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- EMDB-40187: Representative cryo-electron tomogram of pRLB-540 mixed with lipo... -

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Basic information

Entry
Database: EMDB / ID: EMD-40187
TitleRepresentative cryo-electron tomogram of pRLB-540 mixed with liposomes, acidified to pH 5.5 and plunge frozen after 60s incubation.
Map dataCryo-electron tomogram of pRLB-540 mixed with liposomes (pH 5.5, 60s incubation)
Sample
  • Complex: pRLB-540 mixed with DOPC lipids (pH 8.0)
KeywordspH-activated / lipid / lytic / liposome / LIPID BINDING PROTEIN
Biological speciessynthetic construct (others)
Methodelectron tomography / cryo EM
AuthorsCroft JT / Lee KK
Funding support United States, European Union, 3 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)1762114 United States
National Science Foundation (NSF, United States)2140004 United States
European Molecular Biology Organization (EMBO)ALTF 139-2018European Union
CitationJournal: Protein Sci / Year: 2023
Title: De novo design of monomeric helical bundles for pH-controlled membrane lysis.
Authors: Nicolas Goldbach / Issa Benna / Basile I M Wicky / Jacob T Croft / Lauren Carter / Asim K Bera / Hannah Nguyen / Alex Kang / Banumathi Sankaran / Erin C Yang / Kelly K Lee / David Baker /
Abstract: Targeted intracellular delivery via receptor-mediated endocytosis requires the delivered cargo to escape the endosome to prevent lysosomal degradation. This can in principle be achieved by membrane ...Targeted intracellular delivery via receptor-mediated endocytosis requires the delivered cargo to escape the endosome to prevent lysosomal degradation. This can in principle be achieved by membrane lysis tightly restricted to endosomal membranes upon internalization to avoid general membrane insertion and lysis. Here, we describe the design of small monomeric proteins with buried histidine containing pH-responsive hydrogen bond networks and membrane permeating amphipathic helices. Of the 30 designs that were experimentally tested, all expressed in Escherichia coli, 13 were monomeric with the expected secondary structure, and 4 designs disrupted artificial liposomes in a pH-dependent manner. Mutational analysis showed that the buried histidine hydrogen bond networks mediate pH-responsiveness and control lysis of model membranes within a very narrow range of pH (6.0-5.5) with almost no lysis occurring at neutral pH. These tightly controlled lytic monomers could help mediate endosomal escape in designed targeted delivery platforms.
History
DepositionMar 20, 2023-
Header (metadata) releaseSep 13, 2023-
Map releaseSep 13, 2023-
UpdateNov 8, 2023-
Current statusNov 8, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40187.png

Downloads & links

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Map

FileDownload / File: emd_40187.map.gz / Format: CCP4 / Size: 534.9 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationCryo-electron tomogram of pRLB-540 mixed with liposomes (pH 5.5, 60s incubation)
Voxel sizeX=Y=Z: 2.7728 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)3.794937 (±21.613350000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin3052342
Dimensions13901048385
Spacing10481390385
CellA: 2905.8943 Å / B: 3854.192 Å / C: 1067.528 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : pRLB-540 mixed with DOPC lipids (pH 8.0)

EntireName: pRLB-540 mixed with DOPC lipids (pH 8.0)
Components
  • Complex: pRLB-540 mixed with DOPC lipids (pH 8.0)

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Supramolecule #1: pRLB-540 mixed with DOPC lipids (pH 8.0)

SupramoleculeName: pRLB-540 mixed with DOPC lipids (pH 8.0) / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: synthetic construct (others)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 5.5
Component:
ConcentrationFormulaName
50.0 mMTris-HClTris-HCl
150.0 mMNaClNACl

Details: TBS pH 8.0
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: 4 K / Instrument: FEI VITROBOT MARK IV
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 0.3 sec. / Average electron dose: 2.32 e/Å2 / Details: Total tomogram dose 95 e-/A^2
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: OTHER / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 64000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: EMAN2 (ver. 2.99) / Number images used: 41

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