[English] 日本語
Yorodumi
- EMDB-51928: KtrA.ADP prepared on the vitrobot, full particle stack -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-51928
TitleKtrA.ADP prepared on the vitrobot, full particle stack
Map data
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: 3.8 Å
AuthorsHirst IJ / Muench SP
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

-
Structure visualization

Supplemental images

emd_51928.png

Downloads & links

-
Map

FileDownload / File: emd_51928.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 416 pix.
= 357.76 Å		0.86 Å/pix.
x 416 pix.
= 357.76 Å		0.86 Å/pix.
x 416 pix.
= 357.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.36
Minimum - Maximum-1.8826666 - 2.9999654
Average (Standard dev.)0.00075295323 (±0.035664883)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 357.76 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Half map: #2

Fileemd_51928_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: #1

Fileemd_51928_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
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

-
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

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
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, 0.03 mM TCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 6 seconds blot time, blot force 6, no wait time.
DetailsThis sample was monodisperse

-
Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 4502 / Average exposure time: 4.45 sec. / Average electron dose: 34.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination 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: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

+
Image processing

Particle selectionNumber selected: 446773
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: 5 ab initio models / Details: ab initio job in cryoSPARC with 5 classes
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 136774
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)

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more