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- PDB-7f5j: The crystal structure of VyPAL2-I244V, a more efficient mutant of... -

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Basic information

Entry
Database: PDB / ID: 7f5j
TitleThe crystal structure of VyPAL2-I244V, a more efficient mutant of VyPAL2 peptide asparaginyl ligase in its active enzyme form
ComponentsPeptide Asparaginyl Ligases
KeywordsPLANT PROTEIN / AEP / Legumain / Peptide ligase / PAL
Function / homology
Function and homology information


vacuolar protein processing / vacuole / proteolysis involved in protein catabolic process / cysteine-type endopeptidase activity
Similarity search - Function
Asparaginyl endopeptidase / Legumain prodomain superfamily / : / Legumain, prodomain / Peptidase C13, legumain / Peptidase C13 family
Similarity search - Domain/homology
Peptide Asparaginyl Ligases
Similarity search - Component
Biological speciesViola philippica (plant)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.593 Å
AuthorsHu, S. / Sahili, A. / Lescar, J.
Funding support Singapore, 1items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore)MOE2016-T3-1-003 Singapore
CitationJournal: Plant Cell / Year: 2022
Title: Structural basis for proenzyme maturation, substrate recognition, and ligation by a hyperactive peptide asparaginyl ligase.
Authors: Hu, S. / El Sahili, A. / Kishore, S. / Wong, Y.H. / Hemu, X. / Goh, B.C. / Zhipei, S. / Wang, Z. / Tam, J.P. / Liu, C.F. / Lescar, J.
History
DepositionJun 22, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 29, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 30, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Dec 14, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.name
Revision 2.0Nov 15, 2023Group: Atomic model / Data collection
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / chem_comp_atom / chem_comp_bond / pdbx_validate_main_chain_plane / pdbx_validate_peptide_omega / pdbx_validate_rmsd_angle
Item: _atom_site.auth_atom_id / _atom_site.label_atom_id ..._atom_site.auth_atom_id / _atom_site.label_atom_id / _atom_site_anisotrop.pdbx_auth_atom_id / _atom_site_anisotrop.pdbx_label_atom_id / _pdbx_validate_rmsd_angle.angle_deviation / _pdbx_validate_rmsd_angle.angle_standard_deviation / _pdbx_validate_rmsd_angle.angle_target_value / _pdbx_validate_rmsd_angle.angle_value / _pdbx_validate_rmsd_angle.auth_atom_id_1 / _pdbx_validate_rmsd_angle.auth_atom_id_2 / _pdbx_validate_rmsd_angle.auth_atom_id_3 / _pdbx_validate_rmsd_angle.auth_comp_id_1 / _pdbx_validate_rmsd_angle.auth_comp_id_3 / _pdbx_validate_rmsd_angle.auth_seq_id_1 / _pdbx_validate_rmsd_angle.auth_seq_id_3
Revision 2.1Nov 29, 2023Group: Refinement description / Category: pdbx_initial_refinement_model
Revision 2.2May 1, 2024Group: Structure summary / Category: struct / Item: _struct.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Peptide Asparaginyl Ligases
B: Peptide Asparaginyl Ligases
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,65518
Polymers62,2382
Non-polymers2,41716
Water9,008500
1
A: Peptide Asparaginyl Ligases
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,45311
Polymers31,1191
Non-polymers1,33410
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1640 Å2
ΔGint12 kcal/mol
Surface area11440 Å2
MethodPISA
2
B: Peptide Asparaginyl Ligases
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,2027
Polymers31,1191
Non-polymers1,0836
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1180 Å2
ΔGint11 kcal/mol
Surface area11210 Å2
MethodPISA
Unit cell
Length a, b, c (Å)76.27, 78.98, 92.12
Angle α, β, γ (deg.)90, 90, 90
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Peptide Asparaginyl Ligases


Mass: 31118.818 Da / Num. of mol.: 2 / Mutation: I244V
Source method: isolated from a genetically manipulated source
Details: D171 (forming SNN), residue 172 is HD0 (HIS + SNN) / Source: (gene. exp.) Viola philippica (plant) / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A509GV09

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Sugars , 3 types, 6 molecules

#2: Polysaccharide alpha-L-fucopyranose-(1-6)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 367.349 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
LFucpa1-6DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,2,1/[a2122h-1b_1-5_2*NCC/3=O][a1221m-1a_1-5]/1-2/a6-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(6+1)][a-L-Fucp]{}}}LINUCSPDB-CARE
#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 3 types, 510 molecules

#5: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C2H6O2
#6: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H8O3
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 500 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.22 Å3/Da / Density % sol: 44.65 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / Details: 0.1 M Bis-Tris pH 5.5, 25% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.9537 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Oct 20, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9537 Å / Relative weight: 1
ReflectionResolution: 1.593→46.06 Å / Num. obs: 74533 / % possible obs: 99.26 % / Redundancy: 13.4 % / Biso Wilson estimate: 29.35 Å2 / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.08307 / Rpim(I) all: 0.02365 / Rrim(I) all: 0.08643 / Net I/σ(I): 16.5
Reflection shellResolution: 1.593→1.65 Å / Redundancy: 12.5 % / Mean I/σ(I) obs: 0.83 / Num. unique obs: 6869 / CC1/2: 0.449 / CC star: 0.787 / Rpim(I) all: 0.8825 / % possible all: 92.83

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Processing

Software
NameVersionClassification
BUSTER2.10.3refinement
autoPROCdata reduction
XDSdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6IDV

6idv


Resolution: 1.593→46.06 Å / Cor.coef. Fo:Fc: 0.964 / Cor.coef. Fo:Fc free: 0.95 / SU R Cruickshank DPI: 0.081 / Cross valid method: FREE R-VALUE / SU R Blow DPI: 0.086 / SU Rfree Blow DPI: 0.085 / SU Rfree Cruickshank DPI: 0.082
RfactorNum. reflection% reflectionSelection details
Rfree0.1927 3721 -RANDOM
Rwork0.1631 ---
obs-74372 99.26 %-
Displacement parametersBiso mean: 37.91 Å2
Baniso -1Baniso -2Baniso -3
1-6.9977 Å20 Å20 Å2
2---4.1657 Å20 Å2
3----2.832 Å2
Refine analyzeLuzzati coordinate error obs: 0.2 Å
Refinement stepCycle: LAST / Resolution: 1.593→46.06 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4215 0 172 500 4887
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0084538HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.956164HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d1537SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes774HARMONIC5
X-RAY DIFFRACTIONt_it4538HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion612SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact4252SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion3.7
X-RAY DIFFRACTIONt_other_torsion14.9
LS refinement shellResolution: 1.593→1.61 Å
RfactorNum. reflection% reflection
Rfree0.4455 75 -
Rwork0.3941 --
obs--72.06 %
Refinement TLS params.

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)
12.11350.526-0.3571.3338-0.50161.1151-0.0176-0.09980.0366-0.09980.0682-0.07480.0366-0.0748-0.0506-0.08050.0007-0.0088-0.07920.0069-0.07335.4048-8.5187-8.7539
22.33610.9856-0.03261.4241-0.07421.08510.00440.0373-0.06590.0373-0.02240.094-0.06590.0940.018-0.0786-0.0060.0047-0.061-0.0141-0.0864-2.98258.6526-11.1216
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1{ A|* }
2X-RAY DIFFRACTION2{ B|* }

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