Table 1.

Construct designs

Construct name Fusion Mutations Cross-link 
WT GCN4-Kar3Vik1 GCN4  NA 
Cysless GCN4-Kar3Vik1 GCN4 Kar3 (C391L, C469A, C517A, C566V, C655V)Vik1 (C377V, C436A, C536A, C596A, C640A) NA 
10 Å cross-linked GCN4-Kar3Vik1 GCN4 Kar3 (C391L, C469A, C517A, C566V, C655V, E382C)Vik1 (C377V, C436A, C536A, C596A,C640A, K372C) M4M 
20 Å cross-linked GCN4-Kar3Vik1 GCN4 Kar3 (C391L, C469A, C517A, C566V, C655V, E382C)Vik1 (C377V, C436A, C536A, C596A,C640A, K372C) DPDPB 
Non-cross-linked GCN4-Kar3Vik1 GCN4 Kar3 (C391L, C469A, C517A, C566V, C655V, E382C)Vik1 (C377V, C436A, C536A, C596A,C640A, K372C) NA 
WT SHD-Kar3Vik1 SHD  NA 
Cysless SHD-Kar3Vik1 SHD Kar3 (C391L, C469A, C517A, C566V, C655V)Vik1 (C377V, C436A, C536A, C596A, C640A) NA 
Non-cross-linked SHD-Kar3Vik1 SHD Kar3 (C391L, C469A, C517A, C566V, C655V)Vik1 (C377V, C436A, C536A, C596A, C640A, E355C, K423C) NA 
Cross-linked SHD-Kar3Vik1 SHD Kar3 (C391L, C469A, C517A, C566V, C655V)Vik1 (C377V, C436A, C536A, C596A, C640A, E355C, K423C) EBI 

A list of the composition of all constructs including the identity of the stabilizing coiled coil, mutations, and cross-links. Kar3 constructs included a Tobacco etch virus (TEV) protease cleavable His-tag with the sequence: MGSSHHHHHHHHDYDIPTSENLYFQGASM followed by a fusion domain and K353-K729 of Kar3. Vik1 constructs included a TEV protease cleavable Strep tag with the sequence MASWSHPQFEKENLYFQGAS followed by a fusion domain and S341-T647 of Vik1. Amino acids in italics were removed by TEV protease. The sequences of GCN4, basic helix, and acidic helix in the fusions were: ­SVKELEDKVEE­LLSKNYHLENE­VARLKKLV, KIAALKEKIAALKEKIAALKEKI, and EIAALEKEIAALEKEIAALEKEI, respectively. For the SHD fusions, Kar3 was fused to the basic helix, and Vik1 to the acidic helix.

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