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- PDB-7p14: Structure of full-length rXKR9 in complex with a sybody at 3.66A -

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Basic information

Entry
Database: PDB / ID: 7p14
TitleStructure of full-length rXKR9 in complex with a sybody at 3.66A
Components
  • Sybody
  • XK-related protein
KeywordsMEMBRANE PROTEIN / small membrane protein / in complex with sybody / apoptotic lipid scrambling
Function / homology
Function and homology information


phosphatidylserine exposure on apoptotic cell surface / engulfment of apoptotic cell / apoptotic process involved in development / membrane => GO:0016020 / membrane / plasma membrane
Similarity search - Function
XK-related protein / XK-related protein
Similarity search - Domain/homology
Chem-P5S / DIUNDECYL PHOSPHATIDYL CHOLINE / XK-related protein
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsStraub, M.S. / Sawicka, M. / Dutzler, R.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
European Research Council (ERC)339116 Switzerland
University of ZurichFK-20-040 Switzerland
CitationJournal: Elife / Year: 2021
Title: Cryo-EM structures of the caspase-activated protein XKR9 involved in apoptotic lipid scrambling.
Authors: Monique S Straub / Carolina Alvadia / Marta Sawicka / Raimund Dutzler /
Abstract: The exposure of the negatively charged lipid phosphatidylserine on the cell surface, catalyzed by lipid scramblases, is an important signal for the clearance of apoptotic cells by macrophages. The ...The exposure of the negatively charged lipid phosphatidylserine on the cell surface, catalyzed by lipid scramblases, is an important signal for the clearance of apoptotic cells by macrophages. The protein XKR9 is a member of a conserved family that has been associated with apoptotic lipid scrambling. Here, we describe structures of full-length and caspase-treated XKR9 from in complex with a synthetic nanobody determined by cryo-electron microscopy. The 43 kDa monomeric membrane protein can be divided into two structurally related repeats, each containing four membrane-spanning segments and a helix that is partly inserted into the lipid bilayer. In the full-length protein, the C-terminus interacts with a hydrophobic pocket located at the intracellular side acting as an inhibitor of protein function. Cleavage by caspase-3 at a specific site releases 16 residues of the C-terminus, thus making the pocket accessible to the cytoplasm. Collectively, the work has revealed the unknown architecture of the XKR family and has provided initial insight into its activation by caspases.
History
DepositionJul 1, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 28, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: XK-related protein
B: Sybody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)59,2765
Polymers57,2382
Non-polymers2,0383
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3890 Å2
ΔGint-16 kcal/mol
Surface area21660 Å2
MethodPISA

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Components

#1: Protein XK-related protein


Mass: 43563.059 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Xkr9, XRG9 / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q5GH54
#2: Antibody Sybody


Mass: 13675.293 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli MC1061 (bacteria)
#3: Chemical ChemComp-PLC / DIUNDECYL PHOSPHATIDYL CHOLINE


Mass: 622.834 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C32H65NO8P / Comment: phospholipid*YM
#4: Chemical ChemComp-P5S / O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine / phosphatidyl serine / Phosphatidylserine


Mass: 792.075 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C42H82NO10P
Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1rXKR9 with sybodyCOMPLEX#1-#20RECOMBINANT
2rXKR9COMPLEX#11RECOMBINANT
3SybodyCOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.06 MDaNO
210.043 MDaNO
310.017 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Rattus norvegicus (Norway rat)10116
32Rattus norvegicus (Norway rat)10116
43synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Escherichia coli MC1061 (bacteria)1211845
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaClSodium chloride1
225 mMHEPESHEPES1
30.01 %Lauryl Maltose Neopentyl GlycolLMNG1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / 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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 70 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 12396

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2cryoSPARC3.2.0particle selection
3EPU2.9image acquisition
8Coot0.9.5model fitting
10PHENIXmodel refinement
14cryoSPARC3.2.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 866439 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 53.49 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023892
ELECTRON MICROSCOPYf_angle_d0.5065270
ELECTRON MICROSCOPYf_dihedral_angle_d5.272615
ELECTRON MICROSCOPYf_chiral_restr0.036587
ELECTRON MICROSCOPYf_plane_restr0.004624

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