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- EMDB-16296: Cryo-EM structure of native nanodisc-reconstituted wildtype human... -

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Basic information

Entry
Database: EMDB / ID: EMD-16296
TitleCryo-EM structure of native nanodisc-reconstituted wildtype human MRP4 (inward-facing conformation) - no nucleotides/substrates added
Map dataCryo-EM map of wild type human MRP4 in native nanodiscs
Sample
  • Organelle or cellular component: Cryo-EM structure of native nanodisc-reconstituted wildtype human MRP4 (inward-facing conformation) - no nucleotides/substrates added
    • Protein or peptide: Human ATP-binding cassette sub-family C member 4
KeywordsABC transporter / ABCC4 / MRP4 / MEMBRANE PROTEIN
Function / homology
Function and homology information


guanine nucleotide transmembrane transporter activity / 15-hydroxyprostaglandin dehydrogenase (NAD+) activity / purine nucleotide transmembrane transporter activity / cAMP transport / ABC-type bile acid transporter activity / platelet dense granule membrane / leukotriene transport / urate transport / platelet degranulation / prostaglandin transport ...guanine nucleotide transmembrane transporter activity / 15-hydroxyprostaglandin dehydrogenase (NAD+) activity / purine nucleotide transmembrane transporter activity / cAMP transport / ABC-type bile acid transporter activity / platelet dense granule membrane / leukotriene transport / urate transport / platelet degranulation / prostaglandin transport / glutathione transmembrane transporter activity / ABC-type glutathione-S-conjugate transporter / ABC-type glutathione S-conjugate transporter activity / prostaglandin transmembrane transporter activity / ATPase-coupled inorganic anion transmembrane transporter activity / urate transmembrane transporter activity / external side of apical plasma membrane / xenobiotic transmembrane transport / export across plasma membrane / ABC-type xenobiotic transporter / prostaglandin secretion / Paracetamol ADME / Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / ABC-type xenobiotic transporter activity / Azathioprine ADME / bile acid and bile salt transport / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / ATPase-coupled transmembrane transporter activity / transport across blood-brain barrier / cilium assembly / ABC-type transporter activity / xenobiotic metabolic process / ABC-family proteins mediated transport / transmembrane transport / Platelet degranulation / basolateral plasma membrane / apical plasma membrane / nucleolus / Golgi apparatus / ATP hydrolysis activity / ATP binding / membrane / plasma membrane
Similarity search - Function
ATP-binding cassette sub-family C member 4 / : / : / 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 ...ATP-binding cassette sub-family C member 4 / : / : / 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 cassette sub-family C member 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsBloch MB / Pape TH / Raj I / Taylor NMI
Funding support Denmark, 1 items
OrganizationGrant numberCountry
Other government Denmark
CitationJournal: Structure / Year: 2023
Title: Structural and mechanistic basis of substrate transport by the multidrug transporter MRP4.
Authors: Magnus Bloch / Isha Raj / Tillmann Pape / Nicholas M I Taylor /
Abstract: Multidrug resistance-associated protein 4 (MRP4) is an ATP-binding cassette (ABC) transporter expressed at multiple tissue barriers where it actively extrudes a wide variety of drug compounds. ...Multidrug resistance-associated protein 4 (MRP4) is an ATP-binding cassette (ABC) transporter expressed at multiple tissue barriers where it actively extrudes a wide variety of drug compounds. Overexpression of MRP4 provides resistance to clinically used antineoplastic agents, making it a highly attractive therapeutic target for countering multidrug resistance. Here, we report cryo-EM structures of multiple physiologically relevant states of lipid bilayer-embedded human MRP4, including complexes between MRP4 and two widely used chemotherapeutic agents and a complex between MRP4 and its native substrate. The structures display clear similarities and distinct differences in the coordination of these chemically diverse substrates and, in combination with functional and mutational analysis, reveal molecular details of the transport mechanism. Our study provides key insights into the unusually broad substrate specificity of MRP4 and constitutes an important contribution toward a general understanding of multidrug transporters.
History
DepositionDec 7, 2022-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16296.png

Downloads & links

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Map

FileDownload / File: emd_16296.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of wild type human MRP4 in native nanodiscs
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 319.488 Å		0.83 Å/pix.
x 384 pix.
= 319.488 Å		0.83 Å/pix.
x 384 pix.
= 319.488 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-3.3560178 - 4.6126266
Average (Standard dev.)-0.000607498 (±0.060939156)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 319.488 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16296_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of wild type human MRP4 in native nanodiscs

Fileemd_16296_half_map_1.map
AnnotationHalf map 1 of wild type human MRP4 in native nanodiscs
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of wild type human MRP4 in native nanodiscs

Fileemd_16296_half_map_2.map
AnnotationHalf map 2 of wild type human MRP4 in native nanodiscs
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of native nanodisc-reconstituted wildtype human...

EntireName: Cryo-EM structure of native nanodisc-reconstituted wildtype human MRP4 (inward-facing conformation) - no nucleotides/substrates added
Components
  • Organelle or cellular component: Cryo-EM structure of native nanodisc-reconstituted wildtype human MRP4 (inward-facing conformation) - no nucleotides/substrates added
    • Protein or peptide: Human ATP-binding cassette sub-family C member 4

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Supramolecule #1: Cryo-EM structure of native nanodisc-reconstituted wildtype human...

SupramoleculeName: Cryo-EM structure of native nanodisc-reconstituted wildtype human MRP4 (inward-facing conformation) - no nucleotides/substrates added
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Human ATP-binding cassette sub-family C member 4

MacromoleculeName: Human ATP-binding cassette sub-family C member 4 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: ABC-type xenobiotic transporter
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MLPVYQEVKP NPLQDANLCS RVFFWWLNPL FKIGHKRRLE EDDMYSVLPE DRSQHLGEEL QGFWDKEVL RAENDAQKPS LTRAIIKCYW KSYLVLGIFT LIEESAKVIQ PIFLGKIINY F ENYDPMDS VALNTAYAYA TVLTFCTLIL AILHHLYFYH VQCAGMRLRV ...String:
MLPVYQEVKP NPLQDANLCS RVFFWWLNPL FKIGHKRRLE EDDMYSVLPE DRSQHLGEEL QGFWDKEVL RAENDAQKPS LTRAIIKCYW KSYLVLGIFT LIEESAKVIQ PIFLGKIINY F ENYDPMDS VALNTAYAYA TVLTFCTLIL AILHHLYFYH VQCAGMRLRV AMCHMIYRKA LR LSNMAMG KTTTGQIVNL LSNDVNKFDQ VTVFLHFLWA GPLQAIAVTA LLWMEIGISC LAG MAVLII LLPLQSCFGK LFSSLRSKTA TFTDARIRTM NEVITGIRII KMYAWEKSFS NLIT NLRKK EISKILRSSC LRGMNLASFF SASKIIVFVT FTTYVLLGSV ITASRVFVAV TLYGA VRLT VTLFFPSAIE RVSEAIVSIR RIQTFLLLDE ISQRNRQLPS DGKKMVHVQD FTAFWD KAS ETPTLQGLSF TVRPGELLAV VGPVGAGKSS LLSAVLGELA PSHGLVSVHG RIAYVSQ QP WVFSGTLRSN ILFGKKYEKE RYEKVIKACA LKKDLQLLED GDLTVIGDRG TTLSGGQK A RVNLARAVYQ DADIYLLDDP LSAVDAEVSR HLFELCICQI LHEKITILVT HQLQYLKAA SQILILKDGK MVQKGTYTEF LKSGIDFGSL LKKDNEESEQ PPVPGTPTLR NRTFSESSVW SQQSSRPSL KDGALESQDT ENVPVTLSEE NRSEGKVGFQ AYKNYFRAGA HWIVFIFLIL L NTAAQVAY VLQDWWLSYW ANKQSMLNVT VNGGGNVTEK LDLNWYLGIY SGLTVATVLF GI ARSLLVF YVLVNSSQTL HNKMFESILK APVLFFDRNP IGRILNRFSK DIGHLDDLLP LTF LDFIQT LLQVVGVVSV AVAVIPWIAI PLVPLGIIFI FLRRYFLETS RDVKRLESTT RSPV FSHLS SSLQGLWTIR AYKAEERCQE LFDAHQDLHS EAWFLFLTTS RWFAVRLDAI CAMFV IIVA FGSLILAKTL DAGQVGLALS YALTLMGMFQ WCVRQSAEVE NMMISVERVI EYTDLE KEA PWEYQKRPPP AWPHEGVIIF DNVNFMYSPG GPLVLKHLTA LIKSQEKVGI VGRTGAG KS SLISALFRLS EPEGKIWIDK ILTTEIGLHD LRKKMSIIPQ EPVLFTGTMR KNLDPFNE H TDEELWNALQ EVQLKETIED LPGKMDTELA ESGSNFSVGQ RQLVCLARAI LRKNQILII DEATANVDPR TDELIQKKIR EKFAHCTVLT IAHRLNTIID SDKIMVLDSG RLKEYDEPYV LLQNKESLF YKMVQQLGKA EAAALTETAK QVYFKRNYPH IGHTDHMVTN TSNGQPSTLT I FETAL

UniProtKB: ATP-binding cassette sub-family C member 4

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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 / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number real images: 7335 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 96000
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

Particle selectionNumber selected: 1829946
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.2) / Number images used: 242514
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 4.0.2)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 4.0.2)
FSC plot (resolution estimation)

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