National Institutes of Health/Office of the Director
1DP2GM123494
United States
National Institutes of Health/National Institute of General Medical Sciences
R01-GM057045
United States
Citation
Journal: Elife / Year: 2019 Title: The molecular architecture of engulfment during sporulation. Authors: Kanika Khanna / Javier Lopez-Garrido / Ziyi Zhao / Reika Watanabe / Yuan Yuan / Joseph Sugie / Kit Pogliano / Elizabeth Villa / Abstract: The study of bacterial cell biology is limited by difficulties in visualizing cellular structures at high spatial resolution within their native milieu. Here, we visualize sporulation using cryo- ...The study of bacterial cell biology is limited by difficulties in visualizing cellular structures at high spatial resolution within their native milieu. Here, we visualize sporulation using cryo-electron tomography coupled with cryo-focused ion beam milling, allowing the reconstruction of native-state cellular sections at molecular resolution. During sporulation, an asymmetrically-positioned septum generates a larger mother cell and a smaller forespore. Subsequently, the mother cell engulfs the forespore. We show that the septal peptidoglycan is not completely degraded at the onset of engulfment. Instead, the septum is uniformly and only slightly thinned as it curves towards the mother cell. Then, the mother cell membrane migrates around the forespore in tiny finger-like projections, whose formation requires the mother cell SpoIIDMP protein complex. We propose that a limited number of SpoIIDMP complexes tether to and degrade the peptidoglycan ahead of the engulfing membrane, generating an irregular membrane front.
Download / File: emd_20336.map.gz / Format: CCP4 / Size: 37.5 MB / Type: IMAGE STORED AS SIGNED BYTE
Annotation
Cryo-electron tomogram of Bacillus subtilis wild type sporangium with curved septum
Voxel size
X=Y=Z: 24.48 Å
Density
Minimum - Maximum
-128 - 127
Average (Standard dev.)
2.5190954 (±6.83525)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
-244
-176
71
Dimensions
561
580
121
Spacing
580
561
121
Cell
A: 14198.399 Å / B: 13733.279 Å / C: 2962.0798 Å α=β=γ: 90.0 °
CCP4 map header:
mode
envelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z
24.479998275862
24.479998217469
24.48
M x/y/z
580
561
121
origin x/y/z
0.000
0.000
0.000
length x/y/z
14198.399
13733.279
2962.080
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
0
0
0
NX/NY/NZ
162
182
277
MAP C/R/S
1
2
3
start NC/NR/NS
-176
-244
71
NC/NR/NS
580
561
121
D min/max/mean
-128.000
127.000
2.519
-
Supplemental data
-
Sample components
-
Entire : Bacillus subtilis wild type sporangium with flat septum
Entire
Name: Bacillus subtilis wild type sporangium with flat septum
Components
Cell: Bacillus subtilis wild type sporangium with flat septum
-
Supramolecule #1: Bacillus subtilis wild type sporangium with flat septum
Supramolecule
Name: Bacillus subtilis wild type sporangium with flat septum type: cell / ID: 1 / Parent: 0
Source (natural)
Organism: Bacillus subtilis PY79 (bacteria)
-
Experimental details
-
Structure determination
Method
cryo EM
Processing
electron tomography
Aggregation state
cell
-
Sample preparation
Buffer
pH: 7
Grid
Details: unspecified
Vitrification
Cryogen name: ETHANE-PROPANE
Sectioning
Focused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.03 nA / Focused ion beam - Duration: 3600 sec. / Focused ion beam - Temperature: 100 K / Focused ion beam - Initial thickness: 1200 nm / Focused ion beam - Final thickness: 200 nm Focused ion beam - Details: The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Scios Dual Beam. This is not in a list of allowed values set(['DB235', 'OTHER']) so OTHER is written into the XML file.
-
Electron microscopy
Microscope
FEI POLARA 300
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 58.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: OTHER / Imaging mode: OTHER
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
-
Image processing
Final reconstruction
Software - Name: IMOD / Number images used: 68
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