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- PDB-8u2c: Gaussian mixture model based single particle refinement - ABC tra... -

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Basic information

Entry
Database: PDB / ID: 8u2c
TitleGaussian mixture model based single particle refinement - ABC transporter (inhibitor-bound ABCG2 from EMPIAR-10374)
Components
  • 5D3 Fab heavy chain variable domain
  • 5D3 Fab light chain variable domain
  • Broad substrate specificity ATP-binding cassette transporter ABCG2
  • FAB heavy chain constant domain
  • Fab light chain constant domain
KeywordsMEMBRANE PROTEIN / ABC transporter
Function / homology
Function and homology information


biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport / external side of apical plasma membrane ...biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport / external side of apical plasma membrane / organic anion transmembrane transporter activity / xenobiotic transport across blood-brain barrier / export across plasma membrane / ABC-type xenobiotic transporter / Paracetamol ADME / Ciprofloxacin ADME / transepithelial transport / cellular detoxification / ABC-type xenobiotic transporter activity / NFE2L2 regulating MDR associated enzymes / Heme biosynthesis / Heme degradation / lipid transport / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / transport across blood-brain barrier / ATPase-coupled transmembrane transporter activity / mitochondrial membrane / brush border membrane / Iron uptake and transport / transmembrane transport / membrane raft / apical plasma membrane / ATP hydrolysis activity / protein homodimerization activity / nucleoplasm / ATP binding / identical protein binding / plasma membrane
Similarity search - Function
ABC transporter family G domain / ABC-2 type transporter / ABC-2 type transporter / ABC-2 type transporter / 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
Broad substrate specificity ATP-binding cassette transporter ABCG2
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsChen, M. / Pintilie, G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)R21MH125285 United States
CitationJournal: Nat Methods / Year: 2024
Title: Improving resolution and resolvability of single-particle cryoEM structures using Gaussian mixture models.
Authors: Muyuan Chen / Michael F Schmid / Wah Chiu /
Abstract: Cryogenic electron microscopy is widely used in structural biology, but its resolution is often limited by the dynamics of the macromolecule. Here we developed a refinement protocol based on Gaussian ...Cryogenic electron microscopy is widely used in structural biology, but its resolution is often limited by the dynamics of the macromolecule. Here we developed a refinement protocol based on Gaussian mixture models that integrates particle orientation and conformation estimation and improves the alignment for flexible domains of protein structures. We demonstrated this protocol on multiple datasets, resulting in improved resolution and resolvability, locally and globally, by visual and quantitative measures.
History
DepositionSep 5, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 15, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 29, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 24, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Broad substrate specificity ATP-binding cassette transporter ABCG2
B: Broad substrate specificity ATP-binding cassette transporter ABCG2
C: 5D3 Fab light chain variable domain
D: 5D3 Fab heavy chain variable domain
E: 5D3 Fab light chain variable domain
F: 5D3 Fab heavy chain variable domain
I: FAB heavy chain constant domain
K: Fab light chain constant domain
J: FAB heavy chain constant domain
L: Fab light chain constant domain


Theoretical massNumber of molelcules
Total (without water)331,35310
Polymers331,35310
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Broad substrate specificity ATP-binding cassette transporter ABCG2 / ATP-binding cassette sub-family G member 2 / Breast cancer resistance protein / CDw338 / ...ATP-binding cassette sub-family G member 2 / Breast cancer resistance protein / CDw338 / Mitoxantrone resistance-associated protein / Placenta-specific ATP-binding cassette transporter / Urate exporter


Mass: 72385.852 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ABCG2, ABCP, BCRP, BCRP1, MXR / Production host: Homo sapiens (human)
References: UniProt: Q9UNQ0, ABC-type xenobiotic transporter
#2: Antibody 5D3 Fab light chain variable domain


Mass: 23594.016 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#3: Antibody 5D3 Fab heavy chain variable domain


Mass: 23843.633 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#4: Antibody FAB heavy chain constant domain


Mass: 22986.840 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: The Fab domains were not resolved in the original structure and, therefore, were not modeled. With the implementation of an improved method, we can now resolve the domains within the CryoEM ...Details: The Fab domains were not resolved in the original structure and, therefore, were not modeled. With the implementation of an improved method, we can now resolve the domains within the CryoEM density map. However, given the absence of FAB information in the original structure, we opted to fit a representative FAB structure, utilizing PDB ID 7FAB as a reference.
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#5: Antibody Fab light chain constant domain


Mass: 22866.355 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: The Fab domains were not resolved in the original structure and, therefore, were not modeled. With the implementation of an improved method, we can now resolve the domains within the CryoEM ...Details: The Fab domains were not resolved in the original structure and, therefore, were not modeled. With the implementation of an improved method, we can now resolve the domains within the CryoEM density map. However, given the absence of FAB information in the original structure, we opted to fit a representative FAB structure, utilizing PDB ID 7FAB as a reference.
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: inhibitor-bound ABCG2 / Type: COMPLEX / Details: Re-refinement from EMPIAR-10374 / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 30 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
12EMAN2.993D reconstruction
13PHENIX1.2model refinement
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 284831 / Symmetry type: POINT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16ETI

6eti

16ETI1PDBexperimental model
27FAB

7fab

17FABPDBexperimental model

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