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- PDB-6znh: Structure of the Salmonella PrgI needle filament attached to the ... -

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Basic information

Entry
Database: PDB / ID: 6znh
TitleStructure of the Salmonella PrgI needle filament attached to the basal body
ComponentsPrgI
KeywordsPROTEIN TRANSPORT / Type 3 secretion system / Salmonella / Helical reconstruction / Needle filament.
Function / homology
Function and homology information


type III protein secretion system complex / protein secretion by the type III secretion system / cell surface / extracellular region / identical protein binding
Similarity search - Function
Type III secretion, needle-protein-like / Type III secretion, needle-protein-like superfamily / Type III secretion needle MxiH, YscF, SsaG, EprI, PscF, EscF / Type III secretion system, needle protein
Similarity search - Domain/homology
SPI-1 type 3 secretion system needle filament protein
Similarity search - Component
Biological speciesSalmonella typhimurium (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsLunelli, M. / Kotov, V. / Wald, J. / Kolbe, M. / Marlovits, T.C.
CitationJournal: Biochem Biophys Rep / Year: 2021
Title: Helical reconstruction of and needle filaments attached to type 3 basal bodies.
Authors: Vadim Kotov / Michele Lunelli / Jiri Wald / Michael Kolbe / Thomas C Marlovits /
Abstract: Gram-negative pathogens evolved a syringe-like nanomachine, termed type 3 secretion system, to deliver protein effectors into the cytoplasm of host cells. An essential component of this system is a ...Gram-negative pathogens evolved a syringe-like nanomachine, termed type 3 secretion system, to deliver protein effectors into the cytoplasm of host cells. An essential component of this system is a long helical needle filament that protrudes from the bacterial surface and connects the cytoplasms of the bacterium and the eukaryotic cell. Previous structural research was predominantly focused on reconstituted type 3 needle filaments, which lacked the biological context. In this work we introduce a facile procedure to obtain high-resolution cryo-EM structure of needle filaments attached to the basal body of type 3 secretion systems. We validate our approach by solving the structure of PrgI filament and demonstrate its utility by obtaining the first high-resolution cryo-EM reconstruction of Shigella MxiH filament. Our work paves the way to systematic structural characterization of attached type 3 needle filaments in the context of mutagenesis studies, protein structural evolution and drug development.
History
DepositionJul 6, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 7, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 28, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2May 1, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-11311
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  • Superimposition on EM map
  • EMDB-11311
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: PrgI
B: PrgI
C: PrgI
D: PrgI
E: PrgI
F: PrgI
G: PrgI
H: PrgI
I: PrgI
J: PrgI
K: PrgI
L: PrgI
M: PrgI
N: PrgI
O: PrgI
P: PrgI
Q: PrgI
R: PrgI
S: PrgI
T: PrgI
U: PrgI
V: PrgI
W: PrgI


Theoretical massNumber of molelcules
Total (without water)203,89223
Polymers203,89223
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area66220 Å2
ΔGint-356 kcal/mol
Surface area83060 Å2
MethodPISA

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Components

#1: Protein ...
PrgI


Mass: 8864.868 Da / Num. of mol.: 23 / Source method: isolated from a natural source / Source: (natural) Salmonella typhimurium (bacteria) / References: UniProt: P41784

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Needle filament of the type III secretion system / Type: ORGANELLE OR CELLULAR COMPONENT
Details: Structure obtained from needles attached to the basal body
Entity ID: all / Source: NATURAL
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Strain: SB905
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris-HCl1
2500 mMSodium chlorideNaCl1
30.1 %LDAO1
45 mMEDTA1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 31 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum
Image scansMovie frames/image: 25

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Processing

SoftwareName: PHENIX / Version: 1.17_3644: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.14model fitting
11RELION3classification
12RELION33D reconstruction
13Coot0.8.9.2model refinement
14PHENIX1.17-3644model refinementphenix.real_space_refine
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 64.3 ° / Axial rise/subunit: 4.2 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 308303
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34174 / Symmetry type: HELICAL
Atomic model buildingSpace: REAL
Details: Initial automatic modeling with StarMap, a Rosetta plug-in of ChimeraX.
Atomic model buildingPDB-ID: 2LPZ

2lpz


Accession code: 2LPZ / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00614444
ELECTRON MICROSCOPYf_angle_d0.62319665
ELECTRON MICROSCOPYf_dihedral_angle_d17.9825221
ELECTRON MICROSCOPYf_chiral_restr0.0452254
ELECTRON MICROSCOPYf_plane_restr0.0042576

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