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- PDB-1sku: E. coli Aspartate Transcarbamylase 240's Loop Mutant (K244N) -

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Basic information

Entry
Database: PDB / ID: 1sku
TitleE. coli Aspartate Transcarbamylase 240's Loop Mutant (K244N)
Components
  • Aspartate carbamoyltransferase catalytic chain
  • Aspartate carbamoyltransferase regulatory chain
KeywordsTRANSFERASE / allosteric enzyme / loop movements / small-angle X-ray scattering / domain closure / allosteric transition / intersubunit interactions
Function / homology
Function and homology information


aspartate carbamoyltransferase complex / pyrimidine nucleotide biosynthetic process / aspartate carbamoyltransferase / aspartate carbamoyltransferase activity / amino acid binding / glutamine metabolic process / protein homotrimerization / 'de novo' UMP biosynthetic process / 'de novo' pyrimidine nucleobase biosynthetic process / zinc ion binding ...aspartate carbamoyltransferase complex / pyrimidine nucleotide biosynthetic process / aspartate carbamoyltransferase / aspartate carbamoyltransferase activity / amino acid binding / glutamine metabolic process / protein homotrimerization / 'de novo' UMP biosynthetic process / 'de novo' pyrimidine nucleobase biosynthetic process / zinc ion binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Aspartate transcarbamylase regulatory subunit / Aspartate carbamoyltransferase regulatory subunit, C-terminal / Aspartate carbamoyltransferase regulatory subunit, N-terminal / Aspartate carbamoyltransferase regulatory subunit, C-terminal domain superfamily / Aspartate carbamoyltransferase regulatory subunit, N-terminal domain superfamily / Aspartate carbamoyltransferase regulatory chain, allosteric domain / Aspartate carbamoyltransferase regulatory chain, metal binding domain / Aspartate carbamoyltransferase regulatory subunit, N-terminal domain / Aspartate carbamoyltransferase regulatory subunit, C-terminal domain / Aspartate/ornithine carbamoyltransferase ...Aspartate transcarbamylase regulatory subunit / Aspartate carbamoyltransferase regulatory subunit, C-terminal / Aspartate carbamoyltransferase regulatory subunit, N-terminal / Aspartate carbamoyltransferase regulatory subunit, C-terminal domain superfamily / Aspartate carbamoyltransferase regulatory subunit, N-terminal domain superfamily / Aspartate carbamoyltransferase regulatory chain, allosteric domain / Aspartate carbamoyltransferase regulatory chain, metal binding domain / Aspartate carbamoyltransferase regulatory subunit, N-terminal domain / Aspartate carbamoyltransferase regulatory subunit, C-terminal domain / Aspartate/ornithine carbamoyltransferase / Aspartate carbamoyltransferase / Aspartate and ornithine carbamoyltransferases signature. / Aspartate/ornithine carbamoyltransferase / Aspartate/ornithine carbamoyltransferase, Asp/Orn-binding domain / Aspartate/ornithine carbamoyltransferase, carbamoyl-P binding / Aspartate/ornithine carbamoyltransferase superfamily / Aspartate/ornithine carbamoyltransferase, Asp/Orn binding domain / Aspartate/ornithine carbamoyltransferase, carbamoyl-P binding domain / SH3 type barrels. / Roll / Alpha-Beta Plaits / Rossmann fold / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
MALONATE ION / Aspartate carbamoyltransferase catalytic subunit / Aspartate carbamoyltransferase regulatory chain
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsAlam, N. / Stieglitz, K.A. / Caban, M.D. / Gourinath, S. / Tsuruta, H. / Kantrowitz, E.R.
CitationJournal: J.Biol.Chem. / Year: 2004
Title: 240s Loop Interactions Stabilize the T State of Escherichia coli Aspartate Transcarbamoylase.
Authors: Alam, N. / Stieglitz, K.A. / Caban, M.D. / Gourinath, S. / Tsuruta, H. / Kantrowitz, E.R.
History
DepositionMar 5, 2004Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 30, 2004Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Advisory / Derived calculations / Version format compliance
Revision 1.3Oct 27, 2021Group: Database references / Derived calculations
Category: database_2 / pdbx_struct_conn_angle ...database_2 / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Aug 23, 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: Aspartate carbamoyltransferase catalytic chain
B: Aspartate carbamoyltransferase regulatory chain
C: Aspartate carbamoyltransferase catalytic chain
D: Aspartate carbamoyltransferase regulatory chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)103,2668
Polymers102,9314
Non-polymers3354
Water1,18966
1
A: Aspartate carbamoyltransferase catalytic chain
B: Aspartate carbamoyltransferase regulatory chain
C: Aspartate carbamoyltransferase catalytic chain
D: Aspartate carbamoyltransferase regulatory chain
hetero molecules

A: Aspartate carbamoyltransferase catalytic chain
B: Aspartate carbamoyltransferase regulatory chain
C: Aspartate carbamoyltransferase catalytic chain
D: Aspartate carbamoyltransferase regulatory chain
hetero molecules

A: Aspartate carbamoyltransferase catalytic chain
B: Aspartate carbamoyltransferase regulatory chain
C: Aspartate carbamoyltransferase catalytic chain
D: Aspartate carbamoyltransferase regulatory chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)309,79924
Polymers308,79412
Non-polymers1,00512
Water21612
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
Buried area31980 Å2
ΔGint-76 kcal/mol
Surface area111200 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)125.670, 125.670, 198.200
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number146
Space group name H-MH3

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Components

#1: Protein Aspartate carbamoyltransferase catalytic chain / Aspartate transcarbamylase / ATCase


Mass: 34322.031 Da / Num. of mol.: 2 / Mutation: K244N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: PYRB, B4245, C5345, Z5856, ECS5222, SF4245, S4507 / Plasmid: pEK582 / Production host: Escherichia coli (E. coli) / Strain (production host): EK1104 / References: UniProt: P0A786, aspartate carbamoyltransferase
#2: Protein Aspartate carbamoyltransferase regulatory chain


Mass: 17143.625 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: PYRI, B4244, C5344, Z5855, ECS5221 / Plasmid: pEK582 / Production host: Escherichia coli (E. coli) / Strain (production host): EK1104 / References: UniProt: P0A7F3, aspartate carbamoyltransferase
#3: Chemical ChemComp-MLI / MALONATE ION


Mass: 102.046 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H2O4
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 66 / Source method: isolated from a natural source / Formula: H2O

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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: 3.04 Å3/Da / Density % sol: 59.22 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 7
Details: Prior to crystallization the enzyme was dialyzed into 40 mM KH2PO4, 2.0 mM 2-mercaptoethanol, 0.2 mM EDTA, pH 7.0 for 24 hours. Single crystals of the K244N mutant were obtained by mixing a ...Details: Prior to crystallization the enzyme was dialyzed into 40 mM KH2PO4, 2.0 mM 2-mercaptoethanol, 0.2 mM EDTA, pH 7.0 for 24 hours. Single crystals of the K244N mutant were obtained by mixing a 20 mg/ml filtered solution (0.22 m) of the K244N enzyme with a solution of 17% (w/v) PEG 1450, 50 mM malonate, 0.2 mM EDTA, 1 mM sodium azide and 20 mM Bis-Tris buffer pH 7.0 in a 1:1 ratio (v/v), VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X12C / Wavelength: 1.00704 Å
DetectorType: BRANDEIS - B4 / Detector: CCD / Date: Dec 30, 2003
RadiationMonochromator: Si 111 CHANNEL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.00704 Å / Relative weight: 1
ReflectionResolution: 2.59→30 Å / Num. all: 36343 / Num. obs: 34988 / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 6.5 % / Rmerge(I) obs: 0.039 / Net I/σ(I): 13.3
Reflection shellResolution: 2.59→2.68 Å / Redundancy: 2.62 % / Rmerge(I) obs: 0.327 / Mean I/σ(I) obs: 12.2 / % possible all: 89.8

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Processing

Software
NameVersionClassification
REFMAC5.1.24refinement
DENZOdata reduction
SCALEPACKdata scaling
AMoREphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: A consensus model using overlayed previously published pdb entry 1EZZ (mutant P268A catalytic chain) and 1NBE (T82A regulatory chain) and wild type 3AT1 was used.

1ezz

1nbe

3at1


Resolution: 2.6→29.55 Å / Cor.coef. Fo:Fc: 0.888 / Cor.coef. Fo:Fc free: 0.81 / SU B: 10.082 / SU ML: 0.227 / TLS residual ADP flag: LIKELY RESIDUAL
Isotropic thermal model: Isotropic thermal model was retained for individual atoms in final runs but global anisotropic assignment for domains (TLS)
Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 1.267 / ESU R Free: 0.335 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.23117 1589 4.9 %RANDOM
Rwork0.20122 ---
obs0.20186 30529 89.44 %-
all-36343 --
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 34.638 Å2
Baniso -1Baniso -2Baniso -3
1-0.58 Å20.29 Å20 Å2
2--0.58 Å20 Å2
3----0.86 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.35 Å0.31 Å
Luzzati d res low-5 Å
Luzzati sigma a0.46 Å0.45 Å
Refinement stepCycle: LAST / Resolution: 2.6→29.55 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7147 0 16 66 7229
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0630.0217285
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg4.5861.9589875
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg10.4125912
X-RAY DIFFRACTIONr_dihedral_angle_2_deg
X-RAY DIFFRACTIONr_dihedral_angle_3_deg
X-RAY DIFFRACTIONr_dihedral_angle_4_deg
X-RAY DIFFRACTIONr_chiral_restr0.3180.21150
X-RAY DIFFRACTIONr_gen_planes_refined0.020.025472
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined0.3930.24249
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.3250.2322
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.4650.2198
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.4720.221
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_mcbond_it2.3581.54575
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it3.83727389
X-RAY DIFFRACTIONr_scbond_it6.11532710
X-RAY DIFFRACTIONr_scangle_it8.6324.52486
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.6→2.666 Å / Total num. of bins used: 20 /
RfactorNum. reflection
Rfree0.28 92
Rwork0.256 1777
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)
1-0.31210.14530.18780.27830.12650.17010.0031-0.0402-0.0660.02190.0461-0.0342-0.01010.0677-0.04920.10590.0095-0.01380.10430.02350.0869-2.519-16.60226.782
2-0.0623-0.0448-0.127-0.0369-0.13060.04610.0133-0.0013-0.02630.0217-0.00990.04260.0002-0.0373-0.00340.1155-0.0107-0.00240.10020.02920.1033-21.809-23.21418.913
3-0.6043-1.004-0.0649-0.1097-0.43380.61310.02470.03380.1303-0.0945-0.08870.141-0.048-0.02910.0640.0323-0.0272-0.03530.0854-0.01480.070522.027-52.2348.591
4-0.11940.3225-0.1283-0.71270.53371.2443-0.0385-0.0272-0.1520.02630.08020.06970.03780.0547-0.04170.1098-0.0018-0.02380.06240.00820.15613.712-35.49610.51
50.54050.2877-0.06530.2448-0.23060.14510.02170.0201-0.0308-0.0584-0.0087-0.0609-0.0061-0.0049-0.0130.10330.00090.01530.0813-0.03160.07215.556-6.261-26.697
60.2576-0.02250.0435-0.0305-0.143-0.0521-0.0007-0.01840.0182-0.02690.0004-0.0448-0.01320.00090.00030.08620.00950.02080.1079-0.03640.099531.0527.381-18.603
70.3664-0.40290.191-0.69450.3517-0.2597-0.0375-0.0295-0.0126-0.0053-0.0514-0.02210.0947-0.06280.08890.08250.02010.00080.0519-0.06010.125635.094-44.832-8.144
8-0.07410.32680.9229-0.0677-0.03670.25120.04690.0737-0.0948-0.0192-0.0453-0.0326-0.0508-0.0317-0.00160.0627-0.0142-0.01860.0942-0.02780.141628.645-20.931-10.238
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
1X-RAY DIFFRACTION1AA1 - 1501 - 150
2X-RAY DIFFRACTION2AA151 - 310151 - 310
3X-RAY DIFFRACTION3BB6 - 1006 - 100
4X-RAY DIFFRACTION4BB101 - 153101 - 153
5X-RAY DIFFRACTION5CC1 - 1501 - 150
6X-RAY DIFFRACTION6CC151 - 310151 - 310
7X-RAY DIFFRACTION7DD6 - 1006 - 100
8X-RAY DIFFRACTION8DD101 - 153101 - 153

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