SASDDC5: MsbA in stealth nanodisc (SANS, 100% D2O) + 1 mM ADP

Basic information

Entry

Database: SASBDB / ID: SASDDC5

Sample

MsbA in stealth nanodisc (SANS, 100% D2O) + 1 mM ADP

• Lipid A export ATP-binding/permease protein MsbA (protein), MsbA, Escherichia coli (strain K12)
• Membrane scaffold protein 1D1 (deuterated, 75%) (protein), d75-msp1d1
• 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) (other), d(78,92)-POPC, Escherichia coli. (AL95 strain)

Function / homology

Function:

MsbA transporter complex / lipopolysaccharide floppase activity / lipid translocation / ABC-type lipid A-core oligosaccharide transporter / lipopolysaccharide transport / ATPase-coupled lipid transmembrane transporter activity / ABC-type xenobiotic transporter activity / lipid transport / ATP-binding cassette (ABC) transporter complex / transmembrane transport / lipid binding / ATP hydrolysis activity / ATP binding / membrane / identical protein binding / plasma membrane

Domain/homology:

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

Component:

ATP-dependent lipid A-core flippase

• Biological species: Escherichia coli (strain K12) (bacteria); Escherichia coli. (AL95 strain)
• Citation: Journal: Structure / Year: 2018; Title: Conformational States of ABC Transporter MsbA in a Lipid Environment Investigated by Small-Angle Scattering Using Stealth Carrier Nanodiscs.; Authors: Inokentijs Josts / Julius Nitsche / Selma Maric / Haydyn D Mertens / Martine Moulin / Michael Haertlein / Sylvain Prevost / Dmitri I Svergun / Sebastian Busch / V Trevor Forsyth / Henning Tidow / ; Abstract: Structural studies of integral membrane proteins (IMPs) are challenging, as many of them are inactive or insoluble in the absence of a lipid environment. Here, we describe an approach making use of fractionally deuterium labeled "stealth carrier" nanodiscs that are effectively invisible to low-resolution neutron diffraction and enable structural studies of IMPs in a lipidic native-like solution environment. We illustrate the potential of the method in a joint small-angle neutron scattering (SANS) and X-ray scattering (SAXS) study of the ATP-binding cassette (ABC) transporter protein MsbA solubilized in the stealth nanodiscs. The data allow for a direct observation of the signal from the solubilized protein without contribution from the surrounding lipid nanodisc. Not only the overall shape but also differences between conformational states of MsbA can be reliably detected from the scattering data, demonstrating the sensitivity of the approach and its general applicability to structural studies of IMPs.

Contact author

• Haydyn Mertens (EMBL-Hamburg, European Molecular Biology Laboratory (EMBL) - Hamburg outstation, Notkestraße 85, Geb. 25A, 22607 Hamburg, Deutschland, Germany)

Models

Sample

• Sample: Name: MsbA in stealth nanodisc (SANS, 100% D2O) + 1 mM ADP / Contrast: -3.313 / Specific vol: 0.745 / Specimen concentration: 1.00-6.50 / Concentration method: A280 / Entity id: 1016 / 1017 / 1018
• Buffer: Name: 30 mM Tris, 150 mM NaCl, 1 mM ADP / pH: 7.5 / Comment: nanodisc buffer

Entity #1016

Name: MsbA / Type: protein
Description: Lipid A export ATP-binding/permease protein MsbA
Formula weight: 66.279 / Num. of mol.: 2 / Source: Escherichia coli (strain K12) / References: UniProt: P60752
Sequence:

MHHHHHHGEN LYFQGSHNDK DLSTWQTFRR LWPTIAPFKA GLIVAGVALI LNAASDTFML SLLKPLLDDG FGKTDRSVLV WMPLVVIGLM ILRGITSYVS SYCISWVSGK VVMTMRRRLF GHMMGMPVSF FDKQSTGTLL SRITYDSEQV ASSSSGALIT VVREGASIIG LFIMMFYYSW QLSIILIVLA PIVSIAIRVV SKRFRNISKN MQNTMGQVTT SAEQMLKGHK EVLIFGGQEV ETKRFDKVSN RMRLQGMKMV SASSISDPII QLIASLALAF VLYAASFPSV MDSLTAGTIT VVFSSMIALM RPLKSLTNVN AQFQRGMAAC QTLFTILDSE QEKDEGKRVI ERATGDVEFR NVTFTYPGRD VPALRNINLK IPAGKTVALV GRSGSGKSTI ASLITRFYDI DEGEILMDGH DLREYTLASL RNQVALVSQN VHLFNDTVAN NIAYARTEQY SREQIEEAAR MAYAMDFINK MDNGLDTVIG ENGVLLSGGQ RQRIAIARAL LRDSPILILD EATSALDTES ERAIQAALDE LQKNRTSLVI AHRLSTIEKA DEIVVVEDGV IVERGTHNDL LEHRGVYAQL HKMQFGQ

Entity #1017

Name: d75-msp1d1 / Type: protein
Description: Membrane scaffold protein 1D1 (deuterated, 75%)
Formula weight: 24.661 / Num. of mol.: 2
Sequence:

GHHHHHHHDY DIPTTENLYF QGSTFSKLRE QLGPVTQEFW DNLEKETEGL RQEMSKDLEE VKAKVQPYLD DFQKKWQEEM ELYRQKVEPL RAELQEGARQ KLHELQEKLS PLGEEMRDRA RAHVDALRTH LAPYSDELRQ RLAARLEALK ENGGARLAEY HAKATEHLST LSEKAKPALE DLRQGLLPVL ESFKVSFLSA LEEYTKKLNT Q

Entity #1018

Name: d(78,92)-POPC / Type: other
Description: 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl)
Formula weight: 0.76 / Source: Escherichia coli. (AL95 strain)
Sequence:

C42H82NO8P

Experimental information

• Beam: Instrument name: ILL D11 / City: Grenoble / 国: France / Type of source: neutron source / Wavelength: 0.46 Å
• Detector: Name: CERCA / Type: 3-He gas detector / Pixsize x: 7.5 mm

Scan

Title: (100% D2O) MsbA in stealth nanodisc + 1mM ADP / Measurement date: Mar 9, 2017 / Storage temperature: 10 °C / Cell temperature: 10 °C / Exposure time: 4200 sec. / Number of frames: 2 / Unit: 1/A /

	Min	Max
Q	0.0097	0.3022

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 98 /

	Min	Max
Q	0.0199587	0.302247
P(R) point	1	98
R	0	125

Result

Type of curve: single_conc / Standard: water
Comments: 'MsbA ABC transporter protein encapsulated in a partially deuterated

	Experimental	Experimental error	Porod	Porod error
MW	173 kDa	20	109 kDa	10
Volume	-	-	173 nm3	20

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	1.166	0.003	1.14	0.003
Radius of gyration, Rg	3.87 nm	0.1	3.857 nm	0.33

	Min	Max	Error
D	-	12.5	0.5
Guinier point	9	19	-