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- EMDB-15903: Automated simulation-based refinement of maltoporin into a cryo-E... -

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Basic information

Entry
Database: EMDB / ID: EMD-15903
TitleAutomated simulation-based refinement of maltoporin into a cryo-EM density
Map dataHalf map 2, box 128
Sample
  • Complex: Trimeric maltoporin (LamB protein)
    • Protein or peptide: Maltoporin
Keywordsmaltoporin / density fit / automated refinement / cryo-em / MEMBRANE PROTEIN
Function / homology
Function and homology information


maltodextrin transmembrane transporter activity / maltose transporting porin activity / pore complex / monoatomic ion transport / cell outer membrane
Similarity search - Function
Maltoporin / Porin, LamB-type / Porin, LamB-type superfamily / LamB porin
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsYvonnesdotter L / Rovsnik U / Blau C / Lycksell M / Howard RJ / Lindahl E
Funding support Sweden, 2 items
OrganizationGrant numberCountry
Swedish Research Council2019-02433 Sweden
Knut and Alice Wallenberg Foundation1484505 Sweden
CitationJournal: Biophys J / Year: 2023
Title: Automated simulation-based membrane protein refinement into cryo-EM data.
Authors: Linnea Yvonnesdotter / Urška Rovšnik / Christian Blau / Marie Lycksell / Rebecca Joy Howard / Erik Lindahl /
Abstract: The resolution revolution has increasingly enabled single-particle cryogenic electron microscopy (cryo-EM) reconstructions of previously inaccessible systems, including membrane proteins-a category ...The resolution revolution has increasingly enabled single-particle cryogenic electron microscopy (cryo-EM) reconstructions of previously inaccessible systems, including membrane proteins-a category that constitutes a disproportionate share of drug targets. We present a protocol for using density-guided molecular dynamics simulations to automatically refine atomistic models into membrane protein cryo-EM maps. Using adaptive force density-guided simulations as implemented in the GROMACS molecular dynamics package, we show how automated model refinement of a membrane protein is achieved without the need to manually tune the fitting force ad hoc. We also present selection criteria to choose the best-fit model that balances stereochemistry and goodness of fit. The proposed protocol was used to refine models into a new cryo-EM density of the membrane protein maltoporin, either in a lipid bilayer or detergent micelle, and we found that results do not substantially differ from fitting in solution. Fitted structures satisfied classical model-quality metrics and improved the quality and the model-to-map correlation of the x-ray starting structure. Additionally, the density-guided fitting in combination with generalized orientation-dependent all-atom potential was used to correct the pixel-size estimation of the experimental cryo-EM density map. This work demonstrates the applicability of a straightforward automated approach to fitting membrane protein cryo-EM densities. Such computational approaches promise to facilitate rapid refinement of proteins under different conditions or with various ligands present, including targets in the highly relevant superfamily of membrane proteins.
History
DepositionOct 3, 2022-
Header (metadata) releaseJun 21, 2023-
Map releaseJun 21, 2023-
UpdateJul 26, 2023-
Current statusJul 26, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15903.png

Downloads & links

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Map

FileDownload / File: emd_15903.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHalf map 2, box 128
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 128 pix.
= 106.24 Å		0.83 Å/pix.
x 128 pix.
= 106.24 Å		0.83 Å/pix.
x 128 pix.
= 106.24 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: 0.0132
Minimum - Maximum-0.013729883 - 0.032488715
Average (Standard dev.)0.0014090466 (±0.0039026383)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 106.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15903_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Postprocessed sharpened map, box 128

Fileemd_15903_additional_1.map
AnnotationPostprocessed sharpened map, box 128
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 3D refined model used for automated fitting, box 128

Fileemd_15903_half_map_1.map
Annotation3D refined model used for automated fitting, box 128
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1, box 128

Fileemd_15903_half_map_2.map
AnnotationHalf map 1, box 128
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Trimeric maltoporin (LamB protein)

EntireName: Trimeric maltoporin (LamB protein)
Components
  • Complex: Trimeric maltoporin (LamB protein)
    • Protein or peptide: Maltoporin

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Supramolecule #1: Trimeric maltoporin (LamB protein)

SupramoleculeName: Trimeric maltoporin (LamB protein) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Maltoporin

MacromoleculeName: Maltoporin / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 47.425785 KDa
SequenceString: VDFHGYARSG IGWTGSGGEQ QCFQTTGAQS KYRLGNECET YAELKLGQEV WKEGDKSFYF DTNVAYSVAQ QNDWEATDPA FREANVQGK NLIEWLPGST IWAGKRFYQR HDVHMIDFYY WDISGPGAGL ENIDVGFGKL SLAATRSSEA GGSSSFASNN I YDYTNETA ...String:
VDFHGYARSG IGWTGSGGEQ QCFQTTGAQS KYRLGNECET YAELKLGQEV WKEGDKSFYF DTNVAYSVAQ QNDWEATDPA FREANVQGK NLIEWLPGST IWAGKRFYQR HDVHMIDFYY WDISGPGAGL ENIDVGFGKL SLAATRSSEA GGSSSFASNN I YDYTNETA NDVFDVRLAQ MEINPGGTLE LGVDYGRANL RDNYRLVDGA SKDGWLFTAE HTQSVLKGFN KFVVQYATDS MT SQGKGLS QGSGVAFDNE KFAYNINNNG HMLRILDHGA ISMGDNWDMM YVGMYQDINW DNDNGTKWWT VGIRPMYKWT PIM STVMEI GYDNVESQRT GDKNNQYKIT LAQQWQAGDS IWSRPAIRVF ATYAKWDEKW GYDYTGNADN NANFGKAVPA DFNG GSFGR GDSDEWTFGA QMEIWW

UniProtKB: Maltoporin

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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

Concentration3 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE

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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: 2.8000000000000003 µm / Nominal defocus min: 1.4000000000000001 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1mal

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 579000
FSC plot (resolution estimation)

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