KEY RESOURCES TABLE.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
|
Antibodies | ||
| 6× Histag Monoclonal Antibody | Invitrogen | # MA1-21355; PRID: AB_557403 |
| Goat anti-mouse IgG secondary antibody | Invitrogen | Cat# SA5-10276; RRID: AB_2868324 |
|
Bacterial and virus strains | ||
| E.Coli BL21 ΔHflK/C FLAG tag on C terminus of FtsH on chromosome | Ghanbarpour et al.4 | G2 |
| MG1655 ΔHflK/C cells (a generous gift from Sourjik’s lab) | María Isabel Pérez-López et al., PLoS Biol; 23: e3003077. | G88 |
|
Chemicals, peptides, and recombinant proteins | ||
| PrimeSTAR Max Ver.2 | TaKaRa | #AO80337A |
| NEBuilder HiFi DNA Assembly Master Mix 10× KLD enzyme mix | NEW ENGLAND BioLabs NEW ENGLAND BioLabs | #M5520AA; #M0554 |
| Chloramphenicol | GOLDBIO | # C-105-25 |
| Kanamycin | GOLDBIO | # K-120-5 |
| Tobramycin | Sigma-Aldrich | # T4014 |
| Anti-FLAG M2 Affinity Gel | Sigma-Aldrich | # A2220 |
| DYKDDDDK tag peptide | APExBIO | # A6002 |
| LB Broth (Miller) | Sigma-Aldrich | # L3522 |
| 2xYT Medium | Sigma-Aldrich | # Y2627 |
| Agarose | BIO-RAD | # 1613101 |
| Clarity Max Western ECL Substrate | BIO-RAD | # 1705062 |
| Potassium Chloride | Sigma-Aldrich | # P3911 |
| Zinc Chloride | Sigma-Aldrich | # 208086 |
| Magnesium Chloride | Sigma-Aldrich | # M1028 |
| EveryBlot Blocking Buffer | BIO-RAD | #12010020 |
| n-Dodecyl β-D-maltopyranoside (DDM) | GOLDBIO | # DDM5 |
| GDN | Anatrace | # GDN101 |
| Sodium Propionate | Sigma-Aldrich | # P1880 |
| 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine | Avanti Research | # DO-008319 |
| 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) | Avanti Research | # 810143 |
| Surfact-Amps X-100 | Thermo Fisher Scientific | # 28314 |
|
Critical commercial assays | ||
| Gel extraction kit | QIAGEN | # 28704 |
| RNeasy Plus Mini Kit | QIAGEN | # 74134 |
| Plasmid isolation kit | QIAGEN | # 27106 |
|
Deposited data | ||
| Electron Microscopy data | N/A | EMD-71448 |
| Electron Microscopy data | N/A | EMD-71449 |
| Electron Microscopy data | N/A | EMD-71447 |
|
Oligonucleotides and gene blocks | ||
| HflK/C-F primer | GenScript Inc. | N/A |
| GAGCTCAGGAGGAATTGACG TAAGTCTGCAAGTTCG TATG CCGATCGTTGACT |
N/A | N/A |
| HflK/C-R primer | N/A | N/A |
| CTAGAGGATCCCCGGGTACC TCATTATCCTTATAGA AAAAG AAAACCACCGAC |
N/A | N/A |
| Gene block HflC | N/A | N/A |
| AAG GGC AAA GTT CCG GTC ATC AAC CCG AAC AGT ATG GCG GCG CTG GGT ATT GAA GTT GTC GAT GTG CGTATC AAG CAG ATC AAC CTG CCG ACC GAA GTG TCT GAA GCG ATC TAC AAC CGT ATG CGC GCC GAG CGT GAA TGCGTA GCG CGT CGT CAC CGT TCA CAA GGT CAG GAA GAA GCG GAA AAA CTG CGC GCG ACT GCC GAC TAT GAA GTGACC AGA ACG CTG GCA GAA TGC GAG CGT CAG GGC CGC ATC ATG CGT GGT GAA GGC GAT GCC GAA GCA GCC AAACTG TTT GCT GAT GCA TTC AGT AAA GAT CCG GAC TTC TAC GCA TTC ATC CGT AGC CTG CGT GCT TAT GAG AACAGC TTC TCT GG |
GenScript Inc. | N/A |
| Gene block HflK | N/A | N/A |
| ATT CTG ACG GAA GGT CGT ACC GTG ATT CGT AGC GAT ACT CAG CGC GAA CTG GAA GAG ACG ATT CGT CCGTAT GAC ATG GGT ATC ACG CTG CTG GAC GTC AAC TTC CAG GCT GCT CGT CCG CCG GAA GAA GTA AAA GCG GCGTTT GAC GAT GCG ATT GCC GCG CGT GAA AAC GAA CAG CAA TAC ATT CGT GAA GCA GAA TGC TAT ACC AAC GAAGTT CAG CCG CGT GCG AAC GGT CAG TGC CAA CGT ATC CTC GAA GAG GCG CGT GCG TAC AAG GCC CAG ACC ATCCTG GAA GCT CAG GGT GAA GTG GCG CGC TTT GCT AAA CTT CTG CCG GAA TAT AAA GCC GCG CCG GAA ATT ACTCGC GAG CGT CTG TAT ATC GAG ACG ATG GAA AAA |
GenScript Inc. | N/A |
| Gene Block HflK mutant | N/A | N/A |
| ACGAAGTCAAACCGGTGAACGTGGAA GCCGTGGCCGCCCTGGCCGCTTCTG GTGTGATGCTGACGTCGGACGAGAAC GTAGTGCGCGTTGAGATGAACGTGCA GTACCGCGTCACCAATCCGGAAAAAT ATCTGTATAGCGTGACCAGCCCGGAT GACAGCCTGGCCCAGGCTACCGACA GCGCCCTGCGTGGAGTT |
N/A | N/A |
| HflC -Histag on c terminus, primer F | N/A | N/A |
| CACCACCACTAATATAACGACTGC GGTACAGGTC |
GenScript Inc. | N/A |
| HflC -Histag on c terminus, primer R | N/A | N/A |
| ATGATGATGACGCGTTGCGGAAGTCGG | N/A | N/A |
|
Recombinant DNA | ||
| pPRO plasmid (Low copy number plasmid) was used for cloning WT HflK/C and generating all other mutants | Lee and Keasling47 | Addgene Plasmid #17805 |
|
Software and algorithms | ||
| ChimeraX-1.8 | Goddard et al.48 | https://www.rbvi.ucsf.edu/chimerax/ |
| cryoSPARC (v4.7.0) | Punjani et al.49 | https://cryosparc.com/ |
| GraphPad Prism 10 | N/A | https://www.graphpad.com/ |
| EPU Data Collection Software | Thermo Fisher Scientific | https://www.thermofisher.com/ca/en/home/electron-microscopy/products/software-em-3d-vis/epu-software.html |
|
Other | ||
| PVDF membrane 0.2 μM | BIO-RAD | #1704156 |
| Nano Drop one | Thermo Fisher Scientific | N/A |
| C1000 Touch Thermal Cycler | BIO-RAD | N/A |
| Trans-Blot Turbo Transfer System | BIO-RAD | N/A |
| ChemiDoc Imaging System | BIO-RAD | N/A |
| Amicon Ultra-0.5 Centrifugal Filters | Merck Millipore Ltd | UFC510096 |
| R2/1 200 mesh grids | Quantifoil | Q2100CR1 |
| Titan Krios 300 kV | Thermo Fisher Scientific | N/A |
| Talos Arctica 200 kV | Thermo Fisher Scientific | N/A |
| Vitrobot Mark IV system | Thermo Fisher Scientific | N/A |