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- PDB-3k9t: Crystal structure of putative peptidase (NP_348812.1) from CLOSTR... -

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Basic information

Entry
Database: PDB / ID: 3k9t
TitleCrystal structure of putative peptidase (NP_348812.1) from CLOSTRIDIUM ACETOBUTYLICUM at 2.37 A resolution
Componentsputative peptidaseProtease
KeywordsHYDROLASE / putative peptidase / Structural Genomics / Joint Center for Structural Genomics / JCSG / Protein Structure Initiative / PSI-2 / Aminopeptidase
Function / homology
Function and homology information


carbohydrate metabolic process / metal ion binding
Similarity search - Function
Domain of unknown function DUF2172 / Uncharacterised conserved protein UCP01524, polysaccharide biosynthesis aminopeptidase-like / Domain of unknown function DUF4910 / Domain of unknown function DUF2172 / UCP01524, winged helix-turn-helix domain / Domain of unknown function (DUF2172) / winged helix-turn-helix / Domain of unknown function (DUF4910) / Glucose Oxidase; domain 1 / Zn peptidases ...Domain of unknown function DUF2172 / Uncharacterised conserved protein UCP01524, polysaccharide biosynthesis aminopeptidase-like / Domain of unknown function DUF4910 / Domain of unknown function DUF2172 / UCP01524, winged helix-turn-helix domain / Domain of unknown function (DUF2172) / winged helix-turn-helix / Domain of unknown function (DUF4910) / Glucose Oxidase; domain 1 / Zn peptidases / Aminopeptidase / Winged helix-like DNA-binding domain superfamily/Winged helix DNA-binding domain / 3-Layer(bba) Sandwich / Arc Repressor Mutant, subunit A / Winged helix-like DNA-binding domain superfamily / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
IMIDAZOLE / Putative polysaccharide biosynthesis protein with aminopeptidase-like domain
Similarity search - Component
Biological speciesClostridium acetobutylicum (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.37 Å
AuthorsJoint Center for Structural Genomics (JCSG)
CitationJournal: To be published
Title: Crystal structure of putative peptidase (NP_348812.1) from CLOSTRIDIUM ACETOBUTYLICUM at 2.37 A resolution
Authors: Joint Center for Structural Genomics (JCSG)
History
DepositionOct 16, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 10, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Advisory / Version format compliance
Revision 1.2Oct 25, 2017Group: Author supporting evidence / Refinement description / Category: pdbx_struct_assembly_auth_evidence / software / Item: _software.classification / _software.name
Revision 1.3Jul 17, 2019Group: Data collection / Derived calculations / Refinement description
Category: software / struct_conn
Item: _software.classification / _software.contact_author ..._software.classification / _software.contact_author / _software.contact_author_email / _software.language / _software.location / _software.name / _software.type / _software.version / _struct_conn.pdbx_leaving_atom_flag
Revision 1.4Feb 1, 2023Group: Database references / Derived calculations / Category: database_2 / 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 / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: putative peptidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,2649
Polymers50,5861
Non-polymers6788
Water3,981221
1
A: putative peptidase
hetero molecules

A: putative peptidase
hetero molecules

A: putative peptidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)153,79327
Polymers151,7593
Non-polymers2,03424
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
Buried area9390 Å2
ΔGint-161 kcal/mol
Surface area48490 Å2
MethodPISA
Unit cell
Length a, b, c (Å)153.782, 153.782, 168.378
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number155
Space group name H-MH32
DetailsCRYSTAL PACKING ANALYSIS AND SIZE-EXCLUSION CHROMATOGRAPHY COUPLED WITH STATIC LIGHT SCATTERING SUPPORT THE ASSIGNMENT OF A TRIMER AS THE SIGNIFICANT OLIGOMERIC FORM IN SOLUTION.

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Components

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Protein , 1 types, 1 molecules A

#1: Protein putative peptidase / Protease / Protein containing aminopeptidase domain


Mass: 50586.395 Da / Num. of mol.: 1 / Mutation: P309S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium acetobutylicum (bacteria) / Gene: CA_C2195 / Plasmid: SpeedET / Production host: Escherichia Coli (E. coli) / Strain (production host): HK100 / References: UniProt: Q97H19

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Non-polymers , 5 types, 229 molecules

#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-IMD / IMIDAZOLE / Imidazole


Mass: 69.085 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H5N2
#5: Chemical
ChemComp-MRD / (4R)-2-METHYLPENTANE-2,4-DIOL / 2-Methyl-2,4-pentanediol


Mass: 118.174 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H14O2 / Comment: precipitant*YM
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 221 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsTHIS CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH ...THIS CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH TEV PROTEASE LEAVING ONLY A GLYCINE (0) FOLLOWED BY THE TARGET SEQUENCE. DNA SEQUENCING OF THE CLONED CONSTRUCT SHOWS A SERINE AT POSITION 309 INSTEAD OF A PROLINE. THE SERINE AT POSITION 309 IS SUPPORTED BY THE ELECTRON DENSITY.

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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: 3.79 Å3/Da / Density % sol: 67.52 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8
Details: 0.2000M MgCl2, 40.0000% MPD, 0.1M Imidazole pH 8.0, Additive: 0.006M Zinc Chloride, NANODROP', VAPOR DIFFUSION, SITTING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 0.91837,0.97911
DetectorType: MARMOSAIC 325 mm CCD / Detector: CCD / Date: Jul 31, 2009 / Details: Flat collimating mirror, toroid focusing mirror
RadiationMonochromator: Double crystal monochromator / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.918371
20.979111
ReflectionResolution: 2.37→29.062 Å / Num. obs: 31178 / % possible obs: 99.9 % / Redundancy: 5.5 % / Biso Wilson estimate: 38.179 Å2 / Rmerge(I) obs: 0.189 / Rsym value: 0.189 / Net I/σ(I): 9
Reflection shell

Rmerge(I) obs: 0.01 / Diffraction-ID: 1

Resolution (Å)Redundancy (%)Mean I/σ(I) obsNum. measured allNum. unique allRsym value% possible all
2.37-2.435.60.81265122671.017100
2.43-2.55.60.81250122340.907100
2.5-2.575.611211621750.775100
2.57-2.655.61.11177521060.68100
2.65-2.745.61.31138620390.57100
2.74-2.835.61.61104919770.487100
2.83-2.945.61.91059618970.398100
2.94-3.065.62.31029318450.323100
3.06-3.25.63988617770.254100
3.2-3.355.53.7932816810.204100
3.35-3.535.54.6895216170.162100
3.53-3.755.55.7849015330.126100
3.75-4.015.56.5788214310.109100
4.01-4.335.57.4741313430.095100
4.33-4.745.58.6686412440.082100
4.74-5.35.58.2617911280.083100
5.3-6.125.47.653729870.091100
6.12-7.495.4845938580.085100
7.49-10.65.29.434796690.064100
10.6-29.064.88.617803700.06495.3

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Phasing

PhasingMethod: MAD

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Processing

Software
NameVersionClassificationNB
REFMAC5.5.0053refinement
PHENIXrefinement
SHELXphasing
MolProbity3beta29model building
SCALA3.2.5data scaling
PDB_EXTRACT3.006data extraction
MOSFLMdata reduction
SHELXDphasing
autoSHARPphasing
RefinementMethod to determine structure: MAD / Resolution: 2.37→29.062 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.935 / Occupancy max: 1 / Occupancy min: 0.4 / SU B: 11.776 / SU ML: 0.122 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.201 / ESU R Free: 0.18
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: 1.HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2.ATOM RECORDS CONTAIN RESIDUAL B FACTORS ONLY. 3.A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONINE INCORPORATION DURING PROTEIN ...Details: 1.HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2.ATOM RECORDS CONTAIN RESIDUAL B FACTORS ONLY. 3.A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONINE INCORPORATION DURING PROTEIN EXPRESSION. THE OCCUPANCY OF THE SE ATOMS IN THE MSE RESIDUES WAS REDUCED TO 0.75 FOR THE REDUCED SCATTERING POWER DUE TO PARTIAL S-MET INCORPORATION. 4.ZINC (ZN) HAS BEEN MODELED AT THE PUTATIVE ACTIVE SITE BASED ON ITS PRESENCE AS A CO-CRYSTALLIZATION COMPOUND AND ANOMALOUS DIFFERENCE FOURIER MAP. 5.CHLORIDE (CL), IMIDAZOLE (IMD) AND (4R)-2-METHYLPENTANE-2,4-DIOL (MRD) FROM THE CRYSTALLIZATION SOLUTION HAVE BEEN MODELED IN THE SOLVENT STRUCTURE. 6.THE RAMACHANDRAN OUTLIER AT RESIDUE PRO190 IS SUPPORTED BY ELECTRON DENSITY.
RfactorNum. reflection% reflectionSelection details
Rfree0.212 1576 5.1 %RANDOM
Rwork0.169 ---
obs0.171 31177 99.98 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 384.4 Å2 / Biso mean: 29.498 Å2 / Biso min: 2 Å2
Baniso -1Baniso -2Baniso -3
1--1.3 Å2-0.65 Å20 Å2
2---1.3 Å20 Å2
3---1.95 Å2
Refinement stepCycle: LAST / Resolution: 2.37→29.062 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3386 0 40 221 3647
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0223506
X-RAY DIFFRACTIONr_bond_other_d0.0010.022373
X-RAY DIFFRACTIONr_angle_refined_deg1.6091.9724743
X-RAY DIFFRACTIONr_angle_other_deg0.93435784
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.675424
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.36724.479163
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.29315614
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.7171516
X-RAY DIFFRACTIONr_chiral_restr0.0910.2518
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.023856
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02716
X-RAY DIFFRACTIONr_mcbond_it1.65431648
X-RAY DIFFRACTIONr_mcbond_other0.4193677
X-RAY DIFFRACTIONr_mcangle_it3.07852673
X-RAY DIFFRACTIONr_scbond_it5.74181063
X-RAY DIFFRACTIONr_scangle_it7.737111000
LS refinement shellResolution: 2.37→2.431 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.334 108 -
Rwork0.288 2153 -
all-2261 -
obs--99.78 %
Refinement TLS params.Method: refined / Origin x: 32.2688 Å / Origin y: 1.6738 Å / Origin z: 64.0806 Å
111213212223313233
T0.0228 Å20.0107 Å20.0125 Å2-0.0386 Å2-0.0118 Å2--0.0327 Å2
L0.9216 °20.1874 °2-0.0975 °2-0.7499 °2-0.0631 °2--0.4114 °2
S0.0209 Å °0.0462 Å °0.0311 Å °0.0054 Å °0.0275 Å °-0.1023 Å °-0.0296 Å °0.0239 Å °-0.0484 Å °

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