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- PDB-8t14: ADP-bound Bcs1 (C7 symmetrized) -

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Basic information

Entry
Database: PDB / ID: 8t14
TitleADP-bound Bcs1 (C7 symmetrized)
ComponentsMitochondrial chaperone BCS1
KeywordsTRANSLOCASE / Heptamer / AAA-ATPase / ADP-bound
Function / homology
Function and homology information


mitochondrial protein-transporting ATPase activity / protein insertion into mitochondrial inner membrane from matrix / mitochondrial cytochrome c oxidase assembly / mitochondrial respiratory chain complex III assembly / mitochondrial respiratory chain complex I assembly / mitochondrial inner membrane / ATP hydrolysis activity / mitochondrion / ATP binding
Similarity search - Function
BCS1, N-terminal / BCS1 N terminal / BCS1_N / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Mitochondrial chaperone BCS1
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsZhan, J. / Xia, D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: Nat Commun / Year: 2024
Title: Conformations of Bcs1L undergoing ATP hydrolysis suggest a concerted translocation mechanism for folded iron-sulfur protein substrate.
Authors: Jingyu Zhan / Allison Zeher / Rick Huang / Wai Kwan Tang / Lisa M Jenkins / Di Xia /
Abstract: The human AAA-ATPase Bcs1L translocates the fully assembled Rieske iron-sulfur protein (ISP) precursor across the mitochondrial inner membrane, enabling respiratory Complex III assembly. Exactly how ...The human AAA-ATPase Bcs1L translocates the fully assembled Rieske iron-sulfur protein (ISP) precursor across the mitochondrial inner membrane, enabling respiratory Complex III assembly. Exactly how the folded substrate is bound to and released from Bcs1L has been unclear, and there has been ongoing debate as to whether subunits of Bcs1L act in sequence or in unison hydrolyzing ATP when moving the protein cargo. Here, we captured Bcs1L conformations by cryo-EM during active ATP hydrolysis in the presence or absence of ISP substrate. In contrast to the threading mechanism widely employed by AAA proteins in substrate translocation, subunits of Bcs1L alternate uniformly between ATP and ADP conformations without detectable intermediates that have different, co-existing nucleotide states, indicating that the subunits act in concert. We further show that the ISP can be trapped by Bcs1 when its subunits are all in the ADP-bound state, which we propose to be released in the apo form.
History
DepositionJun 1, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Mitochondrial chaperone BCS1
G: Mitochondrial chaperone BCS1
B: Mitochondrial chaperone BCS1
C: Mitochondrial chaperone BCS1
D: Mitochondrial chaperone BCS1
E: Mitochondrial chaperone BCS1
F: Mitochondrial chaperone BCS1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)341,02014
Polymers338,0297
Non-polymers2,9907
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "B"
d_2ens_1chain "A"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "F"
d_7ens_1chain "G"

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11METMETLEULEUBC1 - 4171 - 417
d_12ADPADPADPADPBJ501
d_21METMETLEULEUAA1 - 4171 - 417
d_22ADPADPADPADPAH501
d_31METMETLEULEUCD1 - 4171 - 417
d_32ADPADPADPADPCK501
d_41METMETLEULEUDE1 - 4171 - 417
d_42ADPADPADPADPDL501
d_51METMETLEULEUEF1 - 4171 - 417
d_52ADPADPADPADPEM501
d_61METMETLEULEUFG1 - 4171 - 417
d_62ADPADPADPADPFN501
d_71METMETLEULEUGB1 - 4171 - 417
d_72ADPADPADPADPGI501

NCS oper:
IDCodeMatrixVector
1given(0.623993636714, 0.781428933397, -0.000873722202948), (-0.781428108681, 0.623994247876, 0.00113559759787), (0.00143258644855, -2.58545863745E-5, 0.999998973513)-64.4056989695, 184.252982624, -0.21275056059
2given(0.623152215293, -0.782100139316, -0.000829853397662), (0.782098812652, 0.62315271212, -0.00146445376062), (0.00166247488562, 0.000263550248137, 0.999998583358)184.831900799, -64.2901872534, -0.293456102715
3given(-0.221741005108, -0.975105494133, -0.00044940573119), (0.975105556543, -0.221740838306, -0.000392715142278), (0.000283287089291, -0.000525299075996, 0.999999821905)350.16878482, 39.365894847, 0.0491808291773
4given(-0.900630058265, -0.43458526259, -0.00107130244946), (0.434586230699, -0.900629730621, -0.000946788424953), (-0.000553386540196, -0.00131827940778, 0.999998977951)372.31902663, 233.785411873, 0.300538829014
5given(-0.901536101669, 0.432702481295, -0.00110456746319), (-0.432701539094, -0.901536680589, -0.000995800068467), (-0.00142669324475, -0.000419801670411, 0.999998894156)234.228275214, 372.1761482, 0.272654006595
6given(-0.224282648487, 0.974523511362, -0.00110426052799), (-0.974523084203, -0.224283927797, -0.00121576582269), (-0.00143246026705, 0.000803452196851, 0.99999865126)39.9049347053, 350.605918346, 0.0853510460963

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Components

#1: Protein
Mitochondrial chaperone BCS1 / BCS1-like protein


Mass: 48289.887 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Bcs1l / Production host: Komagataella pastoris (fungus) / References: UniProt: Q9CZP5
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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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: Heptameric Bcs1 in uniform ADP-bound state captured in active ATPase cycle (C7 symmetrized) in the presence of substrate ISP-ED
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.340 MDa / Experimental value: YES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified Bcs1 was mixed with ISP-ED at a molar ratio of 1:4 (Bcs1 heptamer:ISP) and incubated on ice for about 30 min in the presence of 2 mM ATP and 20 mM MgCl2 before vitrification.
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 54.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARCCTF correction
9PHENIX1.20.1-4487-000model refinement
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37681 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 115.15 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002123380
ELECTRON MICROSCOPYf_angle_d0.514231745
ELECTRON MICROSCOPYf_chiral_restr0.03653437
ELECTRON MICROSCOPYf_plane_restr0.00434053
ELECTRON MICROSCOPYf_dihedral_angle_d5.34133220
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2CBELECTRON MICROSCOPYNCS constraints7.59500163277E-13
ens_1d_3CBELECTRON MICROSCOPYNCS constraints4.26492004927E-13
ens_1d_4CBELECTRON MICROSCOPYNCS constraints3.10072736878E-11
ens_1d_5CBELECTRON MICROSCOPYNCS constraints7.93761058509E-11
ens_1d_6CBELECTRON MICROSCOPYNCS constraints6.20875038568E-11
ens_1d_7CBELECTRON MICROSCOPYNCS constraints2.32860356451E-11

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