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- EMDB-51924: KtrA.ADP with cyclic di-AMP prepared on the vitrobot -

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Basic information

Entry
Database: EMDB / ID: EMD-51924
TitleKtrA.ADP with cyclic di-AMP prepared on the vitrobot
Map dataThis is the sharpened map from the refined 35 383 particle stack.
Sample
  • Complex: KtrA, the regulatory subunit from the KtrAB potassium transporter
Keywordssample preparation / air-water interface / potassium transport / RCK C domain / TRANSPORT PROTEIN
Biological speciesBascillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsHirst IJ / Muench SP / Darrow MC / Scarff CA / Thompson RF
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)95519980 United Kingdom
CitationJournal: J Struct Biol / Year: 2025
Title: Untangling the effects of flexibility and the AWI in cryoEM sample preparation: A case study using KtrA.
Authors: Isobel Jackson Hirst / Wesley Tien Chiang / Nien-Jen Hu / Charlotte A Scarff / Rebecca F Thompson / Michele C Darrow / Stephen P Muench /
Abstract: Single particle cryo-electron microscopy (cryoEM) is a powerful tool for elucidating the structures of biological macromolecules without requiring crystallisation or fixation. However, certain ...Single particle cryo-electron microscopy (cryoEM) is a powerful tool for elucidating the structures of biological macromolecules without requiring crystallisation or fixation. However, certain barriers to obtaining high-resolution structures persist, particularly during grid preparation when samples are in a thin liquid film. At this stage, extensive exposure to the air-water interface (AWI) can lead to subunit dissociation, denaturation, and preferred orientation of particles. Another obstacle to high-resolution cryoEM is molecular flexibility, which introduces heterogeneity in the dataset, weakening the signal during image processing. This study explores the effects of AWI interactions and molecular flexibility on the cryoEM density maps of KtrA, the soluble regulatory subunit of the potassium transporter KtrAB from Bacillus subtilis. From grids prepared using a standard blotting technique, we observed a lack of density in the C-lobe domains and preferred orientation. Modifications such as reducing AWI exposure through faster vitrification times (6 s vs ≤100 ms) notably improved C-lobe density. Moreover, the addition of cyclic di-AMP, which binds to the C-lobes, combined with a 100 ms plunge time, further enhanced C-lobe density and eliminated preferred orientation. These findings demonstrate that both AWI interactions and flexibility had to be addressed to obtain density for the C-lobe domains of KtrA. This study underscores the ongoing complexities in achieving high-resolution cryoEM for many samples.
History
DepositionOct 28, 2024-
Header (metadata) releaseMay 21, 2025-
Map releaseMay 21, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51924.png

Downloads & links

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Map

FileDownload / File: emd_51924.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the sharpened map from the refined 35 383 particle stack.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 416 pix.
= 345.28 Å		0.83 Å/pix.
x 416 pix.
= 345.28 Å		0.83 Å/pix.
x 416 pix.
= 345.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.5289903 - 1.0550319
Average (Standard dev.)0.00056750665 (±0.025502697)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 345.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: This is the map after it has been...

Fileemd_51924_additional_1.map
AnnotationThis is the map after it has been low pass filtered in EMAN to 8 A. This map was used for comparison and is the one that is showed in figure 3 in the corresponding paper.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_51924_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_51924_half_map_2.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 : KtrA, the regulatory subunit from the KtrAB potassium transporter

EntireName: KtrA, the regulatory subunit from the KtrAB potassium transporter
Components
  • Complex: KtrA, the regulatory subunit from the KtrAB potassium transporter

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Supramolecule #1: KtrA, the regulatory subunit from the KtrAB potassium transporter

SupramoleculeName: KtrA, the regulatory subunit from the KtrAB potassium transporter
type: complex / ID: 1 / Parent: 0
Details: This is map is part of an investigation into the effects of sample preparation on map quality.
Source (natural)Organism: Bascillus subtilis (bacteria)
Molecular weightTheoretical: 198 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMKClpotassium chloride
50.0 mMTris

Details: 50 mM Tris-HCl, 150 mM KCl, 0.5 mM TCEP with 250 uM CDA
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 6056 / Average exposure time: 3.1 sec. / Average electron dose: 40.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1968748
CTF correctionSoftware - Name: RELION (ver. 4.0)
Software - details: CTFFIND4 was used in relion for CTF estimation
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: 4 ab initio models / Details: ab initio job in cryoSPARC with 4 classes
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 35383
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: Non-uniform refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: Non-uniform refinement
FSC plot (resolution estimation)

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