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- PDB-4kwb: Structure of signal peptide peptidase A with C-termini bound in t... -

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Basic information

Entry
Database: PDB / ID: 4kwb
TitleStructure of signal peptide peptidase A with C-termini bound in the active sites: insights into specificity, self-processing and regulation
ComponentsSignal peptide peptidase SppA
KeywordsHYDROLASE / alpha/beta protein fold / signal peptide digestion / bacterial cell membrane / SELF-COMPARTMENTALIZED
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / serine-type peptidase activity / membrane raft / proteolysis / plasma membrane
Similarity search - Function
peptide peptidase (sppa) fold / peptide peptidase (sppa) fold - #10 / : / Peptidase S49 / Peptidase S49, SppA / Peptidase family S49 / Other non-globular / 2-enoyl-CoA Hydratase; Chain A, domain 1 / 2-enoyl-CoA Hydratase; Chain A, domain 1 / ClpP/crotonase-like domain superfamily ...peptide peptidase (sppa) fold / peptide peptidase (sppa) fold - #10 / : / Peptidase S49 / Peptidase S49, SppA / Peptidase family S49 / Other non-globular / 2-enoyl-CoA Hydratase; Chain A, domain 1 / 2-enoyl-CoA Hydratase; Chain A, domain 1 / ClpP/crotonase-like domain superfamily / Special / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
Putative signal peptide peptidase SppA
Similarity search - Component
Biological speciesBacillus subtilis subsp. subtilis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.39 Å
AuthorsNam, S.E. / Paetzel, M.
CitationJournal: Biochemistry / Year: 2013
Title: Structure of signal peptide peptidase A with C-termini bound in the active sites: insights into specificity, self-processing, and regulation.
Authors: Nam, S.E. / Paetzel, M.
History
DepositionMay 23, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 27, 2013Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2013Group: Structure summary
Revision 1.2Feb 19, 2014Group: Database references
Revision 1.3Nov 15, 2017Group: Refinement description / Category: software / Item: _software.name
Revision 1.4Sep 20, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Signal peptide peptidase SppA
B: Signal peptide peptidase SppA
C: Signal peptide peptidase SppA
D: Signal peptide peptidase SppA
E: Signal peptide peptidase SppA
F: Signal peptide peptidase SppA
G: Signal peptide peptidase SppA
H: Signal peptide peptidase SppA


Theoretical massNumber of molelcules
Total (without water)240,0338
Polymers240,0338
Non-polymers00
Water3,981221
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area35370 Å2
ΔGint-202 kcal/mol
Surface area70500 Å2
MethodPISA
Unit cell
Length a, b, c (Å)87.797, 130.979, 207.099
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein
Signal peptide peptidase SppA


Mass: 30004.143 Da / Num. of mol.: 8 / Fragment: unp residues 51-335 / Mutation: K199A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis subsp. subtilis (bacteria)
Strain: 168
Gene: BSU29530, CLONED SIGNAL PEPTIDE PEPTIDASE A GENE FROM RESIDUES 26 TO 335, LYS199 MUTATED TO ALA, sppA, yteI
Plasmid: PET28B / Production host: Escherichia coli (E. coli) / Strain (production host): TUNER(DE3)
References: UniProt: O34525, Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 221 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsLIMITED PROTEOLYSIS WAS CARRIED OUT ON SPPA_BS DELTA 1-25 K199A USING THERMOLYSIN, FOLLOWED BY SIZE- ...LIMITED PROTEOLYSIS WAS CARRIED OUT ON SPPA_BS DELTA 1-25 K199A USING THERMOLYSIN, FOLLOWED BY SIZE-EXCLUSION CHROMATOGRAPHY. THE CRYSTALLIZED AND SOLVED STRUCTURE IS A PROTEASE RESISTANT FRAGMENT OF SPPA_BS DELTA 1-25 K199A. USING N-TERMINAL SEQUENCING AND POSITIVE ELECTROSPRAY IONIZATION MASS SPECTROMETRY, THE PROTEASE RESISTANT FRAGMENT WAS IDENTIFIED AS SPPA_BS 51-295. THE REFINED OCTAMERIC STRUCTURE REVEALED THE CORE DOMAIN OF SPPA_BS 51-295, WITH THE C-TERMINI OF SPPA_BS BOUND IN THE EIGHT ACTIVE SITES. THE IDENTITY OF THE PEPTIDE WAS CONFIRMED AS 308-335 OF SPPA_BS(FKSEIDFLNMREILSQSGSPRMMYLYAK) BY TANDEM MASS SPECTROMETRY FRAGMENTATION ANALYSIS. CLEAR ELECTRON DESITY IS OBSERVED FOR 326-335 (SPRMMYLYAK). WHAT HAS BEEN CRYSTALLIZED IS SPPA_BS RESIDUES: 51-295 & 308-335

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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: 2.48 Å3/Da / Density % sol: 50.42 %
Crystal growTemperature: 298.15 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 100mM TRIS, 23% TERT-BUTANOL UNDER PARAFFIN OIL, VAPOR DIFFUSION, SITTING DROP, temperature 298.15K, pH 8.5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: CLSI / Beamline: 08ID-1 / Wavelength: 0.97949 / Wavelength: 0.97949 Å
DetectorType: RAYONIX MX-300 / Detector: CCD / Date: Feb 25, 2011 / Details: VERTICAL COLLIMATING MIRROR
RadiationMonochromator: DOUBLE CRYSTAL MONOCHROMATOR (DCM) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97949 Å / Relative weight: 1
ReflectionResolution: 2.39→50 Å / Num. all: 93912 / Num. obs: 93168 / % possible obs: 99.2 % / Observed criterion σ(F): 2 / Observed criterion σ(I): -3 / Redundancy: 6.8 % / Biso Wilson estimate: 51 Å2 / Rmerge(I) obs: 0.06 / Net I/σ(I): 50.3
Reflection shellResolution: 2.39→2.49 Å / Redundancy: 6.3 % / Rmerge(I) obs: 0.302 / Mean I/σ(I) obs: 6.7 / Num. unique all: 9085 / % possible all: 97.5

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Processing

Software
NameVersionClassification
MxDCdata collection
PHASERFOR MR 2.1phasing
REFMAC5.5.0109refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3BFO

3bfo


Resolution: 2.39→50 Å / Cor.coef. Fo:Fc: 0.943 / Cor.coef. Fo:Fc free: 0.926 / SU B: 15.86 / SU ML: 0.17 / Cross valid method: THROUGHOUT / ESU R Free: 0.235 / Stereochemistry target values: MAXIMUM LIKELIHOOD
RfactorNum. reflection% reflectionSelection details
Rfree0.24 4708 5 %RANDOM
Rwork0.206 ---
all0.285 93310 --
obs0.208 88919 98.7 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 50.9 Å2
Baniso -1Baniso -2Baniso -3
1--0.36 Å20 Å20 Å2
2--1.59 Å20 Å2
3----1.23 Å2
Refinement stepCycle: LAST / Resolution: 2.39→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms14103 0 0 221 14324
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.02214250
X-RAY DIFFRACTIONr_angle_refined_deg1.0611.97719135
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.25751841
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.13425.028539
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.578152644
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.9691551
X-RAY DIFFRACTIONr_chiral_restr0.0690.22179
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.02110314
X-RAY DIFFRACTIONr_mcbond_it0.4511.59195
X-RAY DIFFRACTIONr_mcangle_it0.898214663
X-RAY DIFFRACTIONr_scbond_it1.52435055
X-RAY DIFFRACTIONr_scangle_it2.5764.54472
LS refinement shellResolution: 2.39→2.46 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.307 305 -
Rwork0.243 6133 -
obs--93.14 %
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)
11.20120.51870.34491.7322-0.06321.39450.0203-0.0007-0.01750.1339-0.01340.02530.00120.0258-0.00690.01570.00270.01240.17610.04760.030612.585618.9395-23.4787
20.48880.21880.25811.93661.57953.84790.04290.06040.06310.0009-0.1920.2982-0.2908-0.25020.14920.0683-0.05290.01260.24350.03110.155421.560534.0636-1.1891
31.0275-0.1741-0.50421.29460.32922.632-0.02730.1091-0.1112-0.0208-0.12560.19950.0869-0.14140.15280.099-0.1023-0.0420.138-0.0070.106515.673433.36526.617
42.91591.7922-1.82042.6158-0.8792.41250.24130.17760.5050.2298-0.04320.4779-0.1351-0.1734-0.19810.2493-0.0760.03410.1624-0.02580.2120.764516.48244.2149
53.10272.10370.34983.131.12931.6684-0.12320.13910.5559-0.0868-0.12740.4927-0.0903-0.20290.25070.2157-0.02720.02710.1530.00550.1599-17.0332-5.80540.1278
62.4350.65831.30160.93860.7952.5356-0.08590.03730.3855-0.0128-0.03940.0691-0.20190.0110.12520.1053-0.03030.04670.02240.00760.1274-25.8887-21.14317.0644
71.00550.1195-0.15120.7907-0.62184.9629-0.00030.09550.16740.02410.08790.2142-0.3889-0.6097-0.08760.12190.0359-0.00680.14550.01780.1537-20.3456-21.3729-10.9603
81.12040.79530.27841.749-0.60081.6435-0.23850.09180.2287-0.1150.15570.2668-0.2492-0.04270.08280.17070.0266-0.05360.1125-0.00210.0987-4.6205-4.2085-27.6063
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A56 - 335
2X-RAY DIFFRACTION2B58 - 335
3X-RAY DIFFRACTION3C57 - 334
4X-RAY DIFFRACTION4D59 - 333
5X-RAY DIFFRACTION5E57 - 334
6X-RAY DIFFRACTION6F57 - 334
7X-RAY DIFFRACTION7G57 - 334
8X-RAY DIFFRACTION8H58 - 334

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