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Structure paper

TitleRapid small-scale nanobody-assisted purification of ryanodine receptors for cryo-EM.
Journal, issue, pagesJ Biol Chem, Vol. 300, Issue 10, Page 107734, Year 2024
Publish dateSep 2, 2024
AuthorsChenyao Li / Katrien Willegems / Tomasz Uchański / Els Pardon / Jan Steyaert / Rouslan G Efremov /
PubMed AbstractRyanodine receptors (RyRs) are large Ca release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs have been identified in mammals, the disfunction of ...Ryanodine receptors (RyRs) are large Ca release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs have been identified in mammals, the disfunction of which has been associated with a series of life-threatening diseases. The need for large amounts of native tissue or eukaryotic cell cultures limits advances in structural studies of RyRs. Here, we report a method that utilizes nanobodies to purify RyRs from only 5 mg of total protein. The purification process, from isolated membranes to cryo-EM grade protein, is achieved within 4 h on the bench, yielding protein usable for cryo-EM analysis. This is demonstrated by solving the structures of rabbit RyR1, solubilized in detergent, reconstituted into lipid nanodiscs or liposomes, and bovine RyR2 reconstituted in nanodisc, and mouse RyR2 in detergent. The reported method facilitates structural studies of RyRs directed toward drug development and is useful in cases where the amount of starting material is limited.
External linksJ Biol Chem / PubMed:39233227
MethodsEM (single particle)
Resolution3.1 - 4.7 Å
Structure data

19463

8rrs

EMDB-19463, PDB-8rrs:
Structure of mouse RyR2 solubilised in detergent in open state in complex with Ca2+, ATP, caffeine and Nb9657.
Method: EM (single particle) / Resolution: 3.4 Å

19464

8rrt

EMDB-19464: Structure of rabbit RyR1 reconstituted into lipid liposomes in open state in complex with Ca2+, ATP, caffeine and Nb9657.
PDB-8rrt: Structure of rabbit RyR1 reconstituted into lipid liposomes in open state in complex with FKBP and Nb9657
Method: EM (single particle) / Resolution: 4.6 Å

19465

8rru

EMDB-19465: Structure of RyR1 reconstituted into lipid liposomes in primed state in complex with Ca2+, ATP, caffeine and Nb9657.
PDB-8rru: Structure of RyR1 reconstituted into lipid liposomes in primed state in complex with FKBP and Nb9657.
Method: EM (single particle) / Resolution: 4.7 Å

19466

8rrv

EMDB-19466, PDB-8rrv:
Structure of RyR1 in detergent in close state in complex with FKBP and Nb9657.
Method: EM (single particle) / Resolution: 3.2 Å

19467

8rrw

EMDB-19467: Structure of RyR1 in detergent in open state in complex with Ca2+, ATP, caffeine and Nb9657.
PDB-8rrw: Structure of RyR1 in detergent in open state in complex with FKBP and Nb9657.
Method: EM (single particle) / Resolution: 4.2 Å

19468

8rrx

EMDB-19468, PDB-8rrx:
Structure of RyR1 reconstituted into lipid nanodisc in primed state in complex with Ca2+, ATP, caffeine and Nb9657
Method: EM (single particle) / Resolution: 3.1 Å

19472

8rs0

EMDB-19472: Structure of RyR1 solubilised in detergent in primed state in complex with Ca2+, ATP, caffeine and Nb9657
PDB-8rs0: Structure of RyR1 in detergent in primed state in complex with nanobody and FKBP
Method: EM (single particle) / Resolution: 3.3 Å

Chemicals

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

ChemComp-CFF:
CAFFEINE / medication*YM

ChemComp-ZN:
Unknown entry

ChemComp-CA:
Unknown entry

ChemComp-POV:
(2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / phospholipid*YM

Source
  • mus musculus (house mouse)
  • vicugna pacos (alpaca)
  • oryctolagus cuniculus (rabbit)
KeywordsTRANSPORT PROTEIN / Ion channel / Ca2+ / tetramer

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