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- PDB-8b7v: Automated simulation-based refinement of maltoporin into a cryo-E... -

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Basic information

Entry
Database: PDB / ID: 8b7v
TitleAutomated simulation-based refinement of maltoporin into a cryo-EM density
ComponentsMaltoporin
KeywordsMEMBRANE PROTEIN / maltoporin / density fit / automated refinement / cryo-em
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsYvonnesdotter, L. / Rovsnik, U. / Blau, C. / Lycksell, M. / Howard, R.J. / Lindahl, E.
Funding support Sweden, 2items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 26, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Maltoporin
B: Maltoporin
C: Maltoporin


Theoretical massNumber of molelcules
Total (without water)142,2773
Polymers142,2773
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11280 Å2
ΔGint-88 kcal/mol
Surface area47770 Å2

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Components

#1: Protein Maltoporin / / Maltose-inducible porin


Mass: 47425.785 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: A0A2X5NX10

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Trimeric maltoporin (LamB protein) / Type: COMPLEX / Entity ID: all / Source: NATURAL
Source (natural)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2800 nm / Nominal defocus min: 1400 nm
Image recordingElectron dose: 42 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 579000 / Symmetry type: POINT

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