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- PDB-7m6u: Crystal structure of a circular permutation and computationally d... -

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Basic information

Entry
Database: PDB / ID: 7m6u
TitleCrystal structure of a circular permutation and computationally designed pro-enzyme of carboxypeptidase G2
ComponentsCarboxypeptidase G2 circular permuation pro-domain fusion
KeywordsHYDROLASE / pro-enzyme / enzyme design / directed enzyme-prodrug therapy / methotrexate / circular permutation
Function / homology
Function and homology information


glutamate carboxypeptidase / carboxypeptidase activity / metallopeptidase activity / proteolysis / metal ion binding
Similarity search - Function
Peptidase M20, glutamate carboxypeptidase / ArgE / dapE / ACY1 / CPG2 / yscS family signature 1. / ArgE / dapE / ACY1 / CPG2 / yscS family signature 2. / ArgE/DapE/ACY1/CPG2/YscS, conserved site / : / Bacterial exopeptidase dimerisation domain / Peptidase M20, dimerisation domain / Peptidase dimerisation domain / Peptidase M20 / Peptidase family M20/M25/M40
Similarity search - Domain/homology
Biological speciesPseudomonas sp. (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.59 Å
AuthorsYachnin, B.J. / Khare, S.D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM132565 United States
Citation
Journal: Proc.Natl.Acad.Sci.USA / Year: 2022
Title: Massively parallel, computationally guided design of a proenzyme.
Authors: Yachnin, B.J. / Azouz, L.R. / White 3rd, R.E. / Minetti, C.A.S.A. / Remeta, D.P. / Tan, V.M. / Drake, J.M. / Khare, S.D.
#1: Journal: Biorxiv / Year: 2021
Title: Massively parallel, computationally-guided design of a pro-enzyme
Authors: Yachnin, B.J. / Azouz, L.R. / White, R.E. / Minetti, C.A.S.A. / Remeta, D.P. / Tan, V.M. / Drake, J.M. / Khare, S.D.
History
DepositionMar 26, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 14, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 13, 2022Group: Database references / Category: citation / citation_author / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Oct 18, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / pdbx_initial_refinement_model
Item: _citation.journal_id_ISSN

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Carboxypeptidase G2 circular permuation pro-domain fusion
B: Carboxypeptidase G2 circular permuation pro-domain fusion
C: Carboxypeptidase G2 circular permuation pro-domain fusion
D: Carboxypeptidase G2 circular permuation pro-domain fusion
hetero molecules


Theoretical massNumber of molelcules
Total (without water)190,27325
Polymers188,8694
Non-polymers1,40421
Water2,378132
1
A: Carboxypeptidase G2 circular permuation pro-domain fusion
hetero molecules

C: Carboxypeptidase G2 circular permuation pro-domain fusion
hetero molecules


  • defined by author&software
  • Evidence: gel filtration, CPG2 has been well established as a dimer in the literature. Gel filtration experiments with this variant confirm that it also behaves as a dimer, as expected., SAXS, CPG2 ...Evidence: gel filtration, CPG2 has been well established as a dimer in the literature. Gel filtration experiments with this variant confirm that it also behaves as a dimer, as expected., SAXS, CPG2 has been well established as a dimer in the literature. Related SAXS data show good agreement with the model of the dimeric form of CPG2.
  • 95.3 kDa, 2 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)95,25014
Polymers94,4352
Non-polymers81612
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_455-x-1,y+1/2,-z1
Buried area3870 Å2
ΔGint-337 kcal/mol
Surface area31460 Å2
MethodPISA
2
B: Carboxypeptidase G2 circular permuation pro-domain fusion
hetero molecules

D: Carboxypeptidase G2 circular permuation pro-domain fusion
hetero molecules


  • defined by author&software
  • Evidence: gel filtration, CPG2 has been well established as a dimer in the literature. Gel filtration experiments with this variant confirm that it also behaves as a dimer, as expected., SAXS, CPG2 ...Evidence: gel filtration, CPG2 has been well established as a dimer in the literature. Gel filtration experiments with this variant confirm that it also behaves as a dimer, as expected., SAXS, CPG2 has been well established as a dimer in the literature. Related SAXS data show good agreement with the model of the dimeric form of CPG2.
  • 95 kDa, 2 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)95,02311
Polymers94,4352
Non-polymers5899
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation1_556x,y,z+11
Buried area3520 Å2
ΔGint-293 kcal/mol
Surface area32520 Å2
MethodPISA
Unit cell
Length a, b, c (Å)78.54, 106.36, 121.93
Angle α, β, γ (deg.)90, 107.48, 90
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
Carboxypeptidase G2 circular permuation pro-domain fusion / CPDG2 / Folate hydrolase G2 / Glutamate carboxypeptidase / Pteroylmonoglutamic acid hydrolase G2


Mass: 47217.277 Da / Num. of mol.: 4 / Mutation: K177A
Source method: isolated from a genetically manipulated source
Details: Pro-enzyme form
Source: (gene. exp.) Pseudomonas sp. (strain RS-16) (bacteria), (gene. exp.) synthetic construct (others)
Strain: RS-16 / Gene: cpg2 / Plasmid: pET15b / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P06621, glutamate carboxypeptidase
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 132 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.57 Å3/Da / Density % sol: 52.18 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8
Details: Protein was prepared to a concentration of 19 mg/mL in 50 mM Tris 100 mM NaCl pH 7.4 0.2 mM ZnSO4. Reservoirs containing 750 uL of 20 mM Tris pH 8.0, 10% glycerol, and 10% PEG 3350 were ...Details: Protein was prepared to a concentration of 19 mg/mL in 50 mM Tris 100 mM NaCl pH 7.4 0.2 mM ZnSO4. Reservoirs containing 750 uL of 20 mM Tris pH 8.0, 10% glycerol, and 10% PEG 3350 were prepared in 24 well hanging drop vapor diffusion plates. Equal volumes of protein solution and reservoir solution were mixed on a cover slip and suspended over the reservoir

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Data collection

DiffractionMean temperature: 93 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 / Wavelength: 1.54178 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: Jun 6, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54178 Å / Relative weight: 1
ReflectionResolution: 2.59→39.27 Å / Num. obs: 57919 / % possible obs: 97.2 % / Redundancy: 2.9 % / CC1/2: 0.952 / Rmerge(I) obs: 0.102 / Rpim(I) all: 0.071 / Rrim(I) all: 0.125 / Net I/σ(I): 6.8
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) all% possible all
2.59-2.632.480.9460.928330.3880.7191.19595.77
2.63-2.682.60.7361.128610.650.5360.91598.15
2.68-2.732.590.4661.729330.7820.340.57998.32
2.73-2.792.580.8411.429180.7050.4970.84197.36
2.79-2.852.40.6871.826830.6870.4140.68792.01
2.85-2.922.590.4012.129470.8280.2970.50197.84
2.92-2.992.630.2922.629060.9190.2120.36298.44
2.99-3.072.630.244329110.9360.1780.30398.44
3.07-3.162.940.2483.529480.9510.1670.398.56
3.16-3.262.670.243.928240.9440.1690.29595.34
3.26-3.383.020.2054.829410.9530.1380.24998.72
3.38-3.513.040.2135.929160.2620.1590.26898.45
3.51-3.672.890.1069.529180.9390.0750.13197.59
3.67-3.872.950.1049.527840.9550.0750.12893.8
3.87-4.112.970.10510.528040.8160.0770.13193.62
4.11-4.433.220.0612.429720.9940.0390.07299.63
4.43-4.873.240.05313.629690.9960.0340.06399.3
4.87-5.583.250.05413.429640.9950.0350.06598.6
5.58-7.023.250.05214.329540.9960.0330.06298.4
7.02-39.253.210.03519.529330.9980.0220.04195.6

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
xia20.5.277-gc78b72c6-dials-1.5data reduction
xia20.5.277-gc78b72c6-dials-1.5data scaling
PHASER2.7.17phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1CG2

1cg2


Resolution: 2.59→39.27 Å / SU ML: 0.329 / Cross valid method: THROUGHOUT / ESU R: 1.72 / ESU R Free: 0.388
Details: Partway through the refinement, reflections between 4 and 3.4 A were deleted due to low data quality. Refining using the truncated dataset resulted in an improvement in Rfree by ~0.05, and ...Details: Partway through the refinement, reflections between 4 and 3.4 A were deleted due to low data quality. Refining using the truncated dataset resulted in an improvement in Rfree by ~0.05, and so the truncated dataset was used for the rest of refinement.
RfactorNum. reflection% reflectionSelection details
Rfree0.27917 2346 4.9 %Random
Rwork0.22172 ---
obs0.22451 45894 80.89 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 53.598 Å2
Baniso -1Baniso -2Baniso -3
1-5.5 Å20 Å21.54 Å2
2---1.71 Å20 Å2
3----3.97 Å2
Refinement stepCycle: LAST / Resolution: 2.59→39.27 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10948 0 25 132 11105
LS refinement shellResolution: 2.59→2.657 Å
RfactorNum. reflection% reflection
Rfree0.384 219 5.2 %
Rwork0.342 3998 -
obs--96.34 %

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