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- PDB-2h5d: 0.9A resolution crystal structure of alpha-lytic protease complex... -

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Basic information

Entry
Database: PDB / ID: 2h5d
Title0.9A resolution crystal structure of alpha-lytic protease complexed with a transition state analogue, MeOSuc-Ala-Ala-Pro-Val boronic acid
Components
  • ALPHA-LYTIC PROTEASE
  • MEOSUC-ALA-ALA-PRO-ALA BORONIC ACID INHIBITOR
KeywordsHYDROLASE/HYDROLASE INHIBITOR / A-LYTIC PROTEASE / SERINE PROTEASE / ACYLATION TRANSITION STATE / CATALYSIS / PROTEIN FOLDING / PROTEIN STABILITY / PACKING DISTORTION / HYDROLASE-HYDROLASE INHIBITOR COMPLEX
Function / homology
Function and homology information


alpha-lytic endopeptidase / serine-type endopeptidase activity / proteolysis / extracellular region
Similarity search - Function
Alpha-lytic protease prodomain / Streptogrisin prodomain / Peptidase S1A, alpha-lytic prodomain / Alpha-lytic protease prodomain / Peptidase S1A, streptogrisin / Serine proteases, trypsin family, histidine active site. / Serine proteases, trypsin family, serine active site. / Serine proteases, trypsin domain / Trypsin / Trypsin-like serine proteases ...Alpha-lytic protease prodomain / Streptogrisin prodomain / Peptidase S1A, alpha-lytic prodomain / Alpha-lytic protease prodomain / Peptidase S1A, streptogrisin / Serine proteases, trypsin family, histidine active site. / Serine proteases, trypsin family, serine active site. / Serine proteases, trypsin domain / Trypsin / Trypsin-like serine proteases / Thrombin, subunit H / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / Beta Barrel / Mainly Beta
Similarity search - Domain/homology
METHOXYSUCCINYL-ALA-ALA-PRO-VALINE BORONIC ACID / Alpha-lytic protease
Similarity search - Component
Biological speciesLysobacter enzymogenes (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / REFINEMENT OF PREVIOUSLY-SOLVED STRUCTURE OF ALPHA-LYTIC PROTEASE BOUND TO MEOSUC-ALA-ALA-PRO-ALA BORONIC ACID / Resolution: 0.9 Å
AuthorsFuhrmann, C.N. / Agard, D.A.
Citation
Journal: J.Am.Chem.Soc. / Year: 2006
Title: Subangstrom crystallography reveals that short ionic hydrogen bonds, and not a His-Asp low-barrier hydrogen bond, stabilize the transition state in serine protease catalysis
Authors: Fuhrmann, C.N. / Daugherty, M.D. / Agard, D.A.
#1: Journal: J.Mol.Biol. / Year: 2004
Title: The 0.83A resolution crystal structure of alpha-lytic protease reveals the detailed structure of the active site and identifies a source of conformational strain
Authors: Fuhrmann, C.N. / Kelch, B.A. / Ota, N. / Agard, D.A.
#2: Journal: Biochemistry / Year: 1989
Title: Structural analysis of specificity: alpha-lytic protease complexes with analogues of reaction intermediates
Authors: Bone, R. / Frank, D. / Kettner, C.A. / Agard, D.A.
History
DepositionMay 25, 2006Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 26, 2006Provider: repository / Type: Initial release
Revision 1.1May 1, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Atomic model / Database references ...Atomic model / Database references / Derived calculations / Non-polymer description / Structure summary / Version format compliance
Revision 1.3Dec 12, 2012Group: Other
Revision 1.4Aug 30, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _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_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 2.0Nov 15, 2023Group: Atomic model / Data collection / Category: atom_site / chem_comp_atom / chem_comp_bond
Item: _atom_site.auth_atom_id / _atom_site.label_atom_id ..._atom_site.auth_atom_id / _atom_site.label_atom_id / _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ALPHA-LYTIC PROTEASE
B: MEOSUC-ALA-ALA-PRO-ALA BORONIC ACID INHIBITOR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)21,20611
Polymers20,3452
Non-polymers8619
Water7,782432
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2280 Å2
ΔGint-81 kcal/mol
Surface area8060 Å2
MethodPISA
Unit cell
Length a, b, c (Å)65.768, 65.768, 79.554
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221
Components on special symmetry positions
IDModelComponents
11A-595-

HOH

21A-604-

HOH

31A-605-

HOH

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Components

#1: Protein ALPHA-LYTIC PROTEASE / E.C.3.4.21.12 / ALPHA-LYTIC ENDOPEPTIDASE


Mass: 19875.131 Da / Num. of mol.: 1 / Fragment: MATURE PROTEASE DOMAIN (RESIDUES 200-397) / Source method: isolated from a natural source / Details: gene alpha-LP / Source: (natural) Lysobacter enzymogenes (bacteria) / Secretion: SECRETED BY THE NATIVE BACTERIUM / References: UniProt: P00778, alpha-lytic endopeptidase
#2: Protein/peptide MEOSUC-ALA-ALA-PRO-ALA BORONIC ACID INHIBITOR


Type: Peptide-like / Class: Inhibitor / Mass: 470.325 Da / Num. of mol.: 1 / Source method: obtained synthetically / References: METHOXYSUCCINYL-ALA-ALA-PRO-VALINE BORONIC ACID
#3: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 432 / Source method: isolated from a natural source / Formula: H2O
Nonpolymer detailsGLYCEROL BOUND TO THE CATALYTIC ADDUCT, CREATING A MIMIC OF THE ACYLATION TRANSITION STATE INTERMEDIATE

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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: 2.44 Å3/Da / Density % sol: 49.6 %
Crystal growTemperature: 298 K / pH: 8
Details: 1.3M LITHIUM SULFATE, 0.02M TRIS, PH 8.0, VAPOR DIFFUSION, HANGING DROP, TEMPERATURE 298K, pH 8.00

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 0.75
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Jun 19, 2004
RadiationMonochromator: DOUBLE CRYSTAL, SI(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.75 Å / Relative weight: 1
ReflectionResolution: 0.9→28.5 Å / Num. obs: 147100 / % possible obs: 99.9 % / Redundancy: 8.7 % / Rmerge(I) obs: 0.085 / Net I/σ(I): 27.9
Reflection shellResolution: 0.9→0.91 Å / Redundancy: 5.9 % / Rmerge(I) obs: 0.448 / Mean I/σ(I) obs: 4.8 / % possible all: 99.5

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Processing

Software
NameVersionClassification
HKL-2000data collection
SCALEPACKdata scaling
startingmodel (see above)model building
SHELXL-97refinement
HKL-2000data reduction
RefinementMethod to determine structure: REFINEMENT OF PREVIOUSLY-SOLVED STRUCTURE OF ALPHA-LYTIC PROTEASE BOUND TO MEOSUC-ALA-ALA-PRO-ALA BORONIC ACID
Starting model: SAME PROTEIN BOUND TO MEOSUC-ALA-ALA-PRO-ALA BORONIC ACID AT 0.9A RESOLUTION (UNPUBLISHED DATA, BUT RELATED TO 1P02)

1p02


Resolution: 0.9→20 Å / Num. parameters: 20128 / Num. restraintsaints: 23688 / Cross valid method: FREE R / σ(F): 0 / Stereochemistry target values: ENGH & HUBER
Details: HYDROGEN ATOMS WERE INCLUDED IN THE REFINEMENT AS "RIDING HYDROGENS", WITH POSITION AND GEOMETRY FIXED TO THOSE VALUES DEFINED BY SHELXL-97. METHYL AND HYDROXYL HYDROGENS ON SINGLE-CONFORMER ...Details: HYDROGEN ATOMS WERE INCLUDED IN THE REFINEMENT AS "RIDING HYDROGENS", WITH POSITION AND GEOMETRY FIXED TO THOSE VALUES DEFINED BY SHELXL-97. METHYL AND HYDROXYL HYDROGENS ON SINGLE-CONFORMER SIDECHAINS WERE EACH POSITIONED AT A TORSION ANGLE THAT BEST SATISFIED POSITIVE DIFFERENCE ELECTRON DENSITY (USING INSTRUCTIONS HFIX 137 AND HFIX 147, RESPECTIVELY). IT SHOULD BE NOTED THAT THE LENGTH OF DONOR-HYDROGEN BONDS IN THIS STRUCTURE ARE LIKELY SHORTER THAN THEIR TRUE INTERNUCLEAR DISTANCE; THESE BOND LENGTHS ARE DEFINED BY SHELXL-97 PARAMETERS. THE POSITIONS OF SEVEN HYDROGEN ATOMS WERE ALLOWED TO REFINE FREELY: HIS57 HD1, HIS57 HE1, HIS57 HE2, SER214 HG, SER195 HN, GLY193 HN, AND B2V203 H1. DURING THE FINAL STAGES OF REFINEMENT, GEOMETRICAL RESTRAINTS WERE RELEASED FOR ALL NON- HYDROGEN ATOMS IN RESIDUES WITH SINGLE CONFORMATIONS. THE BORON IN RESIDUE B2V WAS REFINED AS A CARBON ATOM, ALLOWING REFINEMENT OF THE OCCUPANCY OF THIS ATOM TO ESTIMATE THE ELECTRON CONTENT (AND NEGATIVE CHARGE) AT THIS LOCATION. TO AID THE READER IN ANALYZING THE STRUCTURE IN THIS PDB FILE, THE ATOM HAS BEEN RE-NAMED TO "B", AND THE OCCUPANCY OF THE BORON ATOM MANUALLY CHANGED TO THE CORRESPONDING OCCUPANCY (1.07; REFINEMENT OF A CARBON IN THIS POSITION RESULTED IN AN OCCUPANCY OF 0.89). SEE PUBLICATION FOR MORE DETAILS.
RfactorNum. reflection% reflectionSelection details
Rfree0.091 7352 5.003 %RANDOM
all0.081 146962 --
obs0.08 -99.9 %-
Solvent computationSolvent model: MOEWS & KRETSINGER, J.MOL.BIOL.91(1973)201-228
Refine analyzeNum. disordered residues: 21 / Occupancy sum hydrogen: 1367.28 / Occupancy sum non hydrogen: 1784.32
Refinement stepCycle: LAST / Resolution: 0.9→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1424 0 45 432 1901
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONs_bond_d0.016
X-RAY DIFFRACTIONs_angle_d0.045
X-RAY DIFFRACTIONs_similar_dist0
X-RAY DIFFRACTIONs_from_restr_planes0.035
X-RAY DIFFRACTIONs_zero_chiral_vol0.081
X-RAY DIFFRACTIONs_non_zero_chiral_vol0.07
X-RAY DIFFRACTIONs_anti_bump_dis_restr0
X-RAY DIFFRACTIONs_rigid_bond_adp_cmpnt0.005
X-RAY DIFFRACTIONs_similar_adp_cmpnt0.038
X-RAY DIFFRACTIONs_approx_iso_adps0.086

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