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- EMDB-11312: Structure of the Shigella MxiH needle filament attached to the ba... -

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Basic information

Entry
Database: EMDB / ID: EMD-11312
TitleStructure of the Shigella MxiH needle filament attached to the basal body
Map data
Sample
  • Organelle or cellular component: Needle filament of the type III secretion system
    • Protein or peptide: Protein MxiH
KeywordsType 3 secretion system / Shigella / Helical reconstruction / Needle filament. / PROTEIN TRANSPORT
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)
Methodhelical reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsLunelli M / Kotov V
Funding support Germany, 2 items
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, 2020-
Header (metadata) releaseJul 7, 2021-
Map releaseJul 7, 2021-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.15
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.15
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6zni
  • Surface level: 0.15
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6zni
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11312.png

Downloads & links

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Map

FileDownload / File: emd_11312.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.38 Å/pix.
x 300 pix.
= 415.107 Å		1.38 Å/pix.
x 300 pix.
= 415.107 Å		1.38 Å/pix.
x 300 pix.
= 415.107 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.38369 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15 / Movie #1: 0.15
Minimum - Maximum-0.22955813 - 0.44250318
Average (Standard dev.)0.00052550255 (±0.01636469)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 415.107 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.383691.383691.38369
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z415.107415.107415.107
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.2300.4430.001

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Supplemental data

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Half map: #2

Fileemd_11312_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11312_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Needle filament of the type III secretion system

EntireName: Needle filament of the type III secretion system
Components
  • Organelle or cellular component: Needle filament of the type III secretion system
    • Protein or peptide: Protein MxiH

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Supramolecule #1: Needle filament of the type III secretion system

SupramoleculeName: Needle filament of the type III secretion system / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Structure obtained from needles attached to the basal body
Source (natural)Organism: Shigella flexneri 5a str. M90T (bacteria)

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Macromolecule #1: Protein MxiH

MacromoleculeName: Protein MxiH / type: protein_or_peptide / ID: 1 / Number of copies: 23 / Enantiomer: LEVO
Source (natural)Organism: Shigella flexneri 5a str. M90T (bacteria)
Molecular weightTheoretical: 11.060279 KDa
Recombinant expressionOrganism: Shigella flexneri 5a str. M90T (bacteria)
SequenceString:
MASWSHPQFE KIEGRMSVTV PNDDWTLSSL SETFDDGTQT LQGELTLALD KLAKNPSNPQ LLAEYQSKLS EYTLYRNAQS NTVKVIKDV DAAIIQNFR

UniProtKB: Type 3 secretion system needle filament protein

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMTris
100.0 mMSodium chlorideNaCl
5.0 mMEDTA
0.02 %DDM
25.0 mMbiotin
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.5 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Frames/image: 1-6 / Number grids imaged: 1 / Number real images: 5238 / Average exposure time: 1.5 sec. / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 100000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 4.322 Å
Applied symmetry - Helical parameters - Δ&Phi: 64.3 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 19348
Segment selectionNumber selected: 101369
Startup modelType of model: OTHER / Details: Cylinder
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2mme


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL
Output model

PDB-6zni:
Structure of the Shigella MxiH needle filament attached to the basal body

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