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- EMDB-4283: Apo form of UIC2 Fab complex of human-mouse chimeric ABCB1 (ABCB1... -

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Basic information

Entry
Database: EMDB / ID: 4283
TitleApo form of UIC2 Fab complex of human-mouse chimeric ABCB1 (ABCB1HM) - Map 2 (kinked TM4 and TM10)
Map data
SampleApo Human-mouse chimeric ABCB1 (ABCB1HM) complex with UIC2 fab
  • Apo Human-mouse chimeric ABCB1 (ABCB1HM)
  • UIC2 fab
SourceHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / 4.47 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 14, 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: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4283.png

Downloads & links

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Map

Fileemd_4283.map.gz (map file in CCP4 format, 32001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
200 pix
1.39 Å/pix.
= 277.4 Å		200 pix
1.39 Å/pix.
= 277.4 Å		200 pix
1.39 Å/pix.
= 277.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.387 Å
Density

Histogram

Histogram (log scale)
Contour Level:0.06 (by author), 0.06 (movie #1):
Minimum - Maximum-0.16936657 - 0.32460624
Average (Standard dev.)0.00028347477 (0.011687097)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions200200200
Origin0.00.00.0
Limit199.0199.0199.0
Spacing200200200
CellA=B=C: 277.4 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3871.3871.387
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z277.400277.400277.400
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/NS200200200
D min/max/mean-0.1690.3250.000

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Supplemental data

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Sample components

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Entire Apo Human-mouse chimeric ABCB1 (ABCB1HM) complex with UIC2 fab

EntireName: Apo Human-mouse chimeric ABCB1 (ABCB1HM) complex with UIC2 fab
Number of components: 3

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Component #1: protein, Apo Human-mouse chimeric ABCB1 (ABCB1HM) complex with UI...

ProteinName: Apo Human-mouse chimeric ABCB1 (ABCB1HM) complex with UIC2 fab
Recombinant expression: No
MassTheoretical: 195 kDa

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Component #2: protein, Apo Human-mouse chimeric ABCB1 (ABCB1HM)

ProteinName: Apo Human-mouse chimeric ABCB1 (ABCB1HM) / Recombinant expression: No
MassTheoretical: 142 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #3: protein, UIC2 fab

ProteinName: UIC2 fab / Recombinant expression: No
MassTheoretical: 48.7 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Mus musculus (house mouse)

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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: 0.9 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 / Number of projections: 153652
3D reconstructionResolution: 4.47 Å / Resolution method: FSC 0.143 CUT-OFF

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