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- EMDB-50107: Artificial membrane protein TMHC4_R (ROCKET) mutant R9A/K10A/R13A -

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Basic information

Entry
Database: EMDB / ID: EMD-50107
TitleArtificial membrane protein TMHC4_R (ROCKET) mutant R9A/K10A/R13A
Map data
Sample
  • Organelle or cellular component: Artificial membrane protein TMHC4_R (ROCKET) mutant R9A/K10A/R13A
    • Protein or peptide: TMHC4_R (ROCKET) mutant R9A/K10A/R13A
Keywordssyntectic construct / membrane protein / artificial protein
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsAbramsson M / Anden O / Howard RJ / Lindahl E / Landreh M
Funding support Sweden, 2 items
OrganizationGrant numberCountry
Swedish Research Council Sweden
Knut and Alice Wallenberg Foundation Sweden
CitationJournal: Elife / Year: 2025
Title: Engineering cardiolipin binding to an artificial membrane protein reveals determinants for lipid-mediated stabilization.
Authors: Mia L Abramsson / Robin A Corey / Jan L Skerle / Louise J Persson / Olivia Anden / Abraham O Oluwole / Rebecca J Howard / Erik Lindahl / Carol V Robinson / Kvido Strisovsky / Erik G Marklund ...Authors: Mia L Abramsson / Robin A Corey / Jan L Skerle / Louise J Persson / Olivia Anden / Abraham O Oluwole / Rebecca J Howard / Erik Lindahl / Carol V Robinson / Kvido Strisovsky / Erik G Marklund / David Drew / Phillip J Stansfeld / Michael Landreh /
Abstract: Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate ...Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.
History
DepositionApr 16, 2024-
Header (metadata) releaseMay 8, 2024-
Map releaseMay 8, 2024-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50107.png

Downloads & links

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Map

FileDownload / File: emd_50107.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.51 Å/pix.
x 512 pix.
= 259.891 Å		0.51 Å/pix.
x 512 pix.
= 259.891 Å		0.51 Å/pix.
x 512 pix.
= 259.891 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.5076 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0013
Minimum - Maximum-0.0034913227 - 0.0055421405
Average (Standard dev.)0.000011102166 (±0.00011995009)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 259.8912 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50107_msk_1.map
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Half map: #1

Fileemd_50107_half_map_1.map
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Half map: #2

Fileemd_50107_half_map_2.map
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Sample components

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Entire : Artificial membrane protein TMHC4_R (ROCKET) mutant R9A/K10A/R13A

EntireName: Artificial membrane protein TMHC4_R (ROCKET) mutant R9A/K10A/R13A
Components
  • Organelle or cellular component: Artificial membrane protein TMHC4_R (ROCKET) mutant R9A/K10A/R13A
    • Protein or peptide: TMHC4_R (ROCKET) mutant R9A/K10A/R13A

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Supramolecule #1: Artificial membrane protein TMHC4_R (ROCKET) mutant R9A/K10A/R13A

SupramoleculeName: Artificial membrane protein TMHC4_R (ROCKET) mutant R9A/K10A/R13A
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 99.56 kDa/nm

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Macromolecule #1: TMHC4_R (ROCKET) mutant R9A/K10A/R13A

MacromoleculeName: TMHC4_R (ROCKET) mutant R9A/K10A/R13A / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSKDTEDSAA IWATIMLLLV FAILLSAIIW YQITTNPDTS QIATLLSMQL LLIALMLVVI ALLLSRQTEQ VAESIRRDVS ALAYVMLGLL LSLLNRLSLA AEAYKKAIEL DPNDALAWLL LGSVLEKLKR LDEAAEAYKK AIELKPNDAS AWKELGKVLE KLGRLDEAAE ...String:
MSKDTEDSAA IWATIMLLLV FAILLSAIIW YQITTNPDTS QIATLLSMQL LLIALMLVVI ALLLSRQTEQ VAESIRRDVS ALAYVMLGLL LSLLNRLSLA AEAYKKAIEL DPNDALAWLL LGSVLEKLKR LDEAAEAYKK AIELKPNDAS AWKELGKVLE KLGRLDEAAE AYKKAIELDP EDAEAWKELG KVLEKLGRLD EAAEAYKKAI ELDPNDLEHH HHHH

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state3D array

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Sample preparation

BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Number images used: 142000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4)
FSC plot (resolution estimation)

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