[English] 日本語
Yorodumi
- EMDB-12816: Enterococcus faecalis EfrCD in complex with a nanobody -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-12816
TitleEnterococcus faecalis EfrCD in complex with a nanobody
Map dataEfrCD volume
Sample
  • Complex: EfrCD in complex with a nanobody
    • Complex: Enterococcus faecalis
      • Protein or peptide: ABC transporter ATP-binding protein
      • Protein or peptide: ABC transporter ATP-binding protein
    • Complex: nanobodySingle-domain antibody
      • Protein or peptide: NanobodySingle-domain antibody
KeywordsABC transporter / Nanobody / Enterococcus faecalis / MEMBRANE PROTEIN
Function / homology
Function and homology information


ABC-type transporter activity / membrane => GO:0016020 / ATP binding
Similarity search - Function
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 / 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
ABC transporter ATP-binding protein / ABC transporter ATP-binding protein
Similarity search - Component
Biological speciesEnterococcus faecalis (bacteria) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.25 Å
AuthorsEhrenbolger K / Hutter CAJ / Meier G / Seeger MA / Barandun J
Funding support Sweden, Switzerland, 3 items
OrganizationGrant numberCountry
Swedish Research Council2019-02011 Sweden
Swiss National Science FoundationPP00P3_144823 Switzerland
Swiss National Science Foundation310030_188817 Switzerland
CitationJournal: Nat Chem Biol / Year: 2023
Title: Deep mutational scan of a drug efflux pump reveals its structure-function landscape.
Authors: Gianmarco Meier / Sujani Thavarasah / Kai Ehrenbolger / Cedric A J Hutter / Lea M Hürlimann / Jonas Barandun / Markus A Seeger /
Abstract: Drug efflux is a common resistance mechanism found in bacteria and cancer cells, but studies providing comprehensive functional insights are scarce. In this study, we performed deep mutational ...Drug efflux is a common resistance mechanism found in bacteria and cancer cells, but studies providing comprehensive functional insights are scarce. In this study, we performed deep mutational scanning (DMS) on the bacterial ABC transporter EfrCD to determine the drug efflux activity profile of more than 1,430 single variants. These systematic measurements revealed that the introduction of negative charges at different locations within the large substrate binding pocket results in strongly increased efflux activity toward positively charged ethidium, whereas additional aromatic residues did not display the same effect. Data analysis in the context of an inward-facing cryogenic electron microscopy structure of EfrCD uncovered a high-affinity binding site, which releases bound drugs through a peristaltic transport mechanism as the transporter transits to its outward-facing conformation. Finally, we identified substitutions resulting in rapid Hoechst influx without affecting the efflux activity for ethidium and daunorubicin. Hence, single mutations can convert EfrCD into a drug-specific ABC importer.
History
DepositionApr 28, 2021-
Header (metadata) releaseMay 18, 2022-
Map releaseMay 18, 2022-
UpdateMay 31, 2023-
Current statusMay 31, 2023Processing site: PDBe / Status: Released

-
Structure visualization

Supplemental images

emd_12816.png

Downloads & links

-
Map

FileDownload / File: emd_12816.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEfrCD volume
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-0.86246914 - 1.7825624
Average (Standard dev.)0.000901812 (±0.047993705)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions296296296
Spacing296296296
CellA=B=C: 307.84 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Half map: EfrCD half map2

Fileemd_12816_half_map_1.map
AnnotationEfrCD half map2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: EfrCD half map1

Fileemd_12816_half_map_2.map
AnnotationEfrCD half map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : EfrCD in complex with a nanobody

EntireName: EfrCD in complex with a nanobody
Components
  • Complex: EfrCD in complex with a nanobody
    • Complex: Enterococcus faecalis
      • Protein or peptide: ABC transporter ATP-binding protein
      • Protein or peptide: ABC transporter ATP-binding protein
    • Complex: nanobodySingle-domain antibody
      • Protein or peptide: NanobodySingle-domain antibody

-
Supramolecule #1: EfrCD in complex with a nanobody

SupramoleculeName: EfrCD in complex with a nanobody / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

-
Supramolecule #2: Enterococcus faecalis

SupramoleculeName: Enterococcus faecalis / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Enterococcus faecalis (bacteria)

-
Supramolecule #3: nanobody

SupramoleculeName: nanobody / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Vicugna pacos (alpaca)

-
Macromolecule #1: ABC transporter ATP-binding protein

MacromoleculeName: ABC transporter ATP-binding protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Enterococcus faecalis (bacteria)
Molecular weightTheoretical: 62.905289 KDa
Recombinant expressionOrganism: Lactococcus lactis (lactic acid bacteria)
SequenceString: MDLIIQHAKK YKGSVVIALL AVIVMVVSAL WQPKLLQQVL EAIMNDDSDK MKNLGIQLIA IAGLGLVAGV INTIFSAKVA QGVSADIRE ATFRKIQTFS FGNIEKFSAG NLVVRLTNDV TQIQNVIMIA LQTLFRIPFL FIGSFILAML TLPQLWWVIV A LVIAVILI ...String:
MDLIIQHAKK YKGSVVIALL AVIVMVVSAL WQPKLLQQVL EAIMNDDSDK MKNLGIQLIA IAGLGLVAGV INTIFSAKVA QGVSADIRE ATFRKIQTFS FGNIEKFSAG NLVVRLTNDV TQIQNVIMIA LQTLFRIPFL FIGSFILAML TLPQLWWVIV A LVIAVILI SMLSFSQMGK HFMIIQNLID KINGIAKENL LGIRVVKSFV QEKNQLSRFT KVSEELTTHN LIVGSLFAVM IP AFMLVAN LAVVGSIFFV SNLVKDDPTL IGGVASFMNY LMQIMMAIII GGMMMMMTSR AAVSIKRIKE VMETEPDVTY KKV PEQELI GSVEFDHVSF RYPGDEEDTL KDISFSIQPG EMIGIVGATG AGKSTLAQLI PRLFDPTEGK IEVGGVDLRE VNEH SLRKT VSFVLQKAIL FSGTIAQNLR HGKRDASEAD MERASGIAQA KEFIEKLAEG YDAPVEERSN NFSGGQKQRL SITRG VIGE PKILILDDST SALDARSERL VREALDKELK ETTTIVIAQK ISSVVHADRI LVLDNGRLVG EGTHEELAAT NPVYQE IYE TQKGKEEA

-
Macromolecule #2: ABC transporter ATP-binding protein

MacromoleculeName: ABC transporter ATP-binding protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Enterococcus faecalis (bacteria)
Molecular weightTheoretical: 66.213734 KDa
Recombinant expressionOrganism: Lactococcus lactis (lactic acid bacteria)
SequenceString: MTDLIKASKF FYHYLKRYKV SFLFIFLAIF AATYLQVKAP QFVGEAIQEL AKYAVNVMQG KDDKSAFVSV IWKLLIFYVL TSAASFIYS ILFTQVVGKS TNRMRIGLFN KLEKLTIRFF DSHQDGEILS RFTSDLDNIQ NSLNQALLQV LTNIALLVGV L IMMFRQNV ...String:
MTDLIKASKF FYHYLKRYKV SFLFIFLAIF AATYLQVKAP QFVGEAIQEL AKYAVNVMQG KDDKSAFVSV IWKLLIFYVL TSAASFIYS ILFTQVVGKS TNRMRIGLFN KLEKLTIRFF DSHQDGEILS RFTSDLDNIQ NSLNQALLQV LTNIALLVGV L IMMFRQNV ELAWATIAST PIAILIAVFV ISKARKYVDL QQDEVGKLNG YMDEKISGQR VIITNGLQEE TIDGFLEQNE KV RAATYKG QVYSGLLFPM MQGMSLVNTA IVIFFGGWLA INGSVDRAAA LGLVVMFVQY SQQYYQPLMQ ISSGYSMIQL AVT GARRLN EMFDEPDEIR PENGEKLEEI NKAVALNHVV FGYNPETPVL KDVSIHVDKG EMVALVGPTG SGKTTIMNLM NRFY DVNEG AVTFDGVDIR EMDLDSLRSH VGIVLQESVL FSGTIRENIA FGKPEATDEE IVQAAKQANI HEFIVNLEQG YDTEI TEEN NLFSTGQKQL VSIARTIITN PELLILDEAT SNVDTVTEAK IQKAMDEAIK GRTSFVIAHR LKTILNADRI IVLRDG EVI EEGNHHELVE QDGFYAELYK NQFVFE

-
Macromolecule #3: Nanobody

MacromoleculeName: Nanobody / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 12.718138 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
GPSQLQLVES GGGLVQAGDT LRLSCEASRS FNRMGWYRQA PGKQRDMVAH IFSDGRTRYA DSVQGRFTIS RDNAKNTVYL QMNNLKPED TAVYYCNGFF IQDFWGQGTP VTVSA

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration8 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
20.0 mMTris
150.0 mMNaClSodium chloride
0.15 %beta-DDM
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
Details: blot force of -5, waiting time of 1 second, blot time of 4.5 seconds at 4C.

-
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 / Cs: 2.7 mm / Nominal defocus max: 3.3000000000000003 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Number real images: 3135 / Average electron dose: 52.1 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

-
Image processing

Particle selectionNumber selected: 720893
Startup modelType of model: OTHER / Details: Initial model CisTEM
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.2)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.2) / Number images used: 254682
FSC plot (resolution estimation)

-
Atomic model buiding 1

Initial modelPDB ID:

4q4h

DetailsInitial docking in Chimera, local adjustments in COOT
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7ocy:
Enterococcus faecalis EfrCD in complex with a nanobody

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more