Journal: Mol Microbiol / Year: 2017 Title: Structural characterization of the NAP; the major adhesion complex of the human pathogen Mycoplasma genitalium. Authors: Margot P Scheffer / Luis Gonzalez-Gonzalez / Anja Seybert / Mercè Ratera / Michael Kunz / José M Valpuesta / Ignacio Fita / Enrique Querol / Jaume Piñol / Jaime Martín-Benito / Achilleas S Frangakis / Abstract: Mycoplasma genitalium, the causative agent of non-gonococcal urethritis and pelvic inflammatory disease in humans, is a small eubacterium that lacks a peptidoglycan cell wall. On the surface of its ...Mycoplasma genitalium, the causative agent of non-gonococcal urethritis and pelvic inflammatory disease in humans, is a small eubacterium that lacks a peptidoglycan cell wall. On the surface of its plasma membrane is the major surface adhesion complex, known as NAP that is essential for adhesion and gliding motility of the organism. Here, we have performed cryo-electron tomography of intact cells and detergent permeabilized M. genitalium cell aggregates, providing sub-tomogram averages of free and cell-attached NAPs respectively, revealing a tetrameric complex with two-fold rotational (C2) symmetry. Each NAP has two pairs of globular lobes (named α and β lobes), arranged as a dimer of heterodimers with each lobe connected by a stalk to the cell membrane. The β lobes are larger than the α lobes by 20%. Classification of NAPs showed that the complex can tilt with respect to the cell membrane. A protein complex containing exclusively the proteins P140 and P110, was purified from M. genitalium and was structurally characterized by negative-stain single particle EM reconstruction. The close structural similarity found between intact NAPs and the isolated P140/P110 complexes, shows that dimers of P140/P110 heterodimers are the only components of the extracellular region of intact NAPs in M. genitalium.
History
Deposition
Jun 7, 2017
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Header (metadata) release
Jul 12, 2017
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Map release
Jul 12, 2017
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Update
Sep 13, 2017
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Current status
Sep 13, 2017
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_3756.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Sub-tomogram average of the surface adhesin (NAP) complex from Mycoplasma genitalium cells by cryo-electron tomography.
Voxel size
X=Y=Z: 6.7 Å
Density
Contour Level
By AUTHOR: 0.00229 / Movie #1: 0.00229
Minimum - Maximum
0. - 0.006781161
Average (Standard dev.)
0.002231048 (±0.00023274026)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
64
64
64
Spacing
64
64
64
Cell
A=B=C: 428.8 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
6.7
6.7
6.7
M x/y/z
64
64
64
origin x/y/z
0.000
0.000
0.000
length x/y/z
428.800
428.800
428.800
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
64
64
64
D min/max/mean
0.000
0.007
0.002
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Supplemental data
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Sample components
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Entire : Sub-tomogram average of the surface adhesin (NAP) complex from My...
Entire
Name: Sub-tomogram average of the surface adhesin (NAP) complex from Mycoplasma genitalium cells by cryo-electron tomography.
Components
Complex: Sub-tomogram average of the surface adhesin (NAP) complex from Mycoplasma genitalium cells by cryo-electron tomography.
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Supramolecule #1: Sub-tomogram average of the surface adhesin (NAP) complex from My...
Supramolecule
Name: Sub-tomogram average of the surface adhesin (NAP) complex from Mycoplasma genitalium cells by cryo-electron tomography. type: complex / ID: 1 / Parent: 0
Source (natural)
Organism: Mycoplasma genitalium G37 (bacteria)
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Experimental details
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Structure determination
Method
cryo EM
Processing
subtomogram averaging
Aggregation state
cell
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Sample preparation
Buffer
pH: 7.4
Vitrification
Cryogen name: ETHANE
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.2 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Final reconstruction
Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Super-sampling SART / Number subtomograms used: 1500
Extraction
Number tomograms: 2 / Number images used: 1500 / Software - Name: MATLAB
CTF correction
Software - Name: Super-sampling SART
Final angle assignment
Type: NOT APPLICABLE
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