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- PDB-2vt1: Crystal structure of the cytoplasmic domain of Spa40, the specifi... -

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Basic information

Entry
Database: PDB / ID: 2vt1
TitleCrystal structure of the cytoplasmic domain of Spa40, the specificity switch for the Shigella flexneri Type III Secretion System
Components(SURFACE PRESENTATION OF ANTIGENS PROTEIN SPAS) x 2
KeywordsMEMBRANE PROTEIN / SHIGELLA FLEXNERI / SPECIFICITY SWITCH / VIRULENCE / TRANSMEMBRANE / INNER MEMBRANE / FLHB / YSCU / T3SS / SPA40 / PLASMID / MEMBRANE / TYPE III SECRETION SYSTEM
Function / homology
Function and homology information


protein secretion / plasma membrane
Similarity search - Function
secretion proteins EscU / name from scop / Type III exporter system, secretion apparatus protein BsaZ / Type III secretion system substrate exporter / Type III secretion system substrate exporter, C-terminal / FlhB HrpN YscU SpaS Family / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Surface presentation of antigens protein SpaS
Similarity search - Component
Biological speciesSHIGELLA FLEXNERI (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsDeane, J.E. / Graham, S.C. / Mitchell, E.P. / Flot, D. / Johnson, S. / Lea, S.M.
CitationJournal: Mol.Microbiol. / Year: 2008
Title: Crystal Structure of Spa40, the Specificity Switch for the Shigella Flexneri Type III Secretion System
Authors: Deane, J.E. / Graham, S.C. / Mitchell, E.P. / Flot, D. / Johnson, S. / Lea, S.M.
History
DepositionMay 8, 2008Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 20, 2008Provider: repository / Type: Initial release
Revision 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3May 8, 2019Group: Data collection / Experimental preparation / Other
Category: database_PDB_rev / database_PDB_rev_record ...database_PDB_rev / database_PDB_rev_record / exptl_crystal_grow / pdbx_database_proc / pdbx_database_status
Item: _exptl_crystal_grow.method / _exptl_crystal_grow.temp / _pdbx_database_status.recvd_author_approval
Revision 1.4Dec 13, 2023Group: Data collection / Database references ...Data collection / Database references / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: SURFACE PRESENTATION OF ANTIGENS PROTEIN SPAS
B: SURFACE PRESENTATION OF ANTIGENS PROTEIN SPAS


Theoretical massNumber of molelcules
Total (without water)17,2852
Polymers17,2852
Non-polymers00
Water52229
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2560 Å2
ΔGint-24.9 kcal/mol
Surface area7250 Å2
MethodPQS
Unit cell
Length a, b, c (Å)25.035, 30.753, 32.099
Angle α, β, γ (deg.)102.52, 110.97, 94.30
Int Tables number1
Space group name H-MP1

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Components

#1: Protein SURFACE PRESENTATION OF ANTIGENS PROTEIN SPAS / SPA40 PROTEIN / SPA40


Mass: 6394.362 Da / Num. of mol.: 1 / Fragment: CYTOPLASMIC DOMAIN, RESIDUES 207-257
Source method: isolated from a genetically manipulated source
Details: A SINGLE CHAIN OF THE CYTOPLASMIC DOMAIN OF SPA40 IS PRESENT, HAVING UNDERGONE AN AUTO-CLEAVAGE EVENT BETWEEN N257 AND P258 TO FORM TWO CHAINS
Source: (gene. exp.) SHIGELLA FLEXNERI (bacteria) / Plasmid: PET-28B / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): B834(DE3) / References: UniProt: P0A1M8
#2: Protein SURFACE PRESENTATION OF ANTIGENS PROTEIN SPAS / SPA40 PROTEIN / SPA40


Mass: 10890.471 Da / Num. of mol.: 1 / Fragment: CYTOPLASMIC DOMAIN, RESIDUES 258-342
Source method: isolated from a genetically manipulated source
Details: A SINGLE CHAIN OF THE CYTOPLASMIC DOMAIN OF SPA40 IS PRESENT, HAVING UNDERGONE AN AUTO-CLEAVAGE EVENT BETWEEN N257 AND P258 TO FORM TWO CHAINS
Source: (gene. exp.) SHIGELLA FLEXNERI (bacteria) / Plasmid: PET-28B / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): B834(DE3) / References: UniProt: P0A1M8
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 29 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsCYTOPLASMIC DOMAIN (207-342). N-TERMINAL START CODON AND C-TERMINAL HIS-TAG (SEQUENCE LEHHHHHH) IS ADDED.

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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: 1.67 Å3/Da / Density % sol: 26.55 % / Description: NONE
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7
Details: SITTING DROPS CONTAINING 200 NL PROTEIN (3.3 MG/ML IN 20 MM TRIS PH 8.0, 500 MM NACL) AND 200 NL RESERVOIR SOLUTION (0.1M HEPES PH7.0, 0.2M NH4CL AND 20% PEG W/V 6000) WERE EQUILIBRATED ...Details: SITTING DROPS CONTAINING 200 NL PROTEIN (3.3 MG/ML IN 20 MM TRIS PH 8.0, 500 MM NACL) AND 200 NL RESERVOIR SOLUTION (0.1M HEPES PH7.0, 0.2M NH4CL AND 20% PEG W/V 6000) WERE EQUILIBRATED AGAINST 100 UL RESERVOIRS AT 20 C. CRYSTALS WERE CRYOPROTECTED IN RESERVOIR SOLUTION SUPPLEMENTED WITH 25% (V/V) GLYCEROL. THE ASYMMETRIC UNIT VOLUME IS NOT SUFFICIENT TO ACCOMMODATE THE ENTIRE SPA40 CONSTRUCT. WE ASSUME THAT THE PROTEIN HAS UNDERGONE SOME PROTEOLYSIS ADDITIONAL TO THE SELF-CLEAVAGE BETWEEN RESIDUES 257 AND 258, REMOVING EITHER THE DISORDERED N-TERMINUS OR THE C-TERMINAL HIS TAG. THE SOLVENT CONTENT QUOTED IS FOR RESIDUE 237 TO THE END OF THE CONSTRUCT.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-1 / Wavelength: 1.0401
DetectorType: ADSC CCD / Detector: CCD / Date: Apr 13, 2008 / Details: MIRRORS
RadiationMonochromator: SI(111) CRYSTAL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0401 Å / Relative weight: 1
ReflectionResolution: 2→30 Å / Num. obs: 5433 / % possible obs: 93.2 % / Observed criterion σ(I): -3 / Redundancy: 2.6 % / Biso Wilson estimate: 21.6 Å2 / Rmerge(I) obs: 0.11 / Net I/σ(I): 3.8
Reflection shellResolution: 2→2.05 Å / Redundancy: 2.7 % / Rmerge(I) obs: 0.4 / Mean I/σ(I) obs: 1.7 / % possible all: 93.3

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Processing

Software
NameVersionClassification
REFMAC5.4.0077refinement
MOSFLMdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 3BZL WITH SIDECHAINS MUTATED TO SER

3bzl


Resolution: 2→29.59 Å / Cor.coef. Fo:Fc: 0.958 / Cor.coef. Fo:Fc free: 0.931 / SU B: 5.042 / SU ML: 0.135 / Cross valid method: THROUGHOUT / ESU R: 0.251 / ESU R Free: 0.19 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES HAVE BEEN REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.227 252 4.6 %RANDOM
Rwork0.178 ---
obs0.18 5181 93.2 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 18.59 Å2
Baniso -1Baniso -2Baniso -3
1--1.29 Å2-0.4 Å2-1.06 Å2
2--1.11 Å20.92 Å2
3----0.24 Å2
Refinement stepCycle: LAST / Resolution: 2→29.59 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms806 0 0 29 835
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.022825
X-RAY DIFFRACTIONr_bond_other_d0.0010.02549
X-RAY DIFFRACTIONr_angle_refined_deg1.1051.9671123
X-RAY DIFFRACTIONr_angle_other_deg0.75231351
X-RAY DIFFRACTIONr_dihedral_angle_1_deg4.85102
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.30924.24233
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.84615145
X-RAY DIFFRACTIONr_dihedral_angle_4_deg13.76154
X-RAY DIFFRACTIONr_chiral_restr0.060.2137
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.021888
X-RAY DIFFRACTIONr_gen_planes_other00.02155
X-RAY DIFFRACTIONr_nbd_refined0.0310.2158
X-RAY DIFFRACTIONr_nbd_other0.0480.2517
X-RAY DIFFRACTIONr_nbtor_refined00.2404
X-RAY DIFFRACTIONr_nbtor_other00.2431
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.0950.223
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.0920.211
X-RAY DIFFRACTIONr_symmetry_vdw_other0.2350.224
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1010.22
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.212515
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it2.2113841
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it3.2734310
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it5.3856281
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2→2.05 Å / Total num. of bins used: 20 /
RfactorNum. reflection
Rfree0.292 20
Rwork0.267 414

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