Changes in the hydrophobic network of the FliGMC domain induce rotational switching of the flagellar motor

. 2023 Jul 11;26(8):107320. doi: 10.1016/j.isci.2023.107320

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains

Vibrio alginolyticus VIO5, see Table S2	Okunishi et al.⁶⁵	N/A
Vibrio alginolyticus NMB198, see Table S2	Yorimitsu et al., 2003⁶⁶	N/A
Escherichia coli DH5α, See Table S2	Grant et al., 1990⁶⁷	N/A
Escherichia coli BL21(DE3), See Table S2	Novagen	Cat#69450-3
Escherichia coli S17-1, See Table S2	Simon et al., 1983⁶⁸	N/A

Chemicals, peptides, and recombinant proteins

Deuterium oxide	Wako	Cat#040-18831
Ammonium ¹⁵N chloride	ISOTEC	Cat#299251-10G
TALON Metal Affinity Resin	Clontech Laboratories, Inc.	Cat#635501
Factor Xa	New England Biolabs	Cat#P8010S
cOmplete™, EDTA-free Protease Inhibitor Cocktail	Roche Life Science	Cat#11873580001
sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS)	Cambridge Isotope Laboratories	Cat#DLM-32-1
α-ketobutyric acid sodium salt (Methyl-¹³C; 3,3-²H₂)	Cambridge Isotope Laboratories	Cat#CDLM-7318-0.5
[δ-¹³C; α, β, γ12, γ13, γ2, γ2, γ2-²H₇; ¹⁵N] isoleucine	SAIL Technologies	Cat#I-004
[0, α, β, δ1, δ2-¹³C₅; β2, ε1, ε2, ζ-²H₄; ¹⁵N] phenylalanine	SAIL Technologies	Cat#F-013a

Critical commercial assays

QuikChange Site-directed Mutagenesis kit	Stratagene	Cat#200516

Deposited data

Structure of FliG-FliM from thermotoga maritima	Vartanian et al.⁶²	PDB: 4FHR
Cryo-ET volume of Vibrio alginolyticus clockwise locked rotor	Carroll et al.⁴⁴	EMDB: EMD-21819
Cryo-ET volume of Vibrio alginolyticus counter-clockwise locked rotor	Carroll et al.⁴⁴	EMDB: EMD-21837

Oligonucleotides

Primer: To generate I140L mutation: CAAGTGGCGAGCTTGATTGTTAACGAACAC	This study	N/A
Primer: To generate I140L mutation: GTGTTCGTTAACAATCAAGCTCGCCACTTG	This study	N/A
Primer: To generate I141L mutation: CAAGTGGCGAGCATCTTGGTTAACGAACACC	This study	N/A
Primer: To generate I141L mutation: GGTGTTCGTTAACCAAGATGCTCGCCACTTG	This study	N/A
Primer: To generate I148L mutation: GAACACCCGCAGTTGCAAACCATCGTATTG	This study	N/A
Primer: To generate I148L mutation: CAATACGATGGTTTGCAACTGCGGGTGTTC	This study	N/A
Primer: To generate I151L mutation: CCGCAGATCCAAACCTTGGTATTGTCTTATTTAG	This study	N/A
Primer: To generate I151L mutation: CTAAATAAGACAATACCAAGGTTTGGATCTGCGG	This study	N/A
Primer: To generate I164L mutation: CCAATCCGCGGAGTTGTTGTCTCAGTTCC	This study	N/A
Primer: To generate I164L mutation: GGAACTGAGACAACAACTCCGCGGATTGG	This study	N/A
Primer: To generate I180L mutation: CCTAATGATGCGTTTGGCCAACCTAGAAG	This study	N/A
Primer: To generate I180L mutation: CTTCTAGGTTGGCCAAACGCATCATTAGG	This study	N/A
Primer: To generate I197L mutation: CAGAGCTGAACGAATTGATGGAGAAACAGTTC	This study	N/A
Primer: To generate I197L mutation: GAACTGTTTCTCCATCAATTCGTTCAGCTCTG	This study	N/A
Primer: To generate I213L mutation: CAAGCAGCCAAGTTAGGCGGCCTGAAAG	This study	N/A
Primer: To generate I213L mutation: CTTTCAGGCCGCCTAACTTGGCTGCTTG	This study	N/A
Primer: To generate G214A mutation: CAGCCAAGATTGCGGGCCTGAAAGCGGCA	This study	N/A
Primer: To generate G214A mutation: TGCCGCTTTCAGGCCCGCAATCTTGGCTG	This study	N/A
Primer: To generate G214S mutation: CAGCCAAGATTAGCGGCCTGAAAGCGG	This study	N/A
Primer: To generate G214S mutation: CCGCTTTCAGGCCGCTAATCTTGGCTG	This study	N/A
Primer: To generate G214C mutation: CAGCCAAGATTTGTGGCCTGAAAGCGGCA	This study	N/A
Primer: To generate G214C mutation: TGCCGCTTTCAGGCCACAAATCTTGGCTG	This study	N/A
Primer: To generate G214P mutation: CAGCCAAGATTCCAGGCCTGAAAGCGGCA	This study	N/A
Primer: To generate G214P mutation: TGCCGCTTTCAGGCCTGGAATCTTGGCTG	This study	N/A
Primer: To generate G215A mutation: CAAGATTGGCGCACTGAAAGCGGCAG	This study	N/A
Primer: To generate G215A mutation: CTGCCGCTTTCAGTGCGCCAATCTTG	This study	N/A
Primer: To generate G215S mutation: CAGCCAAGATTGGCTCTCTGAAAGCGGCA	This study	N/A
Primer: To generate G215S mutation: TGCCGCTTTCAGAGAGCCAATCTTGGCTG	This study	N/A
Primer: To generate G215P mutation: CAGCCAAGATTGGCCCACTGAAAGCGGCA	This study	N/A
Primer: To generate G215P mutation: TGCCGCTTTCAGTGGGCCAATCTTGGCTG	This study	N/A
Primer: To generate G214S/G215A mutation: CAGCCAAGATTAGCGCACTGAAAGCGGCA	This study	N/A
Primer: To generate G214S/G215A mutation: TGCCGCTTTCAGTGCGCTAATCTTGGCTG	This study	N/A
Primer: To generate I222L mutation: CTGAAAGCGGCAGCGGAGCTGATGA ACTATCTAGACAAC	This study	N/A
Primer: To generate I222L mutation: GTTGTCTAGATAGTTCATCAGCTCCG CTGCCGCTTTCAG	This study	N/A
Primer: To generate I238L mutation: GGTTTGTTGATGGAGCAGCTGCGC GATCAAGACGAAGAC	This study	N/A
Primer: To generate I238L mutation: GTCTTCGTCTTGATCGCGCAGCTG CTCCATCAACAAACC	This study	N/A
Primer: To generate I249L mutation: GAAGACATGGCGACGCAACTGCA AGACTTGATGTTTGTC	This study	N/A
Primer: To generate I249L mutation: GACAAACATCAAGTCTTGCAGTT GCGTCGCCATGTCTTC	This study	N/A
Primer: To generate I267L mutation: GAAGTGGACGATCAAGGTCTGCA GAAATTGCTGCGTGAT	This study	N/A
Primer: To generate I267L mutation: ATCACGCAGCAATTTCTGCAGACC TTGATCGTCCACTTC	This study	N/A
Primer: To generate I310L mutation: GAGATGATGCGTGATGACCTGGA AGCGATGCCGCCAGTT	This study	N/A
Primer: To generate I310L mutation: AACTGGCGGCATCGCTTCCAGGT CATCACGCATCATCTC	This study	N/A
Primer: To generate I328L mutation: GAAGCGGCACAGAAAGAACTGC TAGCGATCGCTCGTCGC	This study	N/A
Primer: To generate I328L mutation: GCGACGAGCGATCGCTAGCAGT TCTTTCTGTGCCGCTTC	This study	N/A
Primer: To generate I331L mutation: CAGAAAGAAATCCTAGCGCTGGC TCGTCGCATGGCCGAT	This study	N/A
Primer: To generate I331L mutation: ATCGGCCATGCGACGAGCCAGC GCTAGGATTTCTTTCTG	This study	N/A
Primer: To generate F254 mutation: CAAGACTTGATGTACGTCTTCGA AAACTTAG	This study	N/A
Primer: To generate F254 mutation: CTAAGTTTTCGAAGACGTACAT CAAGTCTTG	This study	N/A
Primer: To generate F256 mutation: GACTTGATGTTTGTCTACGAAAA CTTAGTCGAA	This study	N/A
Primer: To generate F256 mutation: TTCGACTAAGTTTTCGTAGACA AACATCAAGTC	This study	N/A

Recombinant DNA

pMMB206, see Table S2	Morales et al., 1991⁶⁹	N/A
pNT1, see Table S2	Takekawa et al.⁸	N/A
pColdI, see Table S2	Takara	Cat#3361
pColdI-FliG_MC, see Table S2	Onoue et al., 2016⁷⁰	N/A

Software and algorithms

TopSpin version 3.6.2	Bruker BioSpin	https://www.bruker.com/service/supportupgrades/software-downloads/nmr.html
UCSF Chimera X Version 1.5	UCSF	https://www.cgl.ucsf.edu/chimerax/download.html
UCSF Chimera, Microsoft Windows 64-bit, Ver 1.16	UCSF	https://www.cgl.ucsf.edu/chimera/download.html
GROMACS version 2016.5	Pronk et al.⁷¹	http://www.gromacs.org
Pymol, version 2.5.0	Schrödinger LLC	https://pymol.org/2/
MODELLER version 9.6	Marti-Renom et al. 2000⁷²	https://salilab.org/modeller/
NMRFAM-SPARKY	Lee et al.⁷³	https://nmrfam.wisc.edu/nmrfam-sparky-distribution/

Other

Xtreme-60 Syringe pump system	Daedalus Innovations	Cat#Xtreme-60
5 mm high pressure zirconia tube	Daedalus Innovations	https://daedalusinnovations.com/high-pressure-nmr/
Superdex™ 200 Increase 10/300 GL column	Citiva	Cat#28990944