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- EMDB-26396: CryoET of E. coli prepared with the Waffle Method -

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Basic information

Entry
Database: EMDB / ID: EMD-26396
TitleCryoET of E. coli prepared with the Waffle Method
Map dataE. coli waffle lamella tomogram from Appion-Protomo alignment. Pixelsize is actually 8.30616; ignore the filename.
Sample
  • Cell: E. coli BL21 (DE3) cells
Biological speciesEscherichia coli (E. coli)
Methodelectron tomography / cryo EM
AuthorsKelley K / Raczkowski AM / Klykov O / Jaroenlak P / Bobe D / Kopylov M / Eng ET / Bhabha G / Potter CS / Carragher B / Noble AJ
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM128303 United States
Simons FoundationSF349247 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM139171 United States
Citation
Journal: Nat Commun / Year: 2022
Title: Waffle Method: A general and flexible approach for improving throughput in FIB-milling.
Authors: Kotaro Kelley / Ashleigh M Raczkowski / Oleg Klykov / Pattana Jaroenlak / Daija Bobe / Mykhailo Kopylov / Edward T Eng / Gira Bhabha / Clinton S Potter / Bridget Carragher / Alex J Noble /
Abstract: Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in ...Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens with vitrification issues, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and specimens that distribute poorly across grid squares. Here we present a general approach called the 'Waffle Method' which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. We propose the Waffle Method as a way to achieve many advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout.
#1: Journal: BioRxiv / Year: 2021
Title: Waffle Method: A general and flexible approach for improving throughput in FIB-milling
Authors: Kelley K / Raczkowski AM / Klykov O / Jaroenlak P / Bobe D / Kopylov M / Eng ET / Bhabha G / Potter CS / Carragher B / Noble AJ
History
DepositionMar 10, 2022-
Header (metadata) releaseMar 23, 2022-
Map releaseMar 23, 2022-
UpdateApr 20, 2022-
Current statusApr 20, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26396.png

Downloads & links

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Map

FileDownload / File: emd_26396.map.gz / Format: CCP4 / Size: 9.3 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationE. coli waffle lamella tomogram from Appion-Protomo alignment. Pixelsize is actually 8.30616; ignore the filename.
Voxel sizeX=Y=Z: 8.30616 Å
Density
Minimum - Maximum-23.368406 - 10.956615
Average (Standard dev.)-4.6309442e-10 (±0.9999998)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-422
Dimensions14712016844
Spacing20161471844
CellA: 16745.219 Å / B: 12218.361 Å / C: 7010.399 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : E. coli BL21 (DE3) cells

EntireName: E. coli BL21 (DE3) cells
Components
  • Cell: E. coli BL21 (DE3) cells

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Supramolecule #1: E. coli BL21 (DE3) cells

SupramoleculeName: E. coli BL21 (DE3) cells / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli)

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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: 7.4 / Details: PBS
GridModel: Quantifoil R2/2 / Material: COPPER
VitrificationCryogen name: NITROGEN / Details: HPF.
High pressure freezingInstrument: OTHER
Details: The value given for _emd_high_pressure_freezing.instrument is Wohlwend HPF Compact 01. This is not in a list of allowed values {'EMS-002 RAPID IMMERSION FREEZER', 'LEICA EM HPM100', 'OTHER', ...Details: The value given for _emd_high_pressure_freezing.instrument is Wohlwend HPF Compact 01. This is not in a list of allowed values {'EMS-002 RAPID IMMERSION FREEZER', 'LEICA EM HPM100', 'OTHER', 'LEICA EM PACT', 'BAL-TEC HPM 010', 'LEICA EM PACT2'} so OTHER is written into the XML file.
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 3 nA / Focused ion beam - Duration: 3600 sec. / Focused ion beam - Temperature: 70 K / Focused ion beam - Initial thickness: 10000 nm / Focused ion beam - Final thickness: 200 nm
Focused ion beam - Details: Waffle Method. The value given for _emd_sectioning_focused_ion_beam.instrument is TFS Aquilos 2. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is ...Focused ion beam - Details: Waffle Method. The value given for _emd_sectioning_focused_ion_beam.instrument is TFS Aquilos 2. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 6.0 µm / Nominal defocus min: 6.0 µm
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 4.87 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: TOMO3D (ver. Jan 2015) / Software - details: Dose weighted / Number images used: 36

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