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- EMDB-23247: MPER Fluc Bpe in complex with VRC42 -

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Basic information

Entry
Database: EMDB / ID: EMD-23247
TitleMPER Fluc Bpe in complex with VRC42
Map dataMPER-Fluc-Bpe in complex with VRC42
Sample
  • Complex: Complex of MPER fluoride channel (Fluc) Bpe fusion and fab fragment VRC42
Biological speciesBordetella pertussis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.0 Å
AuthorsMcIlwain BC / Erwin AL / Stockbridge RB / Ohi MD
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM128768 United States
CitationJournal: J Mol Biol / Year: 2021
Title: N-terminal Transmembrane-Helix Epitope Tag for X-ray Crystallography and Electron Microscopy of Small Membrane Proteins.
Authors: Benjamin C McIlwain / Amanda L Erwin / Alexander R Davis / B Ben Koff / Louise Chang / Tatsiana Bylund / Gwo-Yu Chuang / Peter D Kwong / Melanie D Ohi / Yen-Ting Lai / Randy B Stockbridge /
Abstract: Structural studies of membrane proteins, especially small membrane proteins, are associated with well-known experimental challenges. Complexation with monoclonal antibody fragments is a common ...Structural studies of membrane proteins, especially small membrane proteins, are associated with well-known experimental challenges. Complexation with monoclonal antibody fragments is a common strategy to augment such proteins; however, generating antibody fragments that specifically bind a target protein is not trivial. Here we identify a helical epitope, from the membrane-proximal external region (MPER) of the gp41-transmembrane subunit of the HIV envelope protein, that is recognized by several well-characterized antibodies and that can be fused as a contiguous extension of the N-terminal transmembrane helix of a broad range of membrane proteins. To analyze whether this MPER-epitope tag might aid structural studies of small membrane proteins, we determined an X-ray crystal structure of a membrane protein target that does not crystallize without the aid of crystallization chaperones, the Fluc fluoride channel, fused to the MPER epitope and in complex with antibody. We also demonstrate the utility of this approach for single particle electron microscopy with Fluc and two additional small membrane proteins that represent different membrane protein folds, AdiC and GlpF. These studies show that the MPER epitope provides a structurally defined, rigid docking site for antibody fragments that is transferable among diverse membrane proteins and can be engineered without prior structural information. Antibodies that bind to the MPER epitope serve as effective crystallization chaperones and electron microscopy fiducial markers, enabling structural studies of challenging small membrane proteins.
History
DepositionJan 6, 2021-
Header (metadata) releaseMar 17, 2021-
Map releaseMar 17, 2021-
UpdateJul 28, 2021-
Current statusJul 28, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

FileDownload / File: emd_23247.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMPER-Fluc-Bpe in complex with VRC42
Voxel sizeX=Y=Z: 0.91 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.056063093 - 0.31465918
Average (Standard dev.)0.0009167895 (±0.017147321)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 327.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.910.910.91
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z327.600327.600327.600
α/β/γ90.00090.00090.000
start NX/NY/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0560.3150.001

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Supplemental data

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Sample components

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Entire : Complex of MPER fluoride channel (Fluc) Bpe fusion and fab fragme...

EntireName: Complex of MPER fluoride channel (Fluc) Bpe fusion and fab fragment VRC42
Components
  • Complex: Complex of MPER fluoride channel (Fluc) Bpe fusion and fab fragment VRC42

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Supramolecule #1: Complex of MPER fluoride channel (Fluc) Bpe fusion and fab fragme...

SupramoleculeName: Complex of MPER fluoride channel (Fluc) Bpe fusion and fab fragment VRC42
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Bordetella pertussis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 80 KDa

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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

Concentration0.5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 65.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER / Software - Name: cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15.0) / Number images used: 35655
FSC plot (resolution estimation)

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