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- EMDB-2105: Single particle electron microscopy of PilQ dodecameric complexes... -

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Basic information

Entry
Database: EMDB / ID: EMD-2105
TitleSingle particle electron microscopy of PilQ dodecameric complexes from Neisseria meningitidis.
Map dataReconstruction of outer membrane protein complex PilQ.
Sample
  • Sample: Outer membrane protein PilQ from Neisseria meningitidis
  • Protein or peptide: PilQ
KeywordsOuter membrane protein / PilQ / pilus biogenesis
Function / homology
Function and homology information


transport / establishment of competence for transformation / protein secretion / cell outer membrane / protein transport / membrane
Similarity search - Function
Type IV pilus inner membrane component PilP / Pilus assembly protein, PilP / Type IV pilus secretin PilQ / Type IV pilus secretin PilQ / AMIN domain / AMIN domain / AMIN domain / Secretin and TonB N terminus short domain / Secretin/TonB, short N-terminal domain / Secretin/TonB, short N-terminal domain ...Type IV pilus inner membrane component PilP / Pilus assembly protein, PilP / Type IV pilus secretin PilQ / Type IV pilus secretin PilQ / AMIN domain / AMIN domain / AMIN domain / Secretin and TonB N terminus short domain / Secretin/TonB, short N-terminal domain / Secretin/TonB, short N-terminal domain / Secretin and TonB N terminus short domain / GspD/PilQ family / GspD/PilQ family / Bacterial type II secretion system protein D signature. / Type II secretion system protein GspD, conserved site / Type II secretion system protein GspD, conserved site / NolW-like / NolW-like / Bacterial type II/III secretion system short domain / NolW-like superfamily / Type II/III secretion system / Type II/III secretion system / Bacterial type II and III secretion system protein / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
Type IV pilus biogenesis and competence protein PilQ / PilP protein
Similarity search - Component
Biological speciesNeisseria meningitidis MC58 (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 26.0 Å
AuthorsBerry JL / Phelan MM / Collins RF / Adomavicius T / Tonjum T / Frye S / Bird L / Owens R / Ford RC / Lian LY / Derrick JP
CitationJournal: PLoS Pathog / Year: 2012
Title: Structure and assembly of a trans-periplasmic channel for type IV pili in Neisseria meningitidis.
Authors: Jamie-Lee Berry / Marie M Phelan / Richard F Collins / Tomas Adomavicius / Tone Tønjum / Stefan A Frye / Louise Bird / Ray Owens / Robert C Ford / Lu-Yun Lian / Jeremy P Derrick /
Abstract: Type IV pili are polymeric fibers which protrude from the cell surface and play a critical role in adhesion and invasion by pathogenic bacteria. The secretion of pili across the periplasm and outer ...Type IV pili are polymeric fibers which protrude from the cell surface and play a critical role in adhesion and invasion by pathogenic bacteria. The secretion of pili across the periplasm and outer membrane is mediated by a specialized secretin protein, PilQ, but the way in which this large channel is formed is unknown. Using NMR, we derived the structures of the periplasmic domains from N. meningitidis PilQ: the N-terminus is shown to consist of two β-domains, which are unique to the type IV pilus-dependent secretins. The structure of the second β-domain revealed an eight-stranded β-sandwich structure which is a novel variant of the HSP20-like fold. The central part of PilQ consists of two α/β fold domains: the structure of the first of these is similar to domains from other secretins, but with an additional α-helix which links it to the second α/β domain. We also determined the structure of the entire PilQ dodecamer by cryoelectron microscopy: it forms a cage-like structure, enclosing a cavity which is approximately 55 Å in internal diameter at its largest extent. Specific regions were identified in the density map which corresponded to the individual PilQ domains: this allowed us to dock them into the cryoelectron microscopy density map, and hence reconstruct the entire PilQ assembly which spans the periplasm. We also show that the C-terminal domain from the lipoprotein PilP, which is essential for pilus assembly, binds specifically to the first α/β domain in PilQ and use NMR chemical shift mapping to generate a model for the PilP:PilQ complex. We conclude that passage of the pilus fiber requires disassembly of both the membrane-spanning and the β-domain regions in PilQ, and that PilP plays an important role in stabilising the PilQ assembly during secretion, through its anchorage in the inner membrane.
History
DepositionMay 23, 2012-
Header (metadata) releaseOct 3, 2012-
Map releaseOct 10, 2012-
UpdateOct 17, 2012-
Current statusOct 17, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.333
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.333
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4av2
  • Surface level: 0.333
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image2105.jpg

Downloads & links

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Map

FileDownload / File: emd_2105.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of outer membrane protein complex PilQ.
Voxel sizeX=Y=Z: 4.53 Å
Density
Contour LevelBy EMDB: 0.28 / Movie #1: 0.333
Minimum - Maximum-0.11259504 - 0.34620619
Average (Standard dev.)0.09523162 (±0.06463852)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 289.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.534.534.53
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z289.920289.920289.920
α/β/γ90.00090.00090.000
start NX/NY/NZ-32-32-32
NX/NY/NZ646464
MAP C/R/S213
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-0.1130.3460.095

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Supplemental data

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Sample components

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Entire : Outer membrane protein PilQ from Neisseria meningitidis

EntireName: Outer membrane protein PilQ from Neisseria meningitidis
Components
  • Sample: Outer membrane protein PilQ from Neisseria meningitidis
  • Protein or peptide: PilQ

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Supramolecule #1000: Outer membrane protein PilQ from Neisseria meningitidis

SupramoleculeName: Outer membrane protein PilQ from Neisseria meningitidis
type: sample / ID: 1000 / Details: Isolated from Neisserial membranes. / Oligomeric state: Dodecamer / Number unique components: 1
Molecular weightExperimental: 960 KDa / Theoretical: 960 KDa / Method: From sequence

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Macromolecule #1: PilQ

MacromoleculeName: PilQ / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: Dodecamer / Recombinant expression: No
Source (natural)Organism: Neisseria meningitidis MC58 (bacteria) / Location in cell: Outer membrane
Molecular weightExperimental: 960 KDa / Theoretical: 960 KDa
SequenceGO: transport, protein secretion, protein transport, establishment of competence for transformation, cell outer membrane, membrane
InterPro: AMIN domain, GspD/PilQ family, NolW-like, Type IV pilus secretin PilQ, Secretin/TonB, short N-terminal domain, Type II/III secretion system, Type II secretion system protein GspD, conserved site

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 7.5
Details: 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 0.1% (w/v) Zwittergent 3-10
StainingType: NEGATIVE
Details: Unstained: Both sides of the grid were briefly blotted dry with Whatman No. 1 filter paper (2 x 1s blots) using a Vitrobot (FEI) device (90% relative humidity), and the grid was then ...Details: Unstained: Both sides of the grid were briefly blotted dry with Whatman No. 1 filter paper (2 x 1s blots) using a Vitrobot (FEI) device (90% relative humidity), and the grid was then immediately plunged into liquid ethane maintained at <100K.
GridDetails: 400 mesh Quantifoil holey carbon grid with 1.2 micron diameter holes.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 100 K / Instrument: FEI VITROBOT MARK III
Method: Both sides of the grid were briefly blotted dry with Whatman No. 1 filter paper in a humidity-controlled chamber using a Vitrobot (FEI) device (90% relative humidity), and the grid was then ...Method: Both sides of the grid were briefly blotted dry with Whatman No. 1 filter paper in a humidity-controlled chamber using a Vitrobot (FEI) device (90% relative humidity), and the grid was then plunged into liquid ethane maintained at <100K.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 33112 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 5.1 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 33000
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 94 K / Max: 100 K / Average: 97 K
Alignment procedureLegacy - Astigmatism: Via FFT
DateNov 27, 2007
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 55 / Average electron dose: 4 e/Å2 / Bits/pixel: 8
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFIT each micrograph
Final two d classificationNumber classes: 101
Final reconstructionApplied symmetry - Point group: C12 (12 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: OTHER / Software - Name: EMAN
Details: Two different starting models were employed with convergence between them. This map was generated from model 1.
Number images used: 25303
Details29000

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