annotation_CSV/PMC4937829.csv

anno_start	anno_end	anno_text	entity_type	sentence	section
12	21	chaperone	protein_type	Visualizing chaperone-assisted protein folding	TITLE
30	40	structures	evidence	Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes.	ABSTRACT
20	29	chaperone	protein_type	One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state.	ABSTRACT
96	105	chaperone	protein_type	One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state.	ABSTRACT
154	164	chaperones	protein_type	One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state.	ABSTRACT
81	102	X-ray crystallography	experimental_method	To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ).	ABSTRACT
111	150	Residual Electron and Anomalous Density	experimental_method	To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ).	ABSTRACT
152	156	READ	experimental_method	To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ).	ABSTRACT
0	4	READ	experimental_method	READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy.	ABSTRACT
92	110	immunity protein 7	protein	READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy.	ABSTRACT
112	115	Im7	protein	READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy.	ABSTRACT
117	132	in complex with	protein_state	READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy.	ABSTRACT
137	144	E. coli	species	READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy.	ABSTRACT
145	154	chaperone	protein_type	READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy.	ABSTRACT
155	158	Spy	protein	READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy.	ABSTRACT
85	88	Im7	protein	This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy.	ABSTRACT
95	103	bound to	protein_state	This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy.	ABSTRACT
104	107	Spy	protein	This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy.	ABSTRACT
24	38	Spy-associated	protein_state	The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone.	ABSTRACT
39	42	Im7	protein	The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone.	ABSTRACT
78	86	unfolded	protein_state	The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone.	ABSTRACT
100	106	folded	protein_state	The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone.	ABSTRACT
111	117	native	protein_state	The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone.	ABSTRACT
199	207	bound to	protein_state	The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone.	ABSTRACT
210	219	chaperone	protein_type	The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone.	ABSTRACT
16	33	structural models	evidence	High-resolution structural models of protein-protein interactions are critical for obtaining mechanistic insights into biological processes.	INTRO
47	61	highly dynamic	protein_state	However, many protein-protein interactions are highly dynamic, making it difficult to obtain high-resolution data.	INTRO
45	86	intrinsically or conditionally disordered	protein_state	Particularly challenging are interactions of intrinsically or conditionally disordered sections of proteins with their partner proteins.	INTRO
19	40	X-ray crystallography	experimental_method	Recent advances in X-ray crystallography and NMR spectroscopy continue to improve our ability to analyze biomolecules that exist in multiple conformations.	INTRO
45	61	NMR spectroscopy	experimental_method	Recent advances in X-ray crystallography and NMR spectroscopy continue to improve our ability to analyze biomolecules that exist in multiple conformations.	INTRO
0	21	X-ray crystallography	experimental_method	X-ray crystallography has historically provided valuable information on small-scale conformational changes, but observing large-amplitude heterogeneous conformational changes often falls beyond the reach of current crystallographic techniques.	INTRO
0	3	NMR	experimental_method	NMR can theoretically be used to determine heterogeneous ensembles, but in practice, this proves to be very challenging.	INTRO
27	37	chaperones	protein_type	It is clear that molecular chaperones aid in protein folding.	INTRO
31	40	chaperone	protein_type	Structural characterization of chaperone-assisted protein folding likely would help bring clarity to this question.	INTRO
0	17	Structural models	evidence	Structural models of chaperone-substrate complexes have recently begun to provide information as to how a chaperone can recognize its substrate.	INTRO
21	30	chaperone	protein_type	Structural models of chaperone-substrate complexes have recently begun to provide information as to how a chaperone can recognize its substrate.	INTRO
106	115	chaperone	protein_type	Structural models of chaperone-substrate complexes have recently begun to provide information as to how a chaperone can recognize its substrate.	INTRO
25	35	chaperones	protein_type	However, the impact that chaperones have on their substrates, and how these interactions affect the folding process remain largely unknown.	INTRO
9	19	chaperones	protein_type	For most chaperones, it is still unclear whether the chaperone actively participates in and affects the folding of the substrate proteins, or merely provides a suitable microenvironment enabling the substrate to fold on its own.	INTRO
53	62	chaperone	protein_type	For most chaperones, it is still unclear whether the chaperone actively participates in and affects the folding of the substrate proteins, or merely provides a suitable microenvironment enabling the substrate to fold on its own.	INTRO
44	53	chaperone	protein_type	This is a truly fundamental question in the chaperone field, and one that has eluded the community largely because of the highly dynamic nature of the chaperone-substrate complexes.	INTRO
122	136	highly dynamic	protein_state	This is a truly fundamental question in the chaperone field, and one that has eluded the community largely because of the highly dynamic nature of the chaperone-substrate complexes.	INTRO
151	160	chaperone	protein_type	This is a truly fundamental question in the chaperone field, and one that has eluded the community largely because of the highly dynamic nature of the chaperone-substrate complexes.	INTRO
46	61	ATP-independent	protein_state	To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy.	INTRO
62	78	Escherichia coli	species	To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy.	INTRO
91	100	chaperone	protein_type	To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy.	INTRO
101	104	Spy	protein	To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy.	INTRO
0	3	Spy	protein	Spy prevents protein aggregation and aids in protein folding under various stress conditions, including treatment with tannin and butanol.	INTRO
119	125	tannin	chemical	Spy prevents protein aggregation and aids in protein folding under various stress conditions, including treatment with tannin and butanol.	INTRO
130	137	butanol	chemical	Spy prevents protein aggregation and aids in protein folding under various stress conditions, including treatment with tannin and butanol.	INTRO
25	28	Spy	protein	We originally discovered Spy by its ability to stabilize the protein-folding model Im7 in vivo and recently demonstrated that Im7 folds while associated with Spy.	INTRO
83	86	Im7	protein	We originally discovered Spy by its ability to stabilize the protein-folding model Im7 in vivo and recently demonstrated that Im7 folds while associated with Spy.	INTRO
126	129	Im7	protein	We originally discovered Spy by its ability to stabilize the protein-folding model Im7 in vivo and recently demonstrated that Im7 folds while associated with Spy.	INTRO
158	161	Spy	protein	We originally discovered Spy by its ability to stabilize the protein-folding model Im7 in vivo and recently demonstrated that Im7 folds while associated with Spy.	INTRO
4	21	crystal structure	evidence	The crystal structure of Spy revealed that it forms a thin α-helical homodimeric cradle.	INTRO
25	28	Spy	protein	The crystal structure of Spy revealed that it forms a thin α-helical homodimeric cradle.	INTRO
69	80	homodimeric	oligomeric_state	The crystal structure of Spy revealed that it forms a thin α-helical homodimeric cradle.	INTRO
81	87	cradle	site	The crystal structure of Spy revealed that it forms a thin α-helical homodimeric cradle.	INTRO
0	36	Crosslinking and genetic experiments	experimental_method	Crosslinking and genetic experiments suggested that Spy interacts with substrates somewhere on its concave side.	INTRO
52	55	Spy	protein	Crosslinking and genetic experiments suggested that Spy interacts with substrates somewhere on its concave side.	INTRO
17	38	X-ray crystallography	experimental_method	By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex.	INTRO
75	93	crystal structures	evidence	By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex.	INTRO
138	146	ensemble	evidence	By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex.	INTRO
154	161	dynamic	protein_state	By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex.	INTRO
162	169	Spy:Im7	complex_assembly	By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex.	INTRO
38	47	chaperone	protein_type	This work provides a detailed view of chaperone-mediated protein folding and shows how substrates like Im7 find their native fold while bound to their chaperones.	INTRO
103	106	Im7	protein	This work provides a detailed view of chaperone-mediated protein folding and shows how substrates like Im7 find their native fold while bound to their chaperones.	INTRO
136	144	bound to	protein_state	This work provides a detailed view of chaperone-mediated protein folding and shows how substrates like Im7 find their native fold while bound to their chaperones.	INTRO
151	161	chaperones	protein_type	This work provides a detailed view of chaperone-mediated protein folding and shows how substrates like Im7 find their native fold while bound to their chaperones.	INTRO
0	13	Crystallizing	experimental_method	Crystallizing the Spy:Im7 complex	RESULTS
18	25	Spy:Im7	complex_assembly	Crystallizing the Spy:Im7 complex	RESULTS
27	35	crystals	evidence	We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence.	RESULTS
57	60	Spy	protein	We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence.	RESULTS
167	193	crystallization conditions	experimental_method	We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence.	RESULTS
207	210	Spy	protein	We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence.	RESULTS
211	219	crystals	evidence	We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence.	RESULTS
227	238	presence of	protein_state	We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence.	RESULTS
275	282	absence	protein_state	We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence.	RESULTS
13	21	screened	experimental_method	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
22	48	crystallization conditions	experimental_method	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
53	56	Spy	protein	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
123	131	unfolded	protein_state	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
132	138	bovine	taxonomy_domain	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
139	147	α-casein	chemical	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
157	166	wild-type	protein_state	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
168	170	WT	protein_state	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
172	179	E. coli	species	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
180	183	Im7	protein	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
188	196	unfolded	protein_state	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
208	211	Im7	protein	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
213	217	L18A	mutant	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
218	222	L19A	mutant	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
223	227	L37A	mutant	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
238	253	N-terminal half	structure_element	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
257	260	Im7	protein	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
262	269	Im76-45	mutant	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
301	320	Spy-binding portion	structure_element	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
324	327	Im7	protein	We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.	RESULTS
49	64	co-crystallized	experimental_method	We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals.	RESULTS
65	69	with	protein_state	We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals.	RESULTS
70	73	Spy	protein	We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals.	RESULTS
88	91	Spy	protein	We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals.	RESULTS
92	97	alone	protein_state	We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals.	RESULTS
112	120	crystals	evidence	We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals.	RESULTS
11	42	crystal washing and dissolution	experimental_method	Subsequent crystal washing and dissolution experiments confirmed the presence of the substrates in the co-crystals (Supplementary Fig. 2).	RESULTS
103	114	co-crystals	experimental_method	Subsequent crystal washing and dissolution experiments confirmed the presence of the substrates in the co-crystals (Supplementary Fig. 2).	RESULTS
4	12	crystals	evidence	The crystals diffracted to ~1.8 Å resolution.	RESULTS
8	19	Spy:Im76-45	complex_assembly	We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes.	RESULTS
20	36	selenomethionine	chemical	We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes.	RESULTS
37	45	crystals	evidence	We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes.	RESULTS
63	102	single-wavelength anomalous diffraction	experimental_method	We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes.	RESULTS
104	107	SAD	experimental_method	We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes.	RESULTS
171	174	Spy	protein	We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes.	RESULTS
95	111	electron density	evidence	However, modeling of the substrate in the complex proved to be a substantial challenge, as the electron density of the substrate was discontinuous and fragmented.	RESULTS
9	32	minimal binding portion	structure_element	Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a).	RESULTS
36	39	Im7	protein	Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a).	RESULTS
41	48	Im76-45	mutant	Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a).	RESULTS
74	90	electron density	evidence	Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a).	RESULTS
36	43	density	evidence	We hypothesized that the fragmented density was due to multiple, partially occupied conformations of the substrate bound within the crystal.	RESULTS
132	139	crystal	evidence	We hypothesized that the fragmented density was due to multiple, partially occupied conformations of the substrate bound within the crystal.	RESULTS
72	93	X-ray crystallography	experimental_method	Such residual density is typically not considered usable by traditional X-ray crystallography methods.	RESULTS
51	66	chaperone-bound	protein_state	Thus, we developed a new approach to interpret the chaperone-bound substrate in multiple conformations.	RESULTS
0	4	READ	experimental_method	READ: a strategy to visualize heterogeneous and dynamic biomolecules	RESULTS
17	26	structure	evidence	To determine the structure of the substrate portion of these Spy:substrate complexes, we conceived of an approach that we term READ, for Residual Electron and Anomalous Density.	RESULTS
61	64	Spy	protein	To determine the structure of the substrate portion of these Spy:substrate complexes, we conceived of an approach that we term READ, for Residual Electron and Anomalous Density.	RESULTS
127	131	READ	experimental_method	To determine the structure of the substrate portion of these Spy:substrate complexes, we conceived of an approach that we term READ, for Residual Electron and Anomalous Density.	RESULTS
137	176	Residual Electron and Anomalous Density	experimental_method	To determine the structure of the substrate portion of these Spy:substrate complexes, we conceived of an approach that we term READ, for Residual Electron and Anomalous Density.	RESULTS
72	75	Spy	protein	We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate.	RESULTS
86	96	co-crystal	evidence	We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate.	RESULTS
122	131	structure	evidence	We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate.	RESULTS
139	145	folded	protein_state	We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate.	RESULTS
146	152	domain	structure_element	We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate.	RESULTS
156	159	Spy	protein	We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate.	RESULTS
186	211	residual electron density	evidence	We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate.	RESULTS
223	230	dynamic	protein_state	We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate.	RESULTS
47	55	flexible	protein_state	(2) We then labeled individual residues in the flexible regions of the substrate with the strong anomalous scatterer iodine, which serves to locate these residues in three-dimensional space using their anomalous density.	RESULTS
117	123	iodine	chemical	(2) We then labeled individual residues in the flexible regions of the substrate with the strong anomalous scatterer iodine, which serves to locate these residues in three-dimensional space using their anomalous density.	RESULTS
202	219	anomalous density	evidence	(2) We then labeled individual residues in the flexible regions of the substrate with the strong anomalous scatterer iodine, which serves to locate these residues in three-dimensional space using their anomalous density.	RESULTS
17	35	molecular dynamics	experimental_method	(3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density.	RESULTS
37	39	MD	experimental_method	(3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density.	RESULTS
41	52	simulations	experimental_method	(3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density.	RESULTS
121	128	dynamic	protein_state	(3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density.	RESULTS
155	182	sample-and-select algorithm	experimental_method	(3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density.	RESULTS
257	287	residual and anomalous density	evidence	(3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density.	RESULTS
73	81	flexible	protein_state	Importantly, even though we only labeled a subset of the residues in the flexible regions of the substrate with iodine, the residual electron density can provide spatial information on many of the other flexible residues.	RESULTS
112	118	iodine	chemical	Importantly, even though we only labeled a subset of the residues in the flexible regions of the substrate with iodine, the residual electron density can provide spatial information on many of the other flexible residues.	RESULTS
124	149	residual electron density	evidence	Importantly, even though we only labeled a subset of the residues in the flexible regions of the substrate with iodine, the residual electron density can provide spatial information on many of the other flexible residues.	RESULTS
203	211	flexible	protein_state	Importantly, even though we only labeled a subset of the residues in the flexible regions of the substrate with iodine, the residual electron density can provide spatial information on many of the other flexible residues.	RESULTS
4	20	electron density	evidence	The electron density then allowed us to connect the labeled residues of the substrate by confining the protein chain within regions of detectable density.	RESULTS
146	153	density	evidence	The electron density then allowed us to connect the labeled residues of the substrate by confining the protein chain within regions of detectable density.	RESULTS
117	124	crystal	evidence	In this way, the two forms of data together were able to describe multiple conformations of the substrate within the crystal.	RESULTS
47	51	READ	experimental_method	As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy.	RESULTS
109	127	Spy-binding domain	structure_element	As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy.	RESULTS
131	134	Im7	protein	As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy.	RESULTS
142	149	Im76-45	mutant	As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy.	RESULTS
164	172	bound to	protein_state	As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy.	RESULTS
173	176	Spy	protein	As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy.	RESULTS
25	29	READ	experimental_method	However, we believe that READ will prove generally applicable to visualizing heterogeneous and dynamic complexes that have previously escaped detailed structural analysis.	RESULTS
11	15	READ	experimental_method	Collecting READ data for the Spy:Im76-45 complex	RESULTS
29	40	Spy:Im76-45	complex_assembly	Collecting READ data for the Spy:Im76-45 complex	RESULTS
13	27	READ technique	experimental_method	To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3).	RESULTS
69	78	chaperone	protein_type	To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3).	RESULTS
79	82	Spy	protein	To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3).	RESULTS
96	103	Im76-45	mutant	To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3).	RESULTS
138	141	NMR	experimental_method	To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3).	RESULTS
162	169	Im76-45	mutant	To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3).	RESULTS
189	197	unfolded	protein_state	To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3).	RESULTS
209	215	folded	protein_state	To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3).	RESULTS
243	246	Im7	protein	To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3).	RESULTS
10	29	binding experiments	experimental_method	Moreover, binding experiments indicated that Im76-45 comprises the entire Spy-binding region.	RESULTS
45	52	Im76-45	mutant	Moreover, binding experiments indicated that Im76-45 comprises the entire Spy-binding region.	RESULTS
74	92	Spy-binding region	site	Moreover, binding experiments indicated that Im76-45 comprises the entire Spy-binding region.	RESULTS
37	43	iodine	chemical	To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe).	RESULTS
48	56	replaced	experimental_method	To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe).	RESULTS
63	70	Im76-45	mutant	To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe).	RESULTS
114	133	4-iodophenylalanine	chemical	To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe).	RESULTS
135	141	pI-Phe	chemical	To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe).	RESULTS
11	31	anomalous scattering	evidence	Its strong anomalous scattering allowed us to track the positions of these individual Im76-45 residues one at a time, potentially even if the residue was found in several locations in the same crystal.	RESULTS
86	93	Im76-45	mutant	Its strong anomalous scattering allowed us to track the positions of these individual Im76-45 residues one at a time, potentially even if the residue was found in several locations in the same crystal.	RESULTS
193	200	crystal	evidence	Its strong anomalous scattering allowed us to track the positions of these individual Im76-45 residues one at a time, potentially even if the residue was found in several locations in the same crystal.	RESULTS
8	23	co-crystallized	experimental_method	We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2).	RESULTS
24	27	Spy	protein	We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2).	RESULTS
42	49	Im76-45	mutant	We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2).	RESULTS
97	103	pI-Phe	chemical	We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2).	RESULTS
104	116	substitution	experimental_method	We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2).	RESULTS
147	156	collected	experimental_method	We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2).	RESULTS
157	171	anomalous data	evidence	We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2).	RESULTS
186	197	Spy:Im76-45	complex_assembly	We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2).	RESULTS
20	40	electron density map	evidence	Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site.	RESULTS
72	89	anomalous signals	evidence	Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site.	RESULTS
105	111	cradle	site	Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site.	RESULTS
115	118	Spy	protein	Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site.	RESULTS
153	156	Im7	protein	Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site.	RESULTS
157	179	substrate binding site	site	Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site.	RESULTS
31	38	density	evidence	Consistent with the fragmented density, however, we observed multiple iodine positions for seven of the eight substituted residues.	RESULTS
70	76	iodine	chemical	Consistent with the fragmented density, however, we observed multiple iodine positions for seven of the eight substituted residues.	RESULTS
43	46	Im7	protein	Together, these results indicated that the Im7 substrate binds Spy in multiple conformations.	RESULTS
63	66	Spy	protein	Together, these results indicated that the Im7 substrate binds Spy in multiple conformations.	RESULTS
0	4	READ	experimental_method	READ sample-and-select procedure	RESULTS
5	22	sample-and-select	experimental_method	READ sample-and-select procedure	RESULTS
42	49	Im76-45	mutant	To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes.	RESULTS
63	71	bound to	protein_state	To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes.	RESULTS
72	75	Spy	protein	To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes.	RESULTS
93	118	residual electron density	evidence	To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes.	RESULTS
127	144	anomalous signals	evidence	To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes.	RESULTS
154	160	pI-Phe	chemical	To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes.	RESULTS
173	184	Spy:Im76-45	complex_assembly	To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes.	RESULTS
41	50	chaperone	protein_type	To generate an accurate depiction of the chaperone-substrate interactions, we devised a selection protocol based on a sample-and-select procedure employed in NMR spectroscopy.	RESULTS
118	135	sample-and-select	experimental_method	To generate an accurate depiction of the chaperone-substrate interactions, we devised a selection protocol based on a sample-and-select procedure employed in NMR spectroscopy.	RESULTS
158	174	NMR spectroscopy	experimental_method	To generate an accurate depiction of the chaperone-substrate interactions, we devised a selection protocol based on a sample-and-select procedure employed in NMR spectroscopy.	RESULTS
38	55	genetic algorithm	experimental_method	During each round of the selection, a genetic algorithm alters the ensemble and its agreement to the experimental data is re-evaluated.	RESULTS
104	129	residual electron density	evidence	If successful, the selection identifies the smallest group of specific conformations that best fits the residual electron density and anomalous signals.	RESULTS
134	151	anomalous signals	evidence	If successful, the selection identifies the smallest group of specific conformations that best fits the residual electron density and anomalous signals.	RESULTS
4	8	READ	experimental_method	The READ sample-and-select algorithm is diagrammed in Fig. 2.	RESULTS
9	36	sample-and-select algorithm	experimental_method	The READ sample-and-select algorithm is diagrammed in Fig. 2.	RESULTS
76	85	chaperone	protein_type	Prior to performing the selection, we generated a large and diverse pool of chaperone-substrate complexes using coarse-grained MD simulations in a pseudo-crystal environment (Fig. 2 and Supplementary Fig. 4).	RESULTS
112	141	coarse-grained MD simulations	experimental_method	Prior to performing the selection, we generated a large and diverse pool of chaperone-substrate complexes using coarse-grained MD simulations in a pseudo-crystal environment (Fig. 2 and Supplementary Fig. 4).	RESULTS
147	173	pseudo-crystal environment	experimental_method	Prior to performing the selection, we generated a large and diverse pool of chaperone-substrate complexes using coarse-grained MD simulations in a pseudo-crystal environment (Fig. 2 and Supplementary Fig. 4).	RESULTS
4	30	coarse-grained simulations	experimental_method	The coarse-grained simulations are based on a single-residue resolution model for protein folding and were extended here to describe Spy-Im76-45 binding events (Online Methods).	RESULTS
133	144	Spy-Im76-45	complex_assembly	The coarse-grained simulations are based on a single-residue resolution model for protein folding and were extended here to describe Spy-Im76-45 binding events (Online Methods).	RESULTS
30	49	binding simulations	experimental_method	The initial conditions of the binding simulations are not biased toward a particular conformation of the substrate or any specific chaperone-substrate interaction (Online Methods).	RESULTS
131	140	chaperone	protein_type	The initial conditions of the binding simulations are not biased toward a particular conformation of the substrate or any specific chaperone-substrate interaction (Online Methods).	RESULTS
0	7	Im76-45	mutant	Im76-45 binds and unbinds to Spy throughout the simulations.	RESULTS
29	32	Spy	protein	Im76-45 binds and unbinds to Spy throughout the simulations.	RESULTS
48	59	simulations	experimental_method	Im76-45 binds and unbinds to Spy throughout the simulations.	RESULTS
82	91	chaperone	protein_type	This strategy allows a wide range of substrate conformations to interact with the chaperone.	RESULTS
9	11	MD	experimental_method	From the MD simulations, we extracted ~10,000 diverse Spy:Im76-45 complexes to be used by the ensuing selection.	RESULTS
12	23	simulations	experimental_method	From the MD simulations, we extracted ~10,000 diverse Spy:Im76-45 complexes to be used by the ensuing selection.	RESULTS
54	65	Spy:Im76-45	complex_assembly	From the MD simulations, we extracted ~10,000 diverse Spy:Im76-45 complexes to be used by the ensuing selection.	RESULTS
44	47	Spy	protein	Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate.	RESULTS
48	53	dimer	oligomeric_state	Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate.	RESULTS
54	62	bound to	protein_state	Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate.	RESULTS
72	79	Im76-45	mutant	Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate.	RESULTS
113	166	residual electron and anomalous crystallographic data	evidence	This pool was then used by the selection algorithm to identify the minimal ensemble that best satisfies both the residual electron and anomalous crystallographic data.	RESULTS
4	24	anomalous scattering	evidence	The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective Cα atom in each coarse-grained conformer by 6.5 Å. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals.	RESULTS
78	84	pI-Phe	chemical	The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective Cα atom in each coarse-grained conformer by 6.5 Å. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals.	RESULTS
99	105	iodine	chemical	The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective Cα atom in each coarse-grained conformer by 6.5 Å. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals.	RESULTS
263	269	iodine	chemical	The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective Cα atom in each coarse-grained conformer by 6.5 Å. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals.	RESULTS
270	287	anomalous signals	evidence	The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective Cα atom in each coarse-grained conformer by 6.5 Å. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals.	RESULTS
28	53	residual electron density	evidence	Simultaneously, it uses the residual electron density to help choose ensembles.	RESULTS
12	38	electron density selection	experimental_method	To make the electron density selection practical, we needed to develop a method to rapidly evaluate the agreement between the selected sub-ensembles and the experimental electron density on-the-fly during the selection procedure.	RESULTS
170	186	electron density	evidence	To make the electron density selection practical, we needed to develop a method to rapidly evaluate the agreement between the selected sub-ensembles and the experimental electron density on-the-fly during the selection procedure.	RESULTS
79	108	2mFo−DFc electron density map	evidence	To accomplish this task, we generated a compressed version of the experimental 2mFo−DFc electron density map for use in the selection.	RESULTS
39	42	map	evidence	This process provided us with a target map that the ensuing selection tried to recapitulate.	RESULTS
65	68	map	evidence	To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations.	RESULTS
95	102	density	evidence	To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations.	RESULTS
150	157	Im76-45	mutant	To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations.	RESULTS
165	176	Spy:Im76-45	complex_assembly	To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations.	RESULTS
177	179	MD	experimental_method	To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations.	RESULTS
180	191	simulations	experimental_method	To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations.	RESULTS
41	55	coarse-grained	experimental_method	For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the Cα positions of Im76-45 was extracted and used to construct an electron density map (Online Methods).	RESULTS
56	58	MD	experimental_method	For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the Cα positions of Im76-45 was extracted and used to construct an electron density map (Online Methods).	RESULTS
69	85	electron density	evidence	For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the Cα positions of Im76-45 was extracted and used to construct an electron density map (Online Methods).	RESULTS
109	116	Im76-45	mutant	For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the Cα positions of Im76-45 was extracted and used to construct an electron density map (Online Methods).	RESULTS
156	176	electron density map	evidence	For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the Cα positions of Im76-45 was extracted and used to construct an electron density map (Online Methods).	RESULTS
17	38	electron density maps	evidence	These individual electron density maps from the separate conformers could then be combined (Fig. 2) and compared to the averaged experimental electron density map as part of the selection algorithm.	RESULTS
142	162	electron density map	evidence	These individual electron density maps from the separate conformers could then be combined (Fig. 2) and compared to the averaged experimental electron density map as part of the selection algorithm.	RESULTS
56	62	iodine	chemical	This approach allowed us to simultaneously use both the iodine anomalous signals and the residual electron density in the selection procedure.	RESULTS
63	80	anomalous signals	evidence	This approach allowed us to simultaneously use both the iodine anomalous signals and the residual electron density in the selection procedure.	RESULTS
89	114	residual electron density	evidence	This approach allowed us to simultaneously use both the iodine anomalous signals and the residual electron density in the selection procedure.	RESULTS
51	53	MD	experimental_method	The selection resulted in small ensembles from the MD pool that best fit the READ data (Fig. 1c,d).	RESULTS
77	81	READ	experimental_method	The selection resulted in small ensembles from the MD pool that best fit the READ data (Fig. 1c,d).	RESULTS
36	47	Spy:Im76-45	complex_assembly	Before analyzing the details of the Spy:Im76-45 complex, we first engaged in a series of validation tests to verify the ensemble and selection procedure (Supplementary Note 1, Figures 1c,d, Supplemental Figures 5-7).	RESULTS
105	110	RFree	evidence	Of note, the final six-membered ensemble was the largest ensemble that could simultaneously decrease the RFree and pass the 10-fold cross-validation test.	RESULTS
124	153	10-fold cross-validation test	experimental_method	Of note, the final six-membered ensemble was the largest ensemble that could simultaneously decrease the RFree and pass the 10-fold cross-validation test.	RESULTS
59	66	Im76-45	mutant	This ensemble depicts the conformations that the substrate Im76-45 adopts while bound to the chaperone Spy (Fig. 3 Supplementary Movie 1, and Table 1).	RESULTS
80	88	bound to	protein_state	This ensemble depicts the conformations that the substrate Im76-45 adopts while bound to the chaperone Spy (Fig. 3 Supplementary Movie 1, and Table 1).	RESULTS
93	102	chaperone	protein_type	This ensemble depicts the conformations that the substrate Im76-45 adopts while bound to the chaperone Spy (Fig. 3 Supplementary Movie 1, and Table 1).	RESULTS
103	106	Spy	protein	This ensemble depicts the conformations that the substrate Im76-45 adopts while bound to the chaperone Spy (Fig. 3 Supplementary Movie 1, and Table 1).	RESULTS
28	31	Im7	protein	Folding and interactions of Im7 while bound to Spy	RESULTS
38	46	bound to	protein_state	Folding and interactions of Im7 while bound to Spy	RESULTS
47	50	Spy	protein	Folding and interactions of Im7 while bound to Spy	RESULTS
44	48	READ	experimental_method	Our results showed that by using this novel READ approach, we were able to obtain structural information about the dynamic interaction of a chaperone with its substrate protein.	RESULTS
140	149	chaperone	protein_type	Our results showed that by using this novel READ approach, we were able to obtain structural information about the dynamic interaction of a chaperone with its substrate protein.	RESULTS
95	104	chaperone	protein_type	We were particularly interested in finding answers to one of the most fundamental questions in chaperone biology—how does chaperone binding affect substrate structure and vice versa.	RESULTS
122	131	chaperone	protein_type	We were particularly interested in finding answers to one of the most fundamental questions in chaperone biology—how does chaperone binding affect substrate structure and vice versa.	RESULTS
28	38	structures	evidence	By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound.	RESULTS
69	76	Im76-45	mutant	By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound.	RESULTS
77	85	bound to	protein_state	By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound.	RESULTS
86	89	Spy	protein	By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound.	RESULTS
108	115	Im76-45	mutant	By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound.	RESULTS
163	168	bound	protein_state	By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound.	RESULTS
71	79	unfolded	protein_state	We found these conformations to be highly heterogeneous and to include unfolded, partially folded, and native-like states (Fig. 3).	RESULTS
81	97	partially folded	protein_state	We found these conformations to be highly heterogeneous and to include unfolded, partially folded, and native-like states (Fig. 3).	RESULTS
103	114	native-like	protein_state	We found these conformations to be highly heterogeneous and to include unfolded, partially folded, and native-like states (Fig. 3).	RESULTS
35	42	Im76-45	mutant	The ensemble primarily encompasses Im76-45 laying diagonally within the Spy cradle in several different orientations, but some conformations traverse as far as the tips or even extend over the side of the cradle (Figs. 3,4a).	RESULTS
72	75	Spy	protein	The ensemble primarily encompasses Im76-45 laying diagonally within the Spy cradle in several different orientations, but some conformations traverse as far as the tips or even extend over the side of the cradle (Figs. 3,4a).	RESULTS
76	82	cradle	site	The ensemble primarily encompasses Im76-45 laying diagonally within the Spy cradle in several different orientations, but some conformations traverse as far as the tips or even extend over the side of the cradle (Figs. 3,4a).	RESULTS
205	211	cradle	site	The ensemble primarily encompasses Im76-45 laying diagonally within the Spy cradle in several different orientations, but some conformations traverse as far as the tips or even extend over the side of the cradle (Figs. 3,4a).	RESULTS
17	28	contact map	evidence	We constructed a contact map of the complex, which shows the frequency of interactions for chaperone-substrate residue pairs (Fig. 4).	RESULTS
91	100	chaperone	protein_type	We constructed a contact map of the complex, which shows the frequency of interactions for chaperone-substrate residue pairs (Fig. 4).	RESULTS
26	43	interaction sites	site	We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46).	RESULTS
47	50	Spy	protein	We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46).	RESULTS
82	88	Arg122	residue_name_number	We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46).	RESULTS
90	96	Thr124	residue_name_number	We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46).	RESULTS
102	107	Phe29	residue_name_number	We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46).	RESULTS
147	156	chaperone	protein_type	We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46).	RESULTS
158	163	Arg61	residue_name_number	We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46).	RESULTS
165	170	Arg43	residue_name_number	We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46).	RESULTS
172	177	Lys47	residue_name_number	We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46).	RESULTS
179	184	His96	residue_name_number	We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46).	RESULTS
190	195	Met46	residue_name_number	We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46).	RESULTS
4	27	Spy-contacting residues	site	The Spy-contacting residues comprise a mixture of charged, polar, and hydrophobic residues.	RESULTS
45	52	Im76-45	mutant	Surprisingly, we noted that in the ensemble, Im76-45 interacts with only 38% of the hydrophobic residues in the Spy cradle, but interacts with 61% of the hydrophilic residues in the cradle.	RESULTS
112	115	Spy	protein	Surprisingly, we noted that in the ensemble, Im76-45 interacts with only 38% of the hydrophobic residues in the Spy cradle, but interacts with 61% of the hydrophilic residues in the cradle.	RESULTS
116	122	cradle	site	Surprisingly, we noted that in the ensemble, Im76-45 interacts with only 38% of the hydrophobic residues in the Spy cradle, but interacts with 61% of the hydrophilic residues in the cradle.	RESULTS
182	188	cradle	site	Surprisingly, we noted that in the ensemble, Im76-45 interacts with only 38% of the hydrophobic residues in the Spy cradle, but interacts with 61% of the hydrophilic residues in the cradle.	RESULTS
101	108	Im76-45	mutant	This mixture suggests the importance of both electrostatic and hydrophobic components in binding the Im76-45 ensemble.	RESULTS
72	79	Im76-45	mutant	With respect to the substrate, we observed that nearly every residue in Im76-45 is in contact with Spy (Fig. 4a).	RESULTS
99	102	Spy	protein	With respect to the substrate, we observed that nearly every residue in Im76-45 is in contact with Spy (Fig. 4a).	RESULTS
64	71	Im76-45	mutant	However, we did notice that despite this uniformity, regions of Im76-45 preferentially interact with different regions in Spy (Fig. 4b).	RESULTS
122	125	Spy	protein	However, we did notice that despite this uniformity, regions of Im76-45 preferentially interact with different regions in Spy (Fig. 4b).	RESULTS
17	32	N-terminal half	structure_element	For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface.	RESULTS
36	43	Im76-45	mutant	For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface.	RESULTS
75	78	Spy	protein	For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface.	RESULTS
79	85	cradle	site	For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface.	RESULTS
99	114	C-terminal half	structure_element	For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface.	RESULTS
157	160	Spy	protein	For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface.	RESULTS
163	178	concave surface	site	For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface.	RESULTS
35	42	Im76-45	mutant	Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly.	RESULTS
63	71	unfolded	protein_state	Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly.	RESULTS
79	85	native	protein_state	Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly.	RESULTS
115	118	Spy	protein	Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly.	RESULTS
12	24	least-folded	protein_state	Whereas the least-folded Im76-45 pose in the ensemble forms the most hydrophobic contacts with Spy (Fig. 3), the two most-folded conformations form the fewest hydrophobic contacts (Fig. 3).	RESULTS
25	32	Im76-45	mutant	Whereas the least-folded Im76-45 pose in the ensemble forms the most hydrophobic contacts with Spy (Fig. 3), the two most-folded conformations form the fewest hydrophobic contacts (Fig. 3).	RESULTS
95	98	Spy	protein	Whereas the least-folded Im76-45 pose in the ensemble forms the most hydrophobic contacts with Spy (Fig. 3), the two most-folded conformations form the fewest hydrophobic contacts (Fig. 3).	RESULTS
117	128	most-folded	protein_state	Whereas the least-folded Im76-45 pose in the ensemble forms the most hydrophobic contacts with Spy (Fig. 3), the two most-folded conformations form the fewest hydrophobic contacts (Fig. 3).	RESULTS
64	71	Im76-45	mutant	This shift in contacts is likely due to hydrophobic residues of Im76-45 preferentially forming intra-molecular contacts upon folding (i.e., hydrophobic collapse), effectively removing themselves from the interaction sites.	RESULTS
204	221	interaction sites	site	This shift in contacts is likely due to hydrophobic residues of Im76-45 preferentially forming intra-molecular contacts upon folding (i.e., hydrophobic collapse), effectively removing themselves from the interaction sites.	RESULTS
35	48	binding sites	site	The diversity of conformations and binding sites observed here emphasizes the dynamic and heterogeneous nature of the chaperone-substrate ensemble.	RESULTS
118	127	chaperone	protein_type	The diversity of conformations and binding sites observed here emphasizes the dynamic and heterogeneous nature of the chaperone-substrate ensemble.	RESULTS
79	86	Im76-45	mutant	Although we do not yet have time resolution data of these various snapshots of Im76-45, this ensemble illustrates how a substrate samples its folding landscape while bound to a chaperone.	RESULTS
166	174	bound to	protein_state	Although we do not yet have time resolution data of these various snapshots of Im76-45, this ensemble illustrates how a substrate samples its folding landscape while bound to a chaperone.	RESULTS
177	186	chaperone	protein_type	Although we do not yet have time resolution data of these various snapshots of Im76-45, this ensemble illustrates how a substrate samples its folding landscape while bound to a chaperone.	RESULTS
0	3	Spy	protein	Spy changes conformation upon substrate binding	RESULTS
14	23	structure	evidence	Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2).	RESULTS
27	30	Spy	protein	Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2).	RESULTS
38	53	substrate-bound	protein_state	Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2).	RESULTS
58	61	apo	protein_state	Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2).	RESULTS
87	90	Spy	protein	Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2).	RESULTS
91	96	dimer	oligomeric_state	Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2).	RESULTS
28	31	Spy	protein	Upon substrate binding, the Spy dimer twists 9° about its center relative to its apo form.	RESULTS
32	37	dimer	oligomeric_state	Upon substrate binding, the Spy dimer twists 9° about its center relative to its apo form.	RESULTS
81	84	apo	protein_state	Upon substrate binding, the Spy dimer twists 9° about its center relative to its apo form.	RESULTS
99	102	Spy	protein	This twist yields asymmetry and results in substantially different interaction patterns in the two Spy monomers (Fig. 4b).	RESULTS
103	111	monomers	oligomeric_state	This twist yields asymmetry and results in substantially different interaction patterns in the two Spy monomers (Fig. 4b).	RESULTS
67	70	Spy	protein	It is possible that this twist serves to increase heterogeneity in Spy by providing more binding poses.	RESULTS
35	48	linker region	structure_element	Additionally, we observed that the linker region (residues 47–57) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate.	RESULTS
59	64	47–57	residue_range	Additionally, we observed that the linker region (residues 47–57) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate.	RESULTS
69	72	Spy	protein	Additionally, we observed that the linker region (residues 47–57) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate.	RESULTS
134	144	disordered	protein_state	Additionally, we observed that the linker region (residues 47–57) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate.	RESULTS
42	45	Spy	protein	This increased disorder might explain how Spy is able to recognize and bind different substrates and/or differing conformations of the same substrate.	RESULTS
56	59	Spy	protein	Importantly, we observed the same structural changes in Spy regardless of which of the four substrates was bound (Fig. 5b, Table 1).	RESULTS
4	8	RMSD	evidence	The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site.	RESULTS
21	32	well-folded	protein_state	The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site.	RESULTS
45	48	Spy	protein	The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site.	RESULTS
61	70	chaperone	protein_type	The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site.	RESULTS
138	161	competition experiments	experimental_method	The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site.	RESULTS
214	217	Spy	protein	The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site.	RESULTS
328	344	Spy binding site	site	The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site.	RESULTS
21	31	chaperones	protein_type	To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate.	DISCUSS
112	116	READ	experimental_method	To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate.	DISCUSS
148	171	conformational ensemble	evidence	To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate.	DISCUSS
179	188	chaperone	protein_type	To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate.	DISCUSS
189	192	Spy	protein	To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate.	DISCUSS
193	211	bound to substrate	protein_state	To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate.	DISCUSS
24	31	Im76-45	mutant	As a substrate, we used Im76-45, the chaperone-interacting portion of the protein-folding model protein Im7.	DISCUSS
37	66	chaperone-interacting portion	structure_element	As a substrate, we used Im76-45, the chaperone-interacting portion of the protein-folding model protein Im7.	DISCUSS
104	107	Im7	protein	As a substrate, we used Im76-45, the chaperone-interacting portion of the protein-folding model protein Im7.	DISCUSS
7	22	chaperone-bound	protein_state	In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states.	DISCUSS
33	40	Im76-45	mutant	In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states.	DISCUSS
49	57	unfolded	protein_state	In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states.	DISCUSS
69	75	folded	protein_state	In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states.	DISCUSS
81	87	native	protein_state	In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states.	DISCUSS
117	126	chaperone	protein_type	The ensemble provides an unprecedented description of the conformations that a substrate assumes while exploring its chaperone-associated folding landscape.	DISCUSS
15	24	chaperone	protein_type	This substrate-chaperone ensemble helps accomplish the longstanding goal of obtaining a detailed view of how a chaperone aids protein folding.	DISCUSS
111	120	chaperone	protein_type	This substrate-chaperone ensemble helps accomplish the longstanding goal of obtaining a detailed view of how a chaperone aids protein folding.	DISCUSS
24	27	Im7	protein	We recently showed that Im7 can fold while remaining continuously bound to Spy.	DISCUSS
53	74	continuously bound to	protein_state	We recently showed that Im7 can fold while remaining continuously bound to Spy.	DISCUSS
75	78	Spy	protein	We recently showed that Im7 can fold while remaining continuously bound to Spy.	DISCUSS
20	28	ensemble	evidence	The high-resolution ensemble obtained here now provides insight into exactly how this occurs.	DISCUSS
4	14	structures	evidence	The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy.	DISCUSS
22	30	ensemble	evidence	The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy.	DISCUSS
103	112	chaperone	protein_type	The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy.	DISCUSS
159	174	concave surface	site	The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy.	DISCUSS
178	181	Spy	protein	The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy.	DISCUSS
4	12	ensemble	evidence	The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone.	DISCUSS
39	42	Spy	protein	The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone.	DISCUSS
55	74	amphipathic surface	site	The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone.	DISCUSS
146	154	bound to	protein_state	The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone.	DISCUSS
159	168	chaperone	protein_type	The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone.	DISCUSS
88	98	chaperones	protein_type	This model is consistent with previous studies postulating that the flexible binding of chaperones allows for substrate protein folding.	DISCUSS
16	31	concave surface	site	The amphipathic concave surface of Spy likely facilitates this flexible binding and may be a crucial feature for Spy and potentially other chaperones, allowing them to bind multiple conformations of many different substrates.	DISCUSS
35	38	Spy	protein	The amphipathic concave surface of Spy likely facilitates this flexible binding and may be a crucial feature for Spy and potentially other chaperones, allowing them to bind multiple conformations of many different substrates.	DISCUSS
113	116	Spy	protein	The amphipathic concave surface of Spy likely facilitates this flexible binding and may be a crucial feature for Spy and potentially other chaperones, allowing them to bind multiple conformations of many different substrates.	DISCUSS
139	149	chaperones	protein_type	The amphipathic concave surface of Spy likely facilitates this flexible binding and may be a crucial feature for Spy and potentially other chaperones, allowing them to bind multiple conformations of many different substrates.	DISCUSS
15	18	Spy	protein	In contrast to Spy’s binding hotspots, Im76-45 displays substantially less specificity in its binding sites.	DISCUSS
21	37	binding hotspots	site	In contrast to Spy’s binding hotspots, Im76-45 displays substantially less specificity in its binding sites.	DISCUSS
39	46	Im76-45	mutant	In contrast to Spy’s binding hotspots, Im76-45 displays substantially less specificity in its binding sites.	DISCUSS
94	107	binding sites	site	In contrast to Spy’s binding hotspots, Im76-45 displays substantially less specificity in its binding sites.	DISCUSS
11	18	Im76-45	mutant	Nearly all Im76-45 residues come in contact with Spy.	DISCUSS
49	52	Spy	protein	Nearly all Im76-45 residues come in contact with Spy.	DISCUSS
0	8	Unfolded	protein_state	Unfolded substrate conformers interact with Spy through both hydrophobic and hydrophilic interactions, whereas the binding of native-like states is mainly hydrophilic.	DISCUSS
44	47	Spy	protein	Unfolded substrate conformers interact with Spy through both hydrophobic and hydrophilic interactions, whereas the binding of native-like states is mainly hydrophilic.	DISCUSS
126	137	native-like	protein_state	Unfolded substrate conformers interact with Spy through both hydrophobic and hydrophilic interactions, whereas the binding of native-like states is mainly hydrophilic.	DISCUSS
54	69	ATP-independent	protein_state	This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate.	DISCUSS
70	79	chaperone	protein_type	This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate.	DISCUSS
88	96	unfolded	protein_state	This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate.	DISCUSS
211	230	hydrophobic regions	site	This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate.	DISCUSS
153	162	chaperone	protein_type	Once the substrate begins to fold within this protected environment, it progressively buries its own hydrophobic residues, and its interactions with the chaperone shift towards becoming more electrostatic.	DISCUSS
44	47	Spy	protein	Notably, the most frequent contacts between Spy and Im76-45 are charge-charge interactions.	DISCUSS
52	59	Im76-45	mutant	Notably, the most frequent contacts between Spy and Im76-45 are charge-charge interactions.	DISCUSS
23	26	Im7	protein	The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states.	DISCUSS
36	41	Glu21	residue_name_number	The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states.	DISCUSS
43	48	Asp32	residue_name_number	The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states.	DISCUSS
54	59	Asp35	residue_name_number	The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states.	DISCUSS
85	88	Im7	protein	The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states.	DISCUSS
116	119	Spy	protein	The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states.	DISCUSS
141	147	cradle	site	The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states.	DISCUSS
160	168	unfolded	protein_state	The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states.	DISCUSS
173	184	native-like	protein_state	The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states.	DISCUSS
9	14	Asp32	residue_name_number	Residues Asp32 and Asp35 are close to each other in the folded state of Im7.	DISCUSS
19	24	Asp35	residue_name_number	Residues Asp32 and Asp35 are close to each other in the folded state of Im7.	DISCUSS
56	62	folded	protein_state	Residues Asp32 and Asp35 are close to each other in the folded state of Im7.	DISCUSS
72	75	Im7	protein	Residues Asp32 and Asp35 are close to each other in the folded state of Im7.	DISCUSS
71	74	Im7	protein	This proximity likely causes electrostatic repulsion that destabilizes Im7’s native state.	DISCUSS
77	83	native	protein_state	This proximity likely causes electrostatic repulsion that destabilizes Im7’s native state.	DISCUSS
17	20	Spy	protein	Interaction with Spy’s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation.	DISCUSS
96	101	Asp32	residue_name_number	Interaction with Spy’s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation.	DISCUSS
106	111	Asp35	residue_name_number	Interaction with Spy’s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation.	DISCUSS
147	167	helical conformation	protein_state	Interaction with Spy’s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation.	DISCUSS
52	55	Spy	protein	As inter-molecular hydrophobic interactions between Spy and the substrate become progressively replaced by intra-molecular interactions within the substrate, the affinity between chaperone and substrates could decrease, eventually leading to release of the folded client protein.	DISCUSS
179	188	chaperone	protein_type	As inter-molecular hydrophobic interactions between Spy and the substrate become progressively replaced by intra-molecular interactions within the substrate, the affinity between chaperone and substrates could decrease, eventually leading to release of the folded client protein.	DISCUSS
257	263	folded	protein_state	As inter-molecular hydrophobic interactions between Spy and the substrate become progressively replaced by intra-molecular interactions within the substrate, the affinity between chaperone and substrates could decrease, eventually leading to release of the folded client protein.	DISCUSS
24	48	genetic selection system	experimental_method	Recently, we employed a genetic selection system to improve the chaperone activity of Spy.	DISCUSS
64	73	chaperone	protein_type	Recently, we employed a genetic selection system to improve the chaperone activity of Spy.	DISCUSS
86	89	Spy	protein	Recently, we employed a genetic selection system to improve the chaperone activity of Spy.	DISCUSS
34	37	Spy	protein	This selection resulted in “Super Spy” variants that were more effective at both preventing aggregation and promoting protein folding.	DISCUSS
39	47	variants	protein_state	This selection resulted in “Super Spy” variants that were more effective at both preventing aggregation and promoting protein folding.	DISCUSS
24	29	bound	protein_state	In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function.	DISCUSS
30	37	Im76-45	mutant	In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function.	DISCUSS
38	46	ensemble	evidence	In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function.	DISCUSS
125	134	chaperone	protein_type	In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function.	DISCUSS
42	45	Spy	protein	Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both.	DISCUSS
46	54	variants	protein_state	Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both.	DISCUSS
62	67	bound	protein_state	Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both.	DISCUSS
68	71	Im7	protein	Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both.	DISCUSS
85	87	WT	protein_state	Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both.	DISCUSS
88	91	Spy	protein	Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both.	DISCUSS
103	112	chaperone	protein_type	Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both.	DISCUSS
141	153	H/D exchange	experimental_method	Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both.	DISCUSS
4	12	ensemble	evidence	Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a).	DISCUSS
44	47	Spy	protein	Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a).	DISCUSS
48	57	mutations	protein_state	Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a).	DISCUSS
59	63	H96L	mutant	Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a).	DISCUSS
68	73	Q100L	mutant	Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a).	DISCUSS
92	117	chaperone contact surface	site	Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a).	DISCUSS
132	139	Im76-45	mutant	Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a).	DISCUSS
14	26	co-structure	evidence	Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker.	DISCUSS
45	49	L32P	mutant	Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker.	DISCUSS
80	83	Spy	protein	Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker.	DISCUSS
130	146	N-terminal helix	structure_element	Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker.	DISCUSS
189	199	disordered	protein_state	Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker.	DISCUSS
200	206	linker	structure_element	Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker.	DISCUSS
37	40	Spy	protein	This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a).	DISCUSS
51	61	structures	evidence	This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a).	DISCUSS
76	89	linker region	structure_element	This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a).	DISCUSS
128	131	apo	protein_state	This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a).	DISCUSS
41	54	linker region	structure_element	By sampling multiple conformations, this linker region may allow diverse substrate conformations to be accommodated.	DISCUSS
12	15	Spy	protein	Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding.	DISCUSS
16	25	mutations	protein_state	Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding.	DISCUSS
27	32	F115I	mutant	Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding.	DISCUSS
37	42	F115L	mutant	Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding.	DISCUSS
39	46	Im76-45	mutant	This residue does not directly contact Im76-45 in our READ-derived ensemble.	DISCUSS
54	58	READ	experimental_method	This residue does not directly contact Im76-45 in our READ-derived ensemble.	DISCUSS
67	75	ensemble	evidence	This residue does not directly contact Im76-45 in our READ-derived ensemble.	DISCUSS
14	17	Spy	protein	Instead, when Spy is bound to substrate, F115 engages in close CH⋯π hydrogen bonds with Tyr104 (Fig. 6b).	DISCUSS
21	29	bound to	protein_state	Instead, when Spy is bound to substrate, F115 engages in close CH⋯π hydrogen bonds with Tyr104 (Fig. 6b).	DISCUSS
41	45	F115	residue_name_number	Instead, when Spy is bound to substrate, F115 engages in close CH⋯π hydrogen bonds with Tyr104 (Fig. 6b).	DISCUSS
88	94	Tyr104	residue_name_number	Instead, when Spy is bound to substrate, F115 engages in close CH⋯π hydrogen bonds with Tyr104 (Fig. 6b).	DISCUSS
56	72	C-terminal helix	structure_element	This interaction presumably reduces the mobility of the C-terminal helix.	DISCUSS
4	9	F115I	mutant	The F115I/L substitutions would replace these hydrogen bonds with hydrophobic interactions that have little angular dependence.	DISCUSS
10	11	L	mutant	The F115I/L substitutions would replace these hydrogen bonds with hydrophobic interactions that have little angular dependence.	DISCUSS
51	59	flexible	protein_state	As a result, the C-terminus, and possibly also the flexible linker, is likely to become more flexible and thus more accommodating of different conformations of substrates.	DISCUSS
60	66	linker	structure_element	As a result, the C-terminus, and possibly also the flexible linker, is likely to become more flexible and thus more accommodating of different conformations of substrates.	DISCUSS
27	35	ensemble	evidence	Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions.	DISCUSS
49	52	Spy	protein	Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions.	DISCUSS
53	61	variants	protein_state	Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions.	DISCUSS
152	161	chaperone	protein_type	Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions.	DISCUSS
47	56	chaperone	protein_type	Despite extensive studies, exactly how complex chaperone machines help proteins fold remains controversial.	DISCUSS
29	38	chaperone	protein_type	Our study indicates that the chaperone Spy employs a simple surface binding approach that allows the substrate to explore various conformations and form transiently favorable interactions while being protected from aggregation.	DISCUSS
39	42	Spy	protein	Our study indicates that the chaperone Spy employs a simple surface binding approach that allows the substrate to explore various conformations and form transiently favorable interactions while being protected from aggregation.	DISCUSS
29	39	chaperones	protein_type	We speculate that many other chaperones could utilize a similar strategy.	DISCUSS
0	3	ATP	chemical	ATP and co-chaperone dependencies may have emerged later through evolution to better modulate and control chaperone action.	DISCUSS
11	20	chaperone	protein_type	ATP and co-chaperone dependencies may have emerged later through evolution to better modulate and control chaperone action.	DISCUSS
106	115	chaperone	protein_type	ATP and co-chaperone dependencies may have emerged later through evolution to better modulate and control chaperone action.	DISCUSS
29	38	chaperone	protein_type	In addition to insights into chaperone function, this work presents a new method for determining heterogeneous structural ensembles via a hybrid methodology of X-ray crystallography and computational modeling.	DISCUSS
160	181	X-ray crystallography	experimental_method	In addition to insights into chaperone function, this work presents a new method for determining heterogeneous structural ensembles via a hybrid methodology of X-ray crystallography and computational modeling.	DISCUSS
186	208	computational modeling	experimental_method	In addition to insights into chaperone function, this work presents a new method for determining heterogeneous structural ensembles via a hybrid methodology of X-ray crystallography and computational modeling.	DISCUSS
35	45	disordered	protein_state	Heterogeneous dynamic complexes or disordered regions of single proteins, once considered solely approachable by NMR spectroscopy, can now be visualized through X-ray crystallography.	DISCUSS
113	129	NMR spectroscopy	experimental_method	Heterogeneous dynamic complexes or disordered regions of single proteins, once considered solely approachable by NMR spectroscopy, can now be visualized through X-ray crystallography.	DISCUSS
161	182	X-ray crystallography	experimental_method	Heterogeneous dynamic complexes or disordered regions of single proteins, once considered solely approachable by NMR spectroscopy, can now be visualized through X-ray crystallography.	DISCUSS
50	67	2mFo−DFc omit map	evidence	Crystallographic data and ensemble selection. (a) 2mFo−DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 σ.	FIG
80	87	Im76-45	mutant	Crystallographic data and ensemble selection. (a) 2mFo−DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 σ.	FIG
92	107	flexible linker	structure_element	Crystallographic data and ensemble selection. (a) 2mFo−DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 σ.	FIG
108	124	electron density	evidence	Crystallographic data and ensemble selection. (a) 2mFo−DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 σ.	FIG
21	28	density	evidence	This is the residual density that is used in the READ selection.	FIG
49	53	READ	experimental_method	This is the residual density that is used in the READ selection.	FIG
18	24	iodine	chemical	(b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence.	FIG
49	66	anomalous signals	evidence	(b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence.	FIG
73	79	pI-Phe	chemical	(b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence.	FIG
80	93	substitutions	experimental_method	(b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence.	FIG
9	15	iodine	chemical	Multiple iodine positions were detected for most residues.	FIG
26	33	Im76-45	mutant	Agreement to the residual Im76-45 electron density (c) and anomalous iodine signals (d) for ensembles of varying size generated by randomly choosing from the MD pool (blue) and from the selection procedure (black).	FIG
34	50	electron density	evidence	Agreement to the residual Im76-45 electron density (c) and anomalous iodine signals (d) for ensembles of varying size generated by randomly choosing from the MD pool (blue) and from the selection procedure (black).	FIG
59	83	anomalous iodine signals	evidence	Agreement to the residual Im76-45 electron density (c) and anomalous iodine signals (d) for ensembles of varying size generated by randomly choosing from the MD pool (blue) and from the selection procedure (black).	FIG
158	160	MD	experimental_method	Agreement to the residual Im76-45 electron density (c) and anomalous iodine signals (d) for ensembles of varying size generated by randomly choosing from the MD pool (blue) and from the selection procedure (black).	FIG
4	17	cost function	evidence	The cost function, χ2, decreases as the agreement to the experimental data increases and is defined in the Online Methods.	FIG
19	21	χ2	evidence	The cost function, χ2, decreases as the agreement to the experimental data increases and is defined in the Online Methods.	FIG
17	21	READ	experimental_method	Flowchart of the READ sample-and-select process.	FIG
22	39	sample-and-select	experimental_method	Flowchart of the READ sample-and-select process.	FIG
0	11	Spy:Im76-45	complex_assembly	Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here.	FIG
34	38	RMSD	evidence	Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here.	FIG
42	48	native	protein_state	Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here.	FIG
58	65	Im76-45	mutant	Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here.	FIG
111	115	READ	experimental_method	Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here.	FIG
0	3	Spy	protein	Spy is depicted as a gray surface and the Im76-45 conformer is shown as orange balls.	FIG
42	49	Im76-45	mutant	Spy is depicted as a gray surface and the Im76-45 conformer is shown as orange balls.	FIG
52	56	READ	experimental_method	Atoms that were either not directly selected in the READ procedure, or whose position could not be justified based on agreement with the residual electron density were removed, leading to non-contiguous sections.	FIG
137	162	residual electron density	evidence	Atoms that were either not directly selected in the READ procedure, or whose position could not be justified based on agreement with the residual electron density were removed, leading to non-contiguous sections.	FIG
52	59	Im76-45	mutant	Dashed lines connect non-contiguous segments of the Im76-45 substrate.	FIG
16	19	Spy	protein	Residues of the Spy flexible linker region that fit the residual electron density are shown as larger gray spheres.	FIG
29	42	linker region	structure_element	Residues of the Spy flexible linker region that fit the residual electron density are shown as larger gray spheres.	FIG
56	81	residual electron density	evidence	Residues of the Spy flexible linker region that fit the residual electron density are shown as larger gray spheres.	FIG
40	44	RMSD	evidence	Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic.	FIG
70	76	native	protein_state	Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic.	FIG
86	93	Im76-45	mutant	Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic.	FIG
141	148	Im76-45	mutant	Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic.	FIG
153	156	Spy	protein	Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic.	FIG
0	12	Contact maps	evidence	Contact maps of Spy:Im76-45 complex.	FIG
16	27	Spy:Im76-45	complex_assembly	Contact maps of Spy:Im76-45 complex.	FIG
4	15	Spy:Im76-45	complex_assembly	(a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures.	FIG
16	27	contact map	evidence	(a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures.	FIG
47	52	bound	protein_state	(a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures.	FIG
53	56	Spy	protein	(a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures.	FIG
57	62	dimer	oligomeric_state	(a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures.	FIG
75	82	Im76-45	mutant	(a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures.	FIG
91	101	structures	evidence	(a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures.	FIG
13	20	Im76-45	mutant	For clarity, Im76-45 is represented with a single conformation.	FIG
51	68	contact frequency	evidence	The frequency plotted is calculated as the average contact frequency from Spy to every residue of Im76-45 and vice-versa.	FIG
74	77	Spy	protein	The frequency plotted is calculated as the average contact frequency from Spy to every residue of Im76-45 and vice-versa.	FIG
98	105	Im76-45	mutant	The frequency plotted is calculated as the average contact frequency from Spy to every residue of Im76-45 and vice-versa.	FIG
68	75	Im76-45	mutant	As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45.	FIG
88	91	Spy	protein	As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45.	FIG
97	108	contact map	evidence	As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45.	FIG
137	149	contact maps	evidence	As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45.	FIG
153	164	Spy:Im76-45	complex_assembly	As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45.	FIG
20	23	Spy	protein	Contacts to the two Spy monomers are depicted separately.	FIG
24	32	monomers	oligomeric_state	Contacts to the two Spy monomers are depicted separately.	FIG
14	22	flexible	protein_state	Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps.	FIG
23	36	linker region	structure_element	Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps.	FIG
40	43	Spy	protein	Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps.	FIG
54	59	47–57	residue_range	Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps.	FIG
90	102	contact maps	evidence	Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps.	FIG
0	3	Spy	protein	Spy conformation changes upon substrate binding.	FIG
4	11	Overlay	experimental_method	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
15	18	apo	protein_state	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
19	22	Spy	protein	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
48	53	bound	protein_state	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
54	57	Spy	protein	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
71	78	Overlay	experimental_method	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
82	84	WT	protein_state	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
85	88	Spy	protein	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
89	97	bound to	protein_state	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
98	105	Im76-45	mutant	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
115	119	H96L	mutant	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
120	123	Spy	protein	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
124	132	bound to	protein_state	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
133	136	Im7	protein	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
137	141	L18A	mutant	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
142	147	L19 A	mutant	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
147	151	L13A	mutant	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
160	164	H96L	mutant	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
165	168	Spy	protein	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
169	177	bound to	protein_state	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
178	180	WT	protein_state	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
181	184	Im7	protein	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
199	201	WT	protein_state	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
202	205	Spy	protein	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
206	214	bound to	protein_state	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
215	221	casein	chemical	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
236	253	Competition assay	experimental_method	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
262	269	Im76-45	mutant	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
284	287	Im7	protein	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
288	292	L18A	mutant	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
293	297	L19A	mutant	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
298	302	L37A	mutant	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
303	307	H40W	mutant	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
321	333	binding site	site	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
337	340	Spy	protein	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
350	378	substrate competition assays	experimental_method	(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).	FIG
15	18	Spy	protein	Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green).	FIG
19	32	linker region	structure_element	Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green).	FIG
53	56	Spy	protein	Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green).	FIG
74	77	Spy	protein	Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green).	FIG
78	91	linker region	structure_element	Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green).	FIG
132	135	apo	protein_state	Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green).	FIG
212	217	bound	protein_state	Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green).	FIG
4	8	F115	residue_name_number	(b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility.	FIG
13	16	L32	residue_name_number	(b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility.	FIG
24	27	Spy	protein	(b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility.	FIG
30	43	linker region	structure_element	(b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility.	FIG
51	57	cradle	site	(b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility.	FIG
70	73	Spy	protein	(b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility.	FIG
95	108	linker region	structure_element	(b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility.	FIG
10	13	Spy	protein	The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region.	FIG
22	27	F115L	mutant	The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region.	FIG
29	34	F115I	mutant	The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region.	FIG
40	44	L32P	mutant	The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region.	FIG
121	134	linker region	structure_element	The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region.	FIG
0	3	L32	residue_name_number	L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes.	FIG
5	9	F115	residue_name_number	L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes.	FIG
15	19	Y104	residue_name_number	L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes.	FIG
98	101	Spy	protein	L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes.	FIG
102	111	mutations	protein_state	L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes.	FIG