Current limits of structural biology: The transient interaction between cytochrome and photosystem I.

Trimeric photosystem I from the cyanobacterium (PSI) is an intrinsic membrane protein, which converts solar energy into electrical energy by oxidizing the soluble redox mediator cytochrome (Cyt ) and reducing ferredoxin. Here, we use cryo-electron microscopy and small angle neutron scattering (SANS) to characterize the transient binding of Cyt to PSI. The structure of PSI cross-linked to Cyt was solved at a resolution of 2.9 Å and shows additional cofactors as well as side chain density for 84% of the peptide chain of subunit PsaK, revealing a hydrophobic, membrane intrinsic loop that enables binding of associated proteins. Due to the poor binding specificity, Cyt could not be localized with certainty in our cryo-EM analysis. SANS measurements confirm that Cyt does not bind to PSI at protein concentrations comparable to those for cross-linking. However, SANS data indicate a complex formation between PSI and the non-native mitochondrial cytochrome from horse heart (Cyt ). Our study pinpoints the difficulty of identifying very small binding partners (less than 5% of the overall size) in EM structures when binding affinities are poor. We relate our results to well resolved co-structures with known binding affinities and recommend confirmatory methods for complexes with values higher than 20 μM.