area25350 Å2
MethodPISA
Unit cell
Length a, b, c (Å)163.940, 163.940, 57.110
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212
Space group name HallP4abw2nw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+1/4
#3: y+1/2,-x+1/2,z+3/4
#4: x+1/2,-y+1/2,-z+3/4
#5: -x+1/2,y+1/2,-z+1/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2
Components on special symmetry positions
IDModelComponents
11A-403-

CA

21A-686-

HOH

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Alanine racemase


Mass: 41049.891 Da / Num. of mol.: 2 / Mutation: D322N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: alr, Rv3423c, MTCY78.06
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P9WQA9, alanine racemase

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Non-polymers , 7 types, 353 molecules

#2: Chemical ChemComp-OJQ / [2-methyl-3-oxidanyl-5-(phosphonooxymethyl)pyridin-4-yl]methyl-(3-oxidanyl-1,2-oxazol-4-yl)azanium


Mass: 332.226 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H15N3O7P / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-L7N / (~{E})-[2-methyl-3-oxidanyl-5-(phosphonooxymethyl)pyridin-4-yl]methylidene-[(4~{R})-3-oxidanylidene-1,2-oxazolidin-4-yl]azanium


Mass: 332.226 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C11H15N3O7P / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#6: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C3H8O3
#7: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6O2
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 343 / 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: 2.34 Å3/Da / Density % sol: 47.37 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 6.2
Details: 100 mM sodium MES buffer pH 6.2, 150 mM CaCl, 10.5% (v/v) PEG Smear Broad, 4.3% (v/v) Glycerol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.9159 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Dec 2, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9159 Å / Relative weight: 1
ReflectionResolution: 1.78→57.96 Å / Num. obs: 74924 / % possible obs: 100 % / Redundancy: 12.3 % / Biso Wilson estimate: 30.21 Å2 / CC1/2: 1 / Net I/σ(I): 10.2
Reflection shellResolution: 1.78→1.91 Å / Rmerge(I) obs: 1.765 / Num. unique obs: 3661 / CC1/2: 0.3 / Rpim(I) all: 0.569

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Processing

Software
NameVersionClassification
DIALSdata reduction
xia2data scaling
PHASERphasing
PHENIX1.20.1_4487refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6SCZ

6scz


Resolution: 1.78→53.93 Å / SU ML: 0.2833 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 22.565
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2229 3789 5.06 %
Rwork0.1992 71059 -
obs0.2004 74848 99.99 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 37.14 Å2
Refinement stepCycle: LAST / Resolution: 1.78→53.93 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5395 0 87 343 5825
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01475698
X-RAY DIFFRACTIONf_angle_d1.10697791
X-RAY DIFFRACTIONf_chiral_restr0.0697910
X-RAY DIFFRACTIONf_plane_restr0.01081023
X-RAY DIFFRACTIONf_dihedral_angle_d7.9147869
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.78-1.80.4361280.41052585X-RAY DIFFRACTION99.96
1.8-1.830.4411310.39472594X-RAY DIFFRACTION100
1.83-1.850.40611250.36612623X-RAY DIFFRACTION100
1.85-1.880.35941330.33092561X-RAY DIFFRACTION99.96
1.88-1.910.34871500.31692622X-RAY DIFFRACTION100
1.91-1.940.31361340.28792575X-RAY DIFFRACTION99.96
1.94-1.970.27481420.2542618X-RAY DIFFRACTION99.96
1.97-20.30361310.23892589X-RAY DIFFRACTION100
2-2.040.24321490.22282617X-RAY DIFFRACTION100
2.04-2.080.25121580.22082570X-RAY DIFFRACTION100
2.08-2.120.25251220.21872643X-RAY DIFFRACTION100
2.12-2.170.26271550.22222553X-RAY DIFFRACTION100
2.17-2.220.26121340.22122639X-RAY DIFFRACTION100
2.22-2.270.31651510.23232601X-RAY DIFFRACTION100
2.27-2.330.22741540.22762575X-RAY DIFFRACTION99.96
2.33-2.40.2451380.20032636X-RAY DIFFRACTION100
2.4-2.480.2431430.19132623X-RAY DIFFRACTION100
2.48-2.570.21961390.18522622X-RAY DIFFRACTION100
2.57-2.670.19951520.1792615X-RAY DIFFRACTION100
2.67-2.790.21561330.17052636X-RAY DIFFRACTION100
2.79-2.940.22341370.17962652X-RAY DIFFRACTION100
2.94-3.120.20571300.182659X-RAY DIFFRACTION100
3.12-3.360.18371390.17572686X-RAY DIFFRACTION100
3.36-3.70.19691550.16412645X-RAY DIFFRACTION100
3.7-4.240.19081410.16552699X-RAY DIFFRACTION100
4.24-5.340.16341410.16692728X-RAY DIFFRACTION99.97
5.34-53.930.22331440.22582893X-RAY DIFFRACTION99.9
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)
10.3714520402260.05536610461350.3406013979351.716783735220.3002332810831.138837058550.07745187678590.134691105837-0.0695445581924-0.0952919505632-0.06655821454180.09857512185640.1623421546920.2736012855580.01411401554870.1807700989210.0672693421781-0.02576038923250.250861621993-0.03167803604450.22425940505227.4643044551.157226232937.5738203467
20.7628858857760.2138974715560.4247716788370.701519600398-0.09107559465871.19746980944-0.04181849747730.311986244326-0.09278114375-0.153429331269-0.0423928181693-0.125643140543-0.1455465966650.5475530752680.06159539030360.206779609373-0.0661301007088-0.0008718973409490.5174385903670.05295473857750.25979141968551.440055752216.917847124410.6535112637
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION / Auth seq-ID: 13 - 383

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDLabel seq-ID
11chain 'A' and (resid 13 through 383 )AA1 - 371
22chain 'B' and (resid 13 through 383 )BD2 - 371

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