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- EMDB-14022: Volta phase plate cryo-ET of Magnetospirillum gryphiswaldense ove... -

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Entry
Database: EMDB / ID: EMD-14022
TitleVolta phase plate cryo-ET of Magnetospirillum gryphiswaldense overproducing PopZ-Mgr
Map dataVolta phase plate cryo-electron tomography of Magnetospirillum gryphiswaldense (wild-type) overproducing PopZ-Mgr
Sample
  • Cell: Magnetospirillum gryphiswaldense (wild-type) overproducing PopZ-Mgr
    • Organelle or cellular component: PopZ
    • Organelle or cellular component: MamK
    • Organelle or cellular component: Cellular envelope
    • Organelle or cellular component: Flagellum
KeywordsPopZ / Alphaproteobacteria / cytoskeleton / polarity / CELL CYCLE
Biological speciesMagnetospirillum gryphiswaldense MSR-1 (magnetotactic)
Methodelectron tomography / cryo EM
AuthorsToro-Nahuelpan M / Plitzko JM / Schueler D / Pfeiffer D
Funding support Germany, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)692637 Germany
German Research Foundation (DFG)Schu1080/9-2 Germany
CitationJournal: J Mol Biol / Year: 2022
Title: In vivo Architecture of the Polar Organizing Protein Z (PopZ) Meshwork in the Alphaproteobacteria Magnetospirillum gryphiswaldense and Caulobacter crescentus.
Authors: Mauricio Toro-Nahuelpan / Jürgen M Plitzko / Dirk Schüler / Daniel Pfeiffer /
Abstract: The polar organizing protein Z (PopZ) forms a polar microdomain that is inaccessible to larger macromolecules such as ribosomes, and selectively sequesters proteins crucial for cell cycle control and ...The polar organizing protein Z (PopZ) forms a polar microdomain that is inaccessible to larger macromolecules such as ribosomes, and selectively sequesters proteins crucial for cell cycle control and polar morphogenesis in various Alphaproteobacteria. However, the in vivo architecture of this microdomain has remained elusive. Here, we analyzed the three-dimensional ultrastructural organization of the PopZ network in Magnetospirillum gryphiswaldense and Caulobacter crescentus by Volta phase plate cryo-electron tomography, which provides high spatial resolution and improved image contrast. Our results suggest that PopZ forms a porous network of disordered short, flexible, and branching filaments.
History
DepositionDec 16, 2021-
Header (metadata) releaseFeb 2, 2022-
Map releaseFeb 2, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

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  • Imaged by UCSF Chimera
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Supplemental images

emd_14022.png

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Map

FileDownload / File: emd_14022.map.gz / Format: CCP4 / Size: 263.6 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationVolta phase plate cryo-electron tomography of Magnetospirillum gryphiswaldense (wild-type) overproducing PopZ-Mgr
Voxel sizeX=Y=Z: 13.68 Å
Density
Minimum - Maximum-128.0 - 126.0
Average (Standard dev.)4.0680575 (±14.1766205)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00171
Dimensions928928321
Spacing928928321
CellA: 12695.04 Å / B: 12695.04 Å / C: 4391.2803 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z13.6813.6813.68
M x/y/z928928321
origin x/y/z0.0000.0000.000
length x/y/z12695.04012695.0404391.280
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS00171
NC/NR/NS928928321
D min/max/mean-128.000126.0004.068

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Supplemental data

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Mask #1

Fileemd_14022_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Magnetospirillum gryphiswaldense (wild-type) overproducing PopZ-Mgr

EntireName: Magnetospirillum gryphiswaldense (wild-type) overproducing PopZ-Mgr
Components
  • Cell: Magnetospirillum gryphiswaldense (wild-type) overproducing PopZ-Mgr
    • Organelle or cellular component: PopZ
    • Organelle or cellular component: MamK
    • Organelle or cellular component: Cellular envelope
    • Organelle or cellular component: Flagellum

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Supramolecule #1: Magnetospirillum gryphiswaldense (wild-type) overproducing PopZ-Mgr

SupramoleculeName: Magnetospirillum gryphiswaldense (wild-type) overproducing PopZ-Mgr
type: cell / ID: 1 / Parent: 0
Details: Individual segmented structures are depicted using different colors in the EMDB entry page image and in the corresponding publication: Putative PopZ filaments are depicted in white. MamK ...Details: Individual segmented structures are depicted using different colors in the EMDB entry page image and in the corresponding publication: Putative PopZ filaments are depicted in white. MamK filaments are green. The flagellum is colored in gold. The cellular envelope inner and outer membranes are depicted in blue.
Source (natural)Organism: Magnetospirillum gryphiswaldense MSR-1 (magnetotactic)

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Supramolecule #2: PopZ

SupramoleculeName: PopZ / type: organelle_or_cellular_component / ID: 2 / Parent: 1
Details: The polar organizing protein Z (PopZ) forms a polar microdomain that is inaccessible to larger macromolecules such as ribosomes, and selectively sequesters proteins crucial for cell cycle ...Details: The polar organizing protein Z (PopZ) forms a polar microdomain that is inaccessible to larger macromolecules such as ribosomes, and selectively sequesters proteins crucial for cell cycle control and polar morphogenesis in various Alphaproteobacteria. In the present strain PopZ overproduction was achieved via insertion of a Tn5-Ptet-based popZ overexpression cassette into the genome of the Magnetospirillum gryphiswaldense wild-type strain.
Source (natural)Organism: Magnetospirillum gryphiswaldense MSR-1 (magnetotactic)

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Supramolecule #3: MamK

SupramoleculeName: MamK / type: organelle_or_cellular_component / ID: 3 / Parent: 1
Details: The filament-forming bacterial actin MamK is important for organizing magnetosome organelles into chains that are used for navigation along geomagnetic field lines. Magnetosomes are ...Details: The filament-forming bacterial actin MamK is important for organizing magnetosome organelles into chains that are used for navigation along geomagnetic field lines. Magnetosomes are membranous organelles containing nanometer-sized crystals of magnetite present in magnetotactic bacteria.
Source (natural)Organism: Magnetospirillum gryphiswaldense MSR-1 (magnetotactic)

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Supramolecule #4: Cellular envelope

SupramoleculeName: Cellular envelope / type: organelle_or_cellular_component / ID: 4 / Parent: 1 / Details: inner and outer membranes
Source (natural)Organism: Magnetospirillum gryphiswaldense MSR-1 (magnetotactic)

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Supramolecule #5: Flagellum

SupramoleculeName: Flagellum / type: organelle_or_cellular_component / ID: 5 / Parent: 1
Details: Flagella are helical protein filaments powered by a rotary motor to mediate motility of bacteria.
Source (natural)Organism: Magnetospirillum gryphiswaldense MSR-1 (magnetotactic)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7 / Details: modified flask standard medium (FSM)
GridModel: Quantifoil R2/1 / Material: MOLYBDENUM / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE
Details: The mixture was blotted and embedded in vitreous ice by plunge freezing into liquid ethane (< - 170 C). The grids were stored in sealed boxes in liquid nitrogen until used..
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Sigma-Aldrich / Diameter: 15 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsTomography was performed under low-dose conditions using a Titan Krios transmission electron microscope (FEI) equipped with a 300 kV field emission gun, and a Gatan Quantum post-column energy filter. Tilt series were acquired using Serial EM software. The specimen was tilted about one axis with 1.5 degree increments over a typical total angular range of -+ 60 degree. To account for the increased specimen thickness at high tilt angles, the exposure time was multiplied by a factor of 1/cos alpha.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.5 e/Å2
Details: Data collection was performed at 300 kV, with the energy filter operated in the zero-loss mode (slit width of 20 eV). The cumulative electron dose during the tilt series was kept below 150 e- A-2.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsTomograms were reconstructed with (15 nm gold fiducials) the IMOD software package (https://bio3d.colorado.edu/imod/) and treated with an anisotropic non-linear diffusion denoising algorithm to improve signal-to-noise ratio (K: 1, Iterations: 10). Segmentation was performed using Amira software on binned volumes with a voxel size of 10.48 A and 13.68 A. Filaments were traced in Amira using an automated segmentation algorithm based on a generic cylinder as a template implemented in the X-Tracing extension. Prior to filament tracing, binned volumes were subjected to nonlocal-means filtering using Amira software (Thermo Fisher Scientific). The cylindrical templates were generated with a diameter and length of 6 and 15 nm, respectively. To reduce background noise, short filamentous structures with lengths below 30 nm were filtered out. Membrane segmentation was done using the software TomoSegMemTV and a complementary package, SynapSegTools, both for Matlab, and refined manually in Amira (Thermo Fisher Scientific).
Final reconstructionNumber images used: 321

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