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- PDB-8c0j: Structure of AmiB enzymatic domain bound to the EnvC LytM domain -

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Basic information

Entry
Database: PDB / ID: 8c0j
TitleStructure of AmiB enzymatic domain bound to the EnvC LytM domain
Components
  • Murein hydrolase activator EnvC
  • N-acetylmuramoyl-L-alanine amidase
KeywordsHYDROLASE / Complex
Function / homology
Function and homology information


N-acetylmuramoyl-L-alanine amidase / N-acetylmuramoyl-L-alanine amidase activity / peptidoglycan catabolic process / outer membrane-bounded periplasmic space / hydrolase activity / metal ion binding
Similarity search - Function
Zn-dependent exopeptidases / : / Ami_3 / N-acetylmuramoyl-L-alanine amidase, catalytic domain / N-acetylmuramoyl-L-alanine amidase / : / Peptidase M23 / Peptidase family M23 / Duplicated hybrid motif / Aminopeptidase ...Zn-dependent exopeptidases / : / Ami_3 / N-acetylmuramoyl-L-alanine amidase, catalytic domain / N-acetylmuramoyl-L-alanine amidase / : / Peptidase M23 / Peptidase family M23 / Duplicated hybrid motif / Aminopeptidase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
PHOSPHATE ION / Murein hydrolase activator EnvC / N-acetylmuramoyl-L-alanine amidase
Similarity search - Component
Biological speciesCitrobacter rodentium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.381 Å
AuthorsCrow, A.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/V017101/1 United Kingdom
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2023
Title: Activator-induced conformational changes regulate division-associated peptidoglycan amidases.
Authors: Cook, J. / Baverstock, T.C. / McAndrew, M.B.L. / Roper, D.I. / Stansfeld, P.J. / Crow, A.
History
DepositionDec 17, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: N-acetylmuramoyl-L-alanine amidase
B: Murein hydrolase activator EnvC
C: N-acetylmuramoyl-L-alanine amidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)67,6337
Polymers67,3133
Non-polymers3214
Water00
1
A: N-acetylmuramoyl-L-alanine amidase
B: Murein hydrolase activator EnvC
hetero molecules


  • defined by author
  • Evidence: gel filtration, EnvC lytm domain and AmiB enzymatic domain were co-purified by IMAC with tag only on EnvC, they stay together on gel filtration. These proteins have also been shown to ...Evidence: gel filtration, EnvC lytm domain and AmiB enzymatic domain were co-purified by IMAC with tag only on EnvC, they stay together on gel filtration. These proteins have also been shown to interact by Bacterial 2-hybrid assay and the interface has been confirmed using site-directed mutagenesis to break the interaction.
  • 41.9 kDa, 2 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)41,8874
Polymers41,7272
Non-polymers1602
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
C: N-acetylmuramoyl-L-alanine amidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,7463
Polymers25,5861
Non-polymers1602
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)237.363, 237.363, 237.363
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number214
Space group name H-MI4132
Components on special symmetry positions
IDModelComponents
11A-502-

PO4

21A-502-

PO4

31C-502-

PO4

41C-502-

PO4

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21A

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: ASP / Beg label comp-ID: ASP / End auth comp-ID: LEU / End label comp-ID: LEU / Auth asym-ID: A / Label asym-ID: A / Auth seq-ID: 190 - 421 / Label seq-ID: 2 - 233

Dom-ID
1
2

NCS ensembles : (Details: Local NCS retraints between domains: 1 2)

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Components

#1: Protein N-acetylmuramoyl-L-alanine amidase


Mass: 25585.688 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Citrobacter rodentium (bacteria) / Gene: amiB, E2R62_12775 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43
References: UniProt: A0A482PQR2, N-acetylmuramoyl-L-alanine amidase
#2: Protein Murein hydrolase activator EnvC


Mass: 16141.135 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Citrobacter rodentium (bacteria) / Gene: envC, E2R62_17750 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: A0A482PID1
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4
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: 4.14 Å3/Da / Density % sol: 70.28 %
Crystal growTemperature: 290 K / Method: vapor diffusion, sitting drop / pH: 6.5 / Details: Na Phosphate pH7.6

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.95373 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Sep 11, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.95373 Å / Relative weight: 1
ReflectionResolution: 3.38→59.34 Å / Num. obs: 16294 / % possible obs: 100 % / Redundancy: 78.2 % / Biso Wilson estimate: 81 Å2 / CC1/2: 0.998 / Rpim(I) all: 0.088 / Net I/σ(I): 9.9
Reflection shellResolution: 3.38→3.65 Å / Mean I/σ(I) obs: 1.6 / Num. unique obs: 3305 / CC1/2: 0.939 / Rpim(I) all: 0.686

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Processing

Software
NameVersionClassification
REFMAC5.8.0352refinement
DIALSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: in silico model

Resolution: 3.381→50.005 Å / Cor.coef. Fo:Fc: 0.891 / Cor.coef. Fo:Fc free: 0.878 / SU B: 26.314 / SU ML: 0.383 / Cross valid method: FREE R-VALUE / ESU R Free: 0.517
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2909 817 5.074 %
Rwork0.2492 15286 -
all0.251 --
obs-16103 98.967 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 119.898 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20 Å2
2--0 Å20 Å2
3---0 Å2
Refinement stepCycle: LAST / Resolution: 3.381→50.005 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4045 0 12 0 4057
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.0124137
X-RAY DIFFRACTIONr_bond_other_d0.0010.0163755
X-RAY DIFFRACTIONr_ext_dist_refined_b0.0070.01333
X-RAY DIFFRACTIONr_angle_refined_deg1.4051.6455600
X-RAY DIFFRACTIONr_angle_other_deg0.4961.5638731
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.4075523
X-RAY DIFFRACTIONr_dihedral_angle_2_deg8.99540
X-RAY DIFFRACTIONr_dihedral_angle_3_deg18.89510669
X-RAY DIFFRACTIONr_dihedral_angle_6_deg13.74610195
X-RAY DIFFRACTIONr_chiral_restr0.0630.2607
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.024852
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02824
X-RAY DIFFRACTIONr_nbd_refined0.2480.21084
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1970.23939
X-RAY DIFFRACTIONr_nbtor_refined0.1850.22004
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0840.22404
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1970.254
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.1020.23
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1520.222
X-RAY DIFFRACTIONr_nbd_other0.1620.282
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.2620.24
X-RAY DIFFRACTIONr_mcbond_it12.0212.2092107
X-RAY DIFFRACTIONr_mcbond_other12.01812.2092107
X-RAY DIFFRACTIONr_mcangle_it17.81218.3582625
X-RAY DIFFRACTIONr_mcangle_other17.80918.362626
X-RAY DIFFRACTIONr_scbond_it10.90112.6372030
X-RAY DIFFRACTIONr_scbond_other10.91312.6492023
X-RAY DIFFRACTIONr_scangle_it17.07618.7252975
X-RAY DIFFRACTIONr_scangle_other17.10118.7422964
X-RAY DIFFRACTIONr_lrange_it22.52161.9895029
X-RAY DIFFRACTIONr_lrange_other22.518162.0045030
X-RAY DIFFRACTIONr_ncsr_local_group_10.0990.056298
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)Weight position
11AX-RAY DIFFRACTIONLocal ncs0.099260.05009
12AX-RAY DIFFRACTIONLocal ncs0.099260.05009
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection all% reflection obs (%)WRfactor Rwork
3.381-3.4680.373680.35910840.359115599.74030.358
3.468-3.5630.348450.37510930.374114099.82460.373
3.563-3.6660.654520.6459390.645112688.01070.678
3.666-3.7780.364500.29910210.302107699.53530.288
3.778-3.9010.344640.3039880.306105699.62120.283
3.901-4.0370.248590.2499660.24910251000.227
4.037-4.1880.296510.2179320.2219831000.191
4.188-4.3580.231410.1758900.17793499.67880.151
4.358-4.550.219440.158820.15492899.78450.126
4.55-4.7710.205360.1458360.1488721000.123
4.771-5.0260.265390.1928010.1968401000.165
5.026-5.3280.224380.1997660.20180599.87580.172
5.328-5.6910.363370.2277120.2337491000.196
5.691-6.140.277320.1796580.18369199.85530.153
6.14-6.7160.282340.1816330.18566899.85030.158
6.716-7.4920.237320.1945590.1965911000.176
7.492-8.6190.21240.2155080.2155321000.199
8.619-10.4780.307380.2214310.2274691000.211
10.478-14.5030.251180.2713550.273731000.261
14.503-50.0050.366150.5082320.4992471000.491

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