Table 2.

Examples of cross-validation of in silico-predicted properties with experiments to specifically probe conformational changes.

System	Simulation technique	MD observation and hypotheses	Observables	Experimental validation	References
Heterotrimeric Gα-GDP	μs-Long MD Mutant simulations	Spontaneous opening/closing of Gα-GDP in absence of GPCR Domain separation disrupts the GDP-site facilitating nucleotide release	Interdomain distances Nucleotide-exchange rates	DEER spectroscopy confirms multiple peaks for inter domain distance distributions with spin labels Fluorescence GTP-binding kinetics of a G-protein tether construct that restricts domain separation slows down nucleotide exchange	Dror et al., 2015
EAAT	Essential dynamics MD	Substrate transport intermediate forms the anion-selective conduction pathway	Anion currents	Trp-scanning mutagenesis and fluorescence quenching of predicted pore-forming residues confirms their interactions with anions Single channel conductance and anion selectivity of mutations of pore-lining residues	Machtens et al., 2015
Importin	sub-μs MD	Spontaneous transition toward extended conformations in water, and compaction in apolar environment	Intramolecular distances	FRET of a dual-fluorophore labeled importin confirms contraction in hydrophobic environment	Halder et al., 2015
SemiSWEET	μs-Long MD	Spontaneous transition from outward-open to inward-open state, through an occluded intermediate	3D-structure of previously unobserved inward-open state Transport activity	Crystallographic validation with structure of a mutant in the inward-open state Alanine mutagenesis of key residues in the extra- and intra-cellular gates and the sugar binding pocket	Latorraca et al., 2017
Arrestin	μs-Long MD	Motions at the two GPCR-binding interfaces (gate-loop and C-loop) are allosterically coupled via interdomain twisting	Separation between labels at the binding interfaces	Mutagenesis Fluorescence spectroscopy	Latorraca et al., 2018
GLIC	μs-Long MD Mutant simulations	Potentiation in Propofol-sensitive mutations is caused by conformational changes expanding transmembrane binding sites	Ion currents	Electrophysiology with voltage-clamp Mutagenesis	Heusser et al., 2018
Enzymatic micromotors	Accelerated MD	Flexibility near the active site mediates catalysis and coupled motion	Enzymatic activity Motor activity	Increased enzyme rigidity upon inhibitor binding reduces catalytic rates and motor speed	Arqué et al., 2019
PTEN	Multirun ns-MD	Conformational change upon phosphorylation that facilitates binding to Ki-67	Protein-protein interaction	Mutation of the predicted interacting sequences abrogates binding and biological effects	Ma et al., 2019
EGFR	μs-Long MD	Local intrinsic disorder of the EGFR kinase Higher dimerization and phosphorylation activity of L834R mutant	Local disorder Dimerization	H/D exchange measurements Light scattering + BN-PAGE	Shan et al., 2012
	μs-Long MD	Hinge-bending motions and overall position in the membrane affected by glycosylation	Epitope accessibility	Antibody C225 binds independent of glycosylation Antibody 2E9 binds preferentially glycosylated EGFR	(Kaszuba et al., 2015)
	ENMs Mutant simulations	Primary observation: Ectomutations untether the receptor and displace a domain from a cancer-specific cryptoepitope Predictions: Epitope-806 is coupled to the kinase Convergence of missense ectomutations and ectodeletions to activate the kinase Oncogenic activity of untested mutations	Conformational shifts Epitope accessibility Kinase activity Tumor growth rates Therapeutic response Protein design	SAXS dynamic equilibrium between tethered and untethered conformers shifted by mutations FACS binding to mAb806 increased in ectomutations mAb806 binding increases and decreases depending on kinase configuration In silico-designed double mutant has synergistic effects in vitro (FACS, SAXS) and in vivo Ectomutations and ectodeletions respond to mAb806 therapy in animal models	(Orellana et al., 2014, 2019b)