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- EMDB-4285: Amphipol reconstituted UIC2 Fab complex of double EQ mutant of hu... -

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Entry
Database: EMDB / ID: 4285
TitleAmphipol reconstituted UIC2 Fab complex of double EQ mutant of human-mouse chimeric ABCB1 (ABCB1HMEQ)
Map dataAmphipol reconstituted ABCB1HMEQ-UIC2 complex
SampleAmphipol reconstituted UIC2 Fab complex of double EQ mutant of human-mouse chimeric ABCB1 (ABCB1HMEQ):
SourceHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 6.25 Å resolution
AuthorsAlam A / Locher KP
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2018
Title: Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1.
Authors: Amer Alam / Raphael Küng / Julia Kowal / Robert A McLeod / Nina Tremp / Eugenia V Broude / Igor B Roninson / Henning Stahlberg / Kaspar P Locher
Abstract: The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer ...The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer cells. Here, we report the near-atomic resolution cryo-EM structure of nucleotide-free ABCB1 trapped by an engineered disulfide cross-link between the nucleotide-binding domains (NBDs) and bound to the antigen-binding fragment of the human-specific inhibitory antibody UIC2 and to the third-generation ABCB1 inhibitor zosuquidar. Our structure reveals the transporter in an occluded conformation with a central, enclosed, inhibitor-binding pocket lined by residues from all transmembrane (TM) helices of ABCB1. The pocket spans almost the entire width of the lipid membrane and is occupied exclusively by two closely interacting zosuquidar molecules. The external, conformational epitope facilitating UIC2 binding is also visualized, providing a basis for its inhibition of substrate efflux. Additional cryo-EM structures suggest concerted movement of TM helices from both halves of the transporters associated with closing the NBD gap, as well as zosuquidar binding. Our results define distinct recognition interfaces of ABCB1 inhibitory agents, which may be exploited for therapeutic purposes.
DateDeposition: Feb 2, 2018 / Header (metadata) release: Feb 21, 2018 / Map release: Feb 21, 2018 / Last update: Mar 7, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

Fileemd_4285.map.gz (map file in CCP4 format, 42593 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
220 pix
1.34 Å/pix.
= 293.92 Å
220 pix
1.34 Å/pix.
= 293.92 Å
220 pix
1.34 Å/pix.
= 293.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.336 Å
Density
Contour Level:0.07 (by author), 0.07 (movie #1):
Minimum - Maximum-0.06141705 - 0.20542075
Average (Standard dev.)0.0008694446 (0.009064368)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions220220220
Origin0.00.00.0
Limit219.0219.0219.0
Spacing220220220
CellA=B=C: 293.91998 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3361.3361.336
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z293.920293.920293.920
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.0610.2050.001

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Supplemental data

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Sample components

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Entire Amphipol reconstituted UIC2 Fab complex of double EQ mutant of hu...

EntireName: Amphipol reconstituted UIC2 Fab complex of double EQ mutant of human-mouse chimeric ABCB1 (ABCB1HMEQ)
Number of components: 1

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Component #1: protein, Amphipol reconstituted UIC2 Fab complex of double EQ mut...

ProteinName: Amphipol reconstituted UIC2 Fab complex of double EQ mutant of human-mouse chimeric ABCB1 (ABCB1HMEQ)
Recombinant expression: No
MassTheoretical: 195 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 78282
3D reconstructionResolution: 6.25 Å / Resolution method: FSC 0.143 CUT-OFF

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