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- PDB-6zni: Structure of the Shigella MxiH needle filament attached to the ba... -

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Basic information

Entry
Database: PDB / ID: 6zni
TitleStructure of the Shigella MxiH needle filament attached to the basal body
ComponentsProtein MxiH
KeywordsPROTEIN TRANSPORT / Type 3 secretion system / Shigella / 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
Type 3 secretion system needle filament protein
Similarity search - Component
Biological speciesShigella flexneri 5a str. M90T (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsLunelli, M. / Kotov, V. / Marlovits, T.C. / Kolbe, M.
Funding support Germany, 2items
OrganizationGrant numberCountry
European Research Council (ERC)311374 Germany
European Commission653706 Germany
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-11312
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  • Superimposition on EM map
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)254,38623
Polymers254,38623
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area66310 Å2
ΔGint-336 kcal/mol
Surface area78510 Å2
MethodPISA

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Components

#1: Protein ...
Protein MxiH


Mass: 11060.279 Da / Num. of mol.: 23
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella flexneri 5a str. M90T (bacteria)
Gene: mxiH, CP0137 / Plasmid: pASK-IBA7plus / Production host: Shigella flexneri 5a str. M90T (bacteria) / References: UniProt: P0A223

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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: RECOMBINANT
Source (natural)Organism: Shigella flexneri 5a str. M90T (bacteria)
Source (recombinant)Organism: Shigella flexneri 5a str. M90T (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris1
2100 mMSodium chlorideNaCl1
35 mMEDTA1
40.02 %DDM1
525 mMbiotin1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
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: 100000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 25 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5238
Image scansWidth: 4096 / Height: 4096 / Movie frames/image: 7 / Used frames/image: 1-6

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Processing

SoftwareName: PHENIX / Version: 1.17_3644: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4CTFFIND4.0.17CTF 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: 4.322 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 101369
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19348 / Symmetry type: HELICAL
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 2MME

2mme


Accession code: 2MME / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00715019
ELECTRON MICROSCOPYf_angle_d0.60920470
ELECTRON MICROSCOPYf_dihedral_angle_d12.215543
ELECTRON MICROSCOPYf_chiral_restr0.0382461
ELECTRON MICROSCOPYf_plane_restr0.0062668

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