| 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 | |
| 61 101 hydrophobic and hydrophilic interactions bond_interaction 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 | |
| 129 153 hydrophobic interactions bond_interaction 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 | |
| 64 90 charge-charge interactions bond_interaction 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 | |
| 19 43 hydrophobic interactions bond_interaction 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 | |
| 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 | |
| 68 82 hydrogen bonds bond_interaction 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 | |
| 46 60 hydrogen bonds bond_interaction The F115I/L substitutions would replace these hydrogen bonds with hydrophobic interactions that have little angular dependence. DISCUSS | |
| 66 90 hydrophobic interactions bond_interaction 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 | |
| 118 132 hydrogen bonds bond_interaction 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 | |