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

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Basic information

Entry
Database: EMDB / ID: EMD-4285
TitleAmphipol reconstituted UIC2 Fab complex of double EQ mutant of human-mouse chimeric ABCB1 (ABCB1HMEQ)
Map dataAmphipol reconstituted ABCB1HMEQ-UIC2 complex
Sample
  • Complex: Amphipol reconstituted UIC2 Fab complex of double EQ mutant of human-mouse chimeric ABCB1 (ABCB1HMEQ)
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.25 Å
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.
History
DepositionFeb 2, 2018-
Header (metadata) releaseFeb 21, 2018-
Map releaseFeb 21, 2018-
UpdateMar 7, 2018-
Current statusMar 7, 2018Processing site: PDBe / Status: Released

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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:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_4285.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAmphipol reconstituted ABCB1HMEQ-UIC2 complex
Voxel sizeX=Y=Z: 1.336 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.06141705 - 0.20542075
Average (Standard dev.)0.0008694446 (±0.009064368)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 293.91998 Å
α=β=γ: 90.0 °

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)
Components
  • Complex: Amphipol reconstituted UIC2 Fab complex of double EQ mutant of human-mouse chimeric ABCB1 (ABCB1HMEQ)

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Supramolecule #1: Amphipol reconstituted UIC2 Fab complex of double EQ mutant of hu...

SupramoleculeName: Amphipol reconstituted UIC2 Fab complex of double EQ mutant of human-mouse chimeric ABCB1 (ABCB1HMEQ)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 195 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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.25 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 78282

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