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- EMDB-20962: Cryo-EM structure of nucleotide-free MsbA reconstituted into pept... -

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Basic information

Entry
Database: EMDB / ID: EMD-20962
TitleCryo-EM structure of nucleotide-free MsbA reconstituted into peptidiscs, conformation 2
Map datanucleotide-free MsbA reconstituted into peptidiscs, conformation 2
Sample
  • Complex: Lipid A export ATP-binding/permease protein MsbA
    • Protein or peptide: Lipid A export ATP-binding/permease protein MsbA
Keywordsmembrane protein / ABC transporter / membrane mimetic / peptidisc / TRANSLOCASE
Function / homology
Function and homology information


MsbA transporter complex / lipopolysaccharide floppase activity / lipid translocation / ABC-type lipid A-core oligosaccharide transporter / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances / lipopolysaccharide transport / ATPase-coupled lipid transmembrane transporter activity / ABC-type xenobiotic transporter activity / lipid transport / ABC-type transporter activity ...MsbA transporter complex / lipopolysaccharide floppase activity / lipid translocation / ABC-type lipid A-core oligosaccharide transporter / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances / lipopolysaccharide transport / ATPase-coupled lipid transmembrane transporter activity / ABC-type xenobiotic transporter activity / lipid transport / ABC-type transporter activity / ATP-binding cassette (ABC) transporter complex / transmembrane transport / lipid binding / ATP hydrolysis activity / ATP binding / identical protein binding / membrane / plasma membrane
Similarity search - Function
Lipid A export ATP-binding/permease protein msbA family profile. / ABC transporter, lipid A-core flippase, MsbA / Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter ...Lipid A export ATP-binding/permease protein msbA family profile. / ABC transporter, lipid A-core flippase, MsbA / Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ATP-binding transport protein MsbA / ATP-dependent lipid A-core flippase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsAngiulli G / Walz T
CitationJournal: Elife / Year: 2020
Title: New approach for membrane protein reconstitution into peptidiscs and basis for their adaptability to different proteins.
Authors: Gabriella Angiulli / Harveer Singh Dhupar / Hiroshi Suzuki / Irvinder Singh Wason / Franck Duong Van Hoa / Thomas Walz /
Abstract: Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present 'on-gradient' reconstitution, a new gentle ...Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present 'on-gradient' reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional 'on-bead' approach, we reconstituted proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.
History
DepositionNov 15, 2019-
Header (metadata) releaseDec 11, 2019-
Map releaseMar 4, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6uzl
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20962.png

Downloads & links

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Map

FileDownload / File: emd_20962.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnucleotide-free MsbA reconstituted into peptidiscs, conformation 2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1 Å/pix.
x 256 pix.
= 256. Å		1 Å/pix.
x 256 pix.
= 256. Å		1 Å/pix.
x 256 pix.
= 256. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0175 / Movie #1: 0.0175
Minimum - Maximum-0.024678076 - 0.0710697
Average (Standard dev.)-0.0000012449137 (±0.0023351947)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 256.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z256.000256.000256.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0250.071-0.000

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Supplemental data

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Sample components

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Entire : Lipid A export ATP-binding/permease protein MsbA

EntireName: Lipid A export ATP-binding/permease protein MsbA
Components
  • Complex: Lipid A export ATP-binding/permease protein MsbA
    • Protein or peptide: Lipid A export ATP-binding/permease protein MsbA

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Supramolecule #1: Lipid A export ATP-binding/permease protein MsbA

SupramoleculeName: Lipid A export ATP-binding/permease protein MsbA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: Cell inner membrane

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Macromolecule #1: Lipid A export ATP-binding/permease protein MsbA

MacromoleculeName: Lipid A export ATP-binding/permease protein MsbA / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: ABC-type lipid A-core oligosaccharide transporter
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 66.833109 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MMHNDKDLST WQTFRRLWPT IAPFKAGLIV AGVALILNAA SDTFMLSLLK PLLDDGFGKT DRSVLVWMP LVVIGLMILR GITSYVSSYC ISWVSGKVVM TMRRRLFGHM MGMPVSFFDK QSTGTLLSRI TYDSEQVASS S SGALITVV ...String:
MGSSHHHHHH SSGLVPRGSH MMHNDKDLST WQTFRRLWPT IAPFKAGLIV AGVALILNAA SDTFMLSLLK PLLDDGFGKT DRSVLVWMP LVVIGLMILR GITSYVSSYC ISWVSGKVVM TMRRRLFGHM MGMPVSFFDK QSTGTLLSRI TYDSEQVASS S SGALITVV REGASIIGLF IMMFYYSWQL SIILIVLAPI VSIAIRVVSK RFRNISKNMQ NTMGQVTTSA EQMLKGHKEV LI FGGQEVE TKRFDKVSNR MRLQGMKMVS ASSISDPIIQ LIASLALAFV LYAASFPSVM DSLTAGTITV VFSSMIALMR PLK SLTNVN AQFQRGMAAC QTLFTILDSE QEKDEGKRVI ERATGDVEFR NVTFTYPGRD VPALRNINLK IPAGKTVALV GRSG SGKST IASLITRFYD IDEGEILMDG HDLREYTLAS LRNQVALVSQ NVHLFNDTVA NNIAYARTEQ YSREQIEEAA RMAYA MDFI NKMDNGLDTV IGENGVLLSG GQRQRIAIAR ALLRDSPILI LDEATSALDT ESERAIQAAL DELQKNRTSL VIAHRL STI EKADEIVVVE DGVIVERGTH NDLLEHRGVY AQLHKMQFGQ

UniProtKB: ATP-binding transport protein MsbA

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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

Concentration1.3 mg/mL
BufferpH: 7.9
Component:
ConcentrationNameFormula
50.0 mMTris-base
200.0 mMsodium chlorideNaCl
Sugar embeddingMaterial: vitreous ice
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Average exposure time: 10.0 sec. / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -2.0 µm / Nominal defocus min: -1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 78589
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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