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- EMDB-20757: Negative stain 3D reconstruction of PilU with C1 symmetry -

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Basic information

Entry
Database: EMDB / ID: EMD-20757
TitleNegative stain 3D reconstruction of PilU with C1 symmetry
Map dataNegative stain 3D reconstruction of PilU with C1 symmetry
Sample
  • Complex: Pil-U
Biological speciesVibrio cholerae (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 16.6 Å
AuthorsChlebek JL / Wang JC / Dalia AB
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR35GM128674 United States
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesAI116566 United States
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesAI118863 United States
CitationJournal: PLoS Genet / Year: 2019
Title: PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili.
Authors: Jennifer L Chlebek / Hannah Q Hughes / Aleksandra S Ratkiewicz / Rasman Rayyan / Joseph Che-Yen Wang / Brittany E Herrin / Triana N Dalia / Nicolas Biais / Ankur B Dalia /
Abstract: Bacterial type IV pili are critical for diverse biological processes including horizontal gene transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic ...Bacterial type IV pili are critical for diverse biological processes including horizontal gene transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic appendages extend and retract from the cell surface. In many type IVa pilus systems, extension occurs through the action of an extension ATPase, often called PilB, while optimal retraction requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog of PilT called PilU. However, the function of this protein has remained unclear because pilU mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is relatively understudied compared to PilT. Here, we study the type IVa competence pilus of Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, however, contribute to pilus retraction in ΔpilT strains. Thus, these data suggest that PilU is a bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also found that a ΔpilU mutant exhibited a reduction in the force of retraction suggesting that PilU is important for generating maximal retraction forces. Additional in vitro and in vivo data show that PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be broadly applicable to other type IVa pilus systems.
History
DepositionSep 24, 2019-
Header (metadata) releaseOct 23, 2019-
Map releaseOct 30, 2019-
UpdateOct 30, 2019-
Current statusOct 30, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0084
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0084
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20757.png

Downloads & links

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Map

FileDownload / File: emd_20757.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain 3D reconstruction of PilU with C1 symmetry
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.56 Å/pix.
x 160 pix.
= 249.6 Å		1.56 Å/pix.
x 160 pix.
= 249.6 Å		1.56 Å/pix.
x 160 pix.
= 249.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.56 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0084 / Movie #1: 0.0084
Minimum - Maximum-0.02226781 - 0.07350547
Average (Standard dev.)0.00028666414 (±0.0037767317)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-79-79-79
Dimensions160160160
Spacing160160160
CellA=B=C: 249.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.561.561.56
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z249.600249.600249.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ307236
MAP C/R/S123
start NC/NR/NS-79-79-79
NC/NR/NS160160160
D min/max/mean-0.0220.0740.000

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Supplemental data

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Sample components

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Entire : Pil-U

EntireName: Pil-U
Components
  • Complex: Pil-U

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Supramolecule #1: Pil-U

SupramoleculeName: Pil-U / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Vibrio cholerae (bacteria) / Strain: JLC398
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3)
Molecular weightTheoretical: 410 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 8.5
Details: 25 mM Tris, pH 8.5, 50 mM KCl, 10% glycerol, 1 mM EDTA, 1 mM MgCl2, 0.5 mM TCEP
StainingType: NEGATIVE / Material: Uranyl Acetate / Details: 1% w/v uranyl acetate, 0.5% w/v trehalose
GridSupport film - Material: CARBON / Support film - topology: CONTINUOUS / Details: unspecified
DetailsThe sample was monodisperse.

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Electron microscopy

MicroscopeJEOL 3200FS
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: DIRECT ELECTRON DE-64 (8k x 8k) / Detector mode: INTEGRATING / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: JEOL

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: OTHER / Details: SGD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 16.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.7) / Number images used: 45204
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)

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