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- EMDB-27656: CryoEM structure of IglD -

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Basic information

Entry
Database: EMDB / ID: EMD-27656
TitleCryoEM structure of IglD
Map dataIglD trimer
Sample
  • Complex: IglD trimer
    • Protein or peptide: Secretion system protein
KeywordsT6SS / baseplate / PROTEIN TRANSPORT
Function / homologyIntracellular growth locus
Function and homology information
Biological speciesFrancisella tularensis subsp. novicida (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsLiu X / Clemens D / Lee B / Yang X / Zhou H / Horwitz M
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI151055 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM071940 United States
CitationJournal: mBio / Year: 2022
Title: Atomic Structure of IglD Demonstrates Its Role as a Component of the Baseplate Complex of the Type VI Secretion System.
Authors: Xiaoyu Liu / Daniel L Clemens / Bai-Yu Lee / Xue Yang / Z Hong Zhou / Marcus A Horwitz /
Abstract: Francisella tularensis, a Tier 1 select agent of bioterrorism, contains a type VI secretion system (T6SS) encoded within the pathogenicity island (FPI), which is critical for its pathogenesis. Among ...Francisella tularensis, a Tier 1 select agent of bioterrorism, contains a type VI secretion system (T6SS) encoded within the pathogenicity island (FPI), which is critical for its pathogenesis. Among the 18 proteins encoded by FPI is IglD, which is essential to 's intracellular growth and virulence, but neither its location within T6SS nor its functional role has been established. Here, we present the cryoEM structure of IglD from Francisella novicida and show that the IglD forms a homotrimer that is structurally homologous to the T6SS baseplate protein TssK in Escherichia coli. Each IglD monomer consists of an N-terminal β-sandwich domain, a 4-helix bundle domain, and a flexible C-terminal domain. While the overall folds of IglD and TssK are similar, the two structures differ in three aspects: the relative orientation between their β-sandwich and the 4-helix bundle domains; two insertion loops present in TssK's β-sandwich domain; and, consequently, a lack of subunit-subunit interaction between insertion loops in the IglD trimer. Phylogenetic analysis indicates that IglD is genetically remote from the TssK orthologs in other T6SSs. While the other components of the baseplate are unknown, we conducted pulldown assays showing IglJ interacts with IglD and IglH, pointing to a model wherein IglD, IglH, and IglJ form the baseplate of the T6SS. Alanine substitution mutagenesis further established that IglD's hydrophobic pocket in the N-terminal β-sandwich domain interacts with two loops of IglJ, reminiscent of the TssK-TssG interaction. These results form a framework for understanding the hitherto unexplored T6SS baseplate. Francisella tularensis is a facultatively intracellular Gram-negative bacterium that causes the serious and potentially fatal zoonotic illness, tularemia. Because of its extraordinarily high infectivity and mortality to humans, especially when inhaled, F. tularensis is considered a potential bioterrorism agent and is classified as a Tier 1 select agent. The type VI secretion system (T6SS) encoded within the pathogenicity island (FPI) is critical to its pathogenesis, but its baseplate components are largely unknown. Here, we report the cryoEM structure of IglD from Francisella novicida and demonstrate its role as a component of the baseplate complex of the T6SS. We further show that IglD interacts with IglJ and IglH, and propose a model in which these proteins interact to form the T6SS baseplate. Elucidation of the structure and composition of the baseplate should facilitate the design of strategies to prevent and treat infections caused by F. tularensis.
History
DepositionJul 19, 2022-
Header (metadata) releaseAug 17, 2022-
Map releaseAug 17, 2022-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27656.png

Downloads & links

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Map

FileDownload / File: emd_27656.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIglD trimer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 300 pix.
= 330. Å		1.1 Å/pix.
x 300 pix.
= 330. Å		1.1 Å/pix.
x 300 pix.
= 330. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0345
Minimum - Maximum-0.1455628 - 0.2351958
Average (Standard dev.)0.0000053705744 (±0.004016979)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 330.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half1

Fileemd_27656_half_map_1.map
Annotationhalf1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half2

Fileemd_27656_half_map_2.map
Annotationhalf2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : IglD trimer

EntireName: IglD trimer
Components
  • Complex: IglD trimer
    • Protein or peptide: Secretion system protein

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Supramolecule #1: IglD trimer

SupramoleculeName: IglD trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Francisella tularensis subsp. novicida (bacteria)

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Macromolecule #1: Secretion system protein

MacromoleculeName: Secretion system protein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Francisella tularensis subsp. novicida (bacteria)
Molecular weightTheoretical: 49.562379 KDa
Recombinant expressionOrganism: Francisella tularensis subsp. novicida (bacteria)
SequenceString: MDYKDHDGDY KDHDIDYKDD DDKELMFLER IYWEDGLRLD SDILDKSNLS VLERLSTASY LPANLNKGIV SFDLDVESLQ TGLILIKDL KLYLDEKNFV FYDKSYPLSL QIMTDKLSDE IPLFLNIREK VIEKNGVKYI YNQLSLSLEH SYGFKHSIQI A LFRLDRGR ...String:
MDYKDHDGDY KDHDIDYKDD DDKELMFLER IYWEDGLRLD SDILDKSNLS VLERLSTASY LPANLNKGIV SFDLDVESLQ TGLILIKDL KLYLDEKNFV FYDKSYPLSL QIMTDKLSDE IPLFLNIREK VIEKNGVKYI YNQLSLSLEH SYGFKHSIQI A LFRLDRGR LVPEIYDFPL LTLNHYYLGD IFVKLNRTVS ELKSFNRFVF SASRSYASIL LVFLINKLER ELKFAESNRA NS SPKQIFD LIDDIYSLIQ LNLDKVEELD SIEFDFQKPL TKLNLLADRL LTLCEYRKIN NFIRFELHGK KYICESFPEE FFV ATRYYL FLKRKATAPA NVRFENKNAL RITSISRNKN IVTLSLSGVK LVDVECSMIN FTTRFDNIDA IYEIQKGSEW DFIL ADSSA VFTAFEGSEN FDFFIAFS

UniProtKB: Intracellular growth locus

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1138462
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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