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- EMDB-8224: Structure of a mutant type IV pilus machinery (delta N1 ring dele... -

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Basic information

Entry
Database: EMDB / ID: EMD-8224
TitleStructure of a mutant type IV pilus machinery (delta N1 ring deletion in PilQ) in the closed state
Map dataNone
Sample
  • Complex: Structure of a mutant type IV pilus machinery (delta N1 ring deletion in PilQ) in the closed state from Thermus thermophilus
Biological speciesThermus thermophilus (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 50.0 Å
AuthorsGold VAM / Salzer R / Averhoff B
CitationJournal: J Biol Chem / Year: 2016
Title: Topology and Structure/Function Correlation of Ring- and Gate-forming Domains in the Dynamic Secretin Complex of Thermus thermophilus.
Authors: Ralf Salzer / Edoardo D'Imprima / Vicki A M Gold / Ilona Rose / Moritz Drechsler / Janet Vonck / Beate Averhoff /
Abstract: Secretins are versatile outer membrane pores used by many bacteria to secrete proteins, toxins, or filamentous phages; extrude type IV pili (T4P); or take up DNA. Extrusion of T4P and natural ...Secretins are versatile outer membrane pores used by many bacteria to secrete proteins, toxins, or filamentous phages; extrude type IV pili (T4P); or take up DNA. Extrusion of T4P and natural transformation of DNA in the thermophilic bacterium Thermus thermophilus requires a unique secretin complex comprising six stacked rings, a membrane-embedded cone structure, and two gates that open and close a central channel. To investigate the role of distinct domains in ring and gate formation, we examined a set of deletion derivatives by cryomicroscopy techniques. Here we report that maintaining the N0 ring in the deletion derivatives led to stable PilQ complexes. Analyses of the variants unraveled that an N-terminal domain comprising a unique βββαβ fold is essential for the formation of gate 2. Furthermore, we identified four βαββα domains essential for the formation of the N2 to N5 rings. Mutant studies revealed that deletion of individual ring domains significantly reduces piliation. The N1, N2, N4, and N5 deletion mutants were significantly impaired in T4P-mediated twitching motility, whereas the motility of the N3 mutant was comparable with that of wild-type cells. This indicates that the deletion of the N3 ring leads to increased pilus dynamics, thereby compensating for the reduced number of pili of the N3 mutant. All mutants exhibit a wild-type natural transformation phenotype, leading to the conclusion that DNA uptake is independent of functional T4P.
History
DepositionJun 1, 2016-
Header (metadata) releaseJun 15, 2016-
Map releaseJun 15, 2016-
UpdateJul 12, 2017-
Current statusJul 12, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 158
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 158
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8224.png

Downloads & links

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Map

FileDownload / File: emd_8224.map.gz / Format: CCP4 / Size: 501 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 11.56 Å
Density
Contour LevelBy AUTHOR: 158. / Movie #1: 158
Minimum - Maximum151. - 163.999979999999994
Average (Standard dev.)157.50030000000001 (±0.7866444)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-13-13-17
Dimensions404080
Spacing404080
CellA: 462.40002 Å / B: 462.40002 Å / C: 924.80005 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z11.5611.5611.56
M x/y/z404080
origin x/y/z0.0000.0000.000
length x/y/z462.400462.400924.800
α/β/γ90.00090.00090.000
start NX/NY/NZ00-41
NX/NY/NZ737384
MAP C/R/S123
start NC/NR/NS-13-13-17
NC/NR/NS404080
D min/max/mean151.000164.000157.500

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Supplemental data

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Sample components

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Entire : Structure of a mutant type IV pilus machinery (delta N1 ring dele...

EntireName: Structure of a mutant type IV pilus machinery (delta N1 ring deletion in PilQ) in the closed state from Thermus thermophilus
Components
  • Complex: Structure of a mutant type IV pilus machinery (delta N1 ring deletion in PilQ) in the closed state from Thermus thermophilus

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Supramolecule #1: Structure of a mutant type IV pilus machinery (delta N1 ring dele...

SupramoleculeName: Structure of a mutant type IV pilus machinery (delta N1 ring deletion in PilQ) in the closed state from Thermus thermophilus
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB27

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationName
20.0 mMTris
100.0 mMEDTAEthylenediaminetetraacetic acid
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 20 % / Instrument: HOMEMADE PLUNGER
Details: Cell solutions were mixed 1:1 (v/v) with 10 nm ProteinA-gold particles. 3 microlitres of sample were applied to grids and blotted on one side for ~5s before plunging.
DetailsCellular suspension

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Electron microscopy

MicroscopeJEOL 3200FSC
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 7.0 µm / Nominal magnification: 10000
Specialist opticsEnergy filter - Name: In-column Omega Filter
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 3.0 e/Å2
Details: Each image in every tilt series is a drift corrected sum of 3-5 frames

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Image processing

ExtractionNumber tomograms: 8 / Number images used: 36 / Software - Name: IMOD (ver. 4.8.46)
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C12 (12 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 50.0 Å / Resolution method: FSC 0.5 CUT-OFF
Details: Resolution estimate was calculated using a mask to exclude the membrane and peptidoglycan. C12 symmetry was applied to the masked central PilQ core by rotation of each subvolume by 30 ...Details: Resolution estimate was calculated using a mask to exclude the membrane and peptidoglycan. C12 symmetry was applied to the masked central PilQ core by rotation of each subvolume by 30 degrees (360 degrees/12) prior to alignment search.
Number subtomograms used: 432

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