Key Resources Table
| REAGENT or RESOURCES | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Rabbit polyclonal antibody to K5 (AF-138) | Covance | Cat # PRB-160P |
| Mouse monoclonal antibody to K14 (LL001) | Covance | Cat # PRB-155P |
| Alexa Fluor 488-conjugated goat anti-rabbit antibody | Invitrogen | Cat # A11034 |
| Alexa Fluor 594-conjugated goat anti-mouse antibody | Invitrogen | Cat # A11032 |
| Bacterial and Virus Strains | ||
| DH5α | Thermo Fisher | Cat # 18265017 |
| BL21 (DE3) pLysS | Promega | Cat # CMC0015 |
| Rosetta2(DE3) pLysS | Novagen | Cat # 71403 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| Site-directed Mutagenesis Kit | Stratagene | Cat # 200519 |
| DMEM | Gibco | Cat # 11995–065 |
| Calf Serum | Atlanta Biologicals | Cat # S11550 |
| Lipofectamine 2000 | Thermo-Fisher | Cat # 11668019 |
| Crystallization solution modified from Crystal Screen Cryo 46 with 12%(w/v) PEG-8000, 0.1M sodium cacodylate pH 6.5, 0.2 M calcium acetate hydrate | Hampton Research | Cat # HR2-914-46 |
| Oligonucleotides (5’->3’ orientation) | ||
| K14-C367A Primers for mutagenesis | This study | N/A |
| Forward: CAAGTCGACAGCAGAGAAGAACCGC | ||
| Reverse: CCAAAGCTTAGTTCTTGGTGCGCAGGACC | ||
| ID1_K5E_PvuI Primers to produce ID1-deficient mutant of K5 | This study | N/A |
| Forward: CCAGAGGCTGAGAGCCGCGATCGACAATGTCAAGAAACAGTGC | ||
| Reverse: GCACTGTTTCTTGACATTGTCGATCGCGGCTCTCAGCCTCTGGATCATCCGG | ||
| ID1_K14REQ Primers to produce ID1-deficient mutant of K14 | This study | N/A |
| Forward: CGAGATCTCGGAGCTCGCGCGCACCATGCAGAACCTGGCGATTGAGCTGGCGTCCCAGCTCAGCATGAAAGC | ||
| Reverse : CCAGGTTCTGCATGGTGCGCGCGAGCTCCGAGATCTCGCTCTTGCCGC TCTGC | ||
| ID3_K5QNE_NotI Primers to produce ID3-deficient mutant of K5 | This study | N/A |
| Forward: GCCGAGATTGACAATGTCAAGAAACAGTGCGCCAATCTGGCGGCCGCCATTGCGGATGCCGAGCAGCGTGGGGCGCTGGCCCTCAAGGATGCC | ||
| Reverse: GCGGCCGCCAGATTGGCGCACTGTTTCTTGACATTGTCAATCTCGGCTCTCAGCC TCTGGATCATCCGG | ||
| ID3_K14RYQ Primers to produce ID3-deficient mutant of K14 | This study | N/A |
| Forward: GGAGGAGACCAAAGGT GCCTTCTGCATGCAGCTGGCCGCGATCCAGGAGATGATTGG | ||
| Reverse: GGCCAGCTGCATGCAGAAGGCACCTTTGGTCTCCTCCAGGCTGTTCTCCAGGG | ||
| ID7_K5AAA Primers to produce ID7-deficient mutant of K5 | This study | N/A |
| Forward: GGATGCCAGGAACAAGCTGGCCGAGCTGGAGGCAGCGCTGGCGAAGGCCAAGGCGGACATGGCCCGGCTG CT GCGTGAGTACCAGG | ||
| Reverse: CCTGGTACTCACGCAGCAGCCGGGCCATGTCCGCCTTGGCCTTCGCCAGCGCTGCCTCCAGCTCGGCCAGCTTGTTCCTGGCATCC | ||
| Recombinant DNA | ||
| pET24a-K5 | (Lee et al., 2012) | N/A |
| pET3d-K14 | (Lee et al., 2012) | N/A |
| pET24a-K5-ID1 | This paper | N/A |
| pET3d-K14-ID1 | This paper | N/A |
| pET24a-K5-ID3 | This paper | N/A |
| pET3d-K14-ID3 | This paper | N/A |
| pET24a-K5-ID7 | This paper | N/A |
| pCMV-K5 | This paper | N/A |
| pCMV-K14 | This paper | N/A |
| pCMV-K5-ID1 | This paper | N/A |
| pCMV-K14-ID1 | This paper | N/A |
| pCMV-K5-ID3 | This paper | N/A |
| pCMV-K14-ID3 | This paper | N/A |
| pCMV-K5-ID7 | This paper | N/A |
| Deposited Data | ||
| Coordinates of 2B heterocomplex of K5 and K14 wildtype | (Lee et al., 2012) | PDB: 3TNU |
| Coordinates of 2B heterocomplex of K5 and K14-C367A | This paper | PDB: 6JFV |
| Software and Algorithms | ||
| HKL2000 | (Otwinowski and Minor, 1997) | http://www.hkl-xray.com/ |
| PHENIX | (Adams et al., 2010) | http://www.phenix-online.org/ |
| COOT | (Emsley et al., 2010) | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ |
| MOLPROBITY | (Kirmse et al., 2010) | http://molprobity.biochem.duke.edu/ |
| PyMOL | DeLano Scientific LLC | http://www.pymol.org/ |
| PISA | (Krissinel and Henrick, 2007) | http://www.ebi.ac.uk/pdbe/pisa/pistart.html |
| ImageJ | Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA | https://imagej.nih.gov/ij/ |
| Experimental Models: Cell Lines | ||
| NIH-3T3 | ATCC | ATCC CRL-1658 |