PMC 20201220 pmc.key 4937829 NO-CC CODE no 0 0 10.1038/nsmb.3237 NIHMS785109 4937829 27239796 691 7 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: 697 surname:Horowitz;given-names:Scott surname:Salmon;given-names:Loïc surname:Trievel;given-names:Raymond C. surname:Brooks;given-names:Charles L.;suffix:III surname:Bardwell;given-names:James CA surname:Koldewey;given-names:Philipp surname:Ahlstrom;given-names:Logan S. surname:Martin;given-names:Raoul surname:Quan;given-names:Shu surname:Afonine;given-names:Pavel V. surname:van den Bedem;given-names:Henry surname:Wang;given-names:Lili surname:Xu;given-names:Qingping TITLE front 23 2016 0 Visualizing chaperone-assisted protein folding protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone ABSTRACT abstract 47 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. 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. To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ). 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. This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy. 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. 0.98602355 evidence cleaner0 2023-06-29T11:53:47Z DUMMY: structures protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone protein_type MESH: cleaner0 2023-06-29T09:59:04Z chaperone protein_type MESH: cleaner0 2023-06-29T10:01:05Z chaperones 0.9951129 experimental_method cleaner0 2023-06-29T09:58:23Z MESH: X-ray crystallography experimental_method MESH: cleaner0 2023-06-29T09:52:13Z Residual Electron and Anomalous Density experimental_method MESH: cleaner0 2023-06-29T12:00:22Z READ 0.9784461 experimental_method cleaner0 2023-06-29T09:51:47Z MESH: READ 0.9859038 protein cleaner0 2023-06-29T09:56:57Z PR: immunity protein 7 0.9981862 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.8496159 protein_state cleaner0 2023-06-29T12:12:05Z DUMMY: in complex with 0.9949854 species cleaner0 2023-06-29T09:49:55Z MESH: E. coli 0.5430761 protein_type cleaner0 2023-06-29T09:57:10Z MESH: chaperone 0.99860436 protein cleaner0 2023-06-29T09:57:24Z PR: Spy 0.9972229 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.9905349 protein_state cleaner0 2023-06-29T12:12:11Z DUMMY: bound to 0.9983197 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9952988 protein_state cleaner0 2023-06-29T12:12:15Z DUMMY: Spy-associated 0.99446774 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.97574776 protein_state cleaner0 2023-06-29T12:12:18Z DUMMY: unfolded 0.7940124 protein_state cleaner0 2023-06-29T12:12:20Z DUMMY: folded 0.8012317 protein_state cleaner0 2023-06-29T12:12:23Z DUMMY: native 0.9822854 protein_state cleaner0 2023-06-29T12:12:25Z DUMMY: bound to protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone INTRO paragraph 1106 High-resolution structural models of protein-protein interactions are critical for obtaining mechanistic insights into biological processes. However, many protein-protein interactions are highly dynamic, making it difficult to obtain high-resolution data. Particularly challenging are interactions of intrinsically or conditionally disordered sections of proteins with their partner proteins. Recent advances in X-ray crystallography and NMR spectroscopy continue to improve our ability to analyze biomolecules that exist in multiple conformations. 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. NMR can theoretically be used to determine heterogeneous ensembles, but in practice, this proves to be very challenging. evidence DUMMY: cleaner0 2023-06-29T09:59:29Z structural models 0.83736765 protein_state cleaner0 2023-06-29T12:12:29Z DUMMY: highly dynamic 0.9667534 protein_state cleaner0 2023-06-29T12:12:32Z DUMMY: intrinsically or conditionally disordered 0.99611044 experimental_method cleaner0 2023-06-29T09:58:23Z MESH: X-ray crystallography 0.98872364 experimental_method cleaner0 2023-06-29T09:59:38Z MESH: NMR spectroscopy 0.99599904 experimental_method cleaner0 2023-06-29T09:58:23Z MESH: X-ray crystallography 0.99427587 experimental_method cleaner0 2023-06-29T09:59:43Z MESH: NMR INTRO paragraph 2020 Despite the importance of understanding how proteins fold into their native state within the cell, our knowledge about this critical process remains limited. It is clear that molecular chaperones aid in protein folding. However, exactly how they facilitate the folding process is still being debated. Structural characterization of chaperone-assisted protein folding likely would help bring clarity to this question. Structural models of chaperone-substrate complexes have recently begun to provide information as to how a chaperone can recognize its substrate. However, the impact that chaperones have on their substrates, and how these interactions affect the folding process remain largely unknown. 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. 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. protein_type MESH: cleaner0 2023-06-29T10:01:05Z chaperones protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.9913229 evidence cleaner0 2023-06-29T11:53:52Z DUMMY: Structural models protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone protein_type MESH: cleaner0 2023-06-29T10:01:05Z chaperones protein_type MESH: cleaner0 2023-06-29T10:01:05Z chaperones protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.9763981 protein_state cleaner0 2023-06-29T12:12:37Z DUMMY: highly dynamic protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone INTRO paragraph 3133 To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy. Spy prevents protein aggregation and aids in protein folding under various stress conditions, including treatment with tannin and butanol. 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. The crystal structure of Spy revealed that it forms a thin α-helical homodimeric cradle. Crosslinking and genetic experiments suggested that Spy interacts with substrates somewhere on its concave side. 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. 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. 0.99309033 protein_state cleaner0 2023-06-29T12:12:41Z DUMMY: ATP-independent 0.99527353 species cleaner0 2023-06-29T09:50:02Z MESH: Escherichia coli 0.88843155 protein_type cleaner0 2023-06-29T09:57:11Z MESH: chaperone 0.9985643 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9985098 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9877836 chemical cleaner0 2023-06-29T12:04:53Z CHEBI: tannin 0.9702978 chemical cleaner0 2023-06-29T12:04:57Z CHEBI: butanol 0.9984925 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9975439 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.9958406 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.99794847 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9975363 evidence cleaner0 2023-06-29T11:53:59Z DUMMY: crystal structure 0.9985667 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.7065689 oligomeric_state cleaner0 2023-06-29T12:18:00Z DUMMY: homodimeric 0.97890526 site cleaner0 2023-06-29T10:23:09Z SO: cradle 0.98890847 experimental_method cleaner0 2023-06-29T12:00:37Z MESH: Crosslinking and genetic experiments 0.9981775 protein cleaner0 2023-06-29T09:57:25Z PR: Spy experimental_method MESH: cleaner0 2023-06-29T09:58:23Z X-ray crystallography 0.9977272 evidence cleaner0 2023-06-29T11:54:04Z DUMMY: crystal structures 0.77756804 evidence cleaner0 2023-06-29T11:54:08Z DUMMY: ensemble 0.99241126 protein_state cleaner0 2023-06-29T12:12:45Z DUMMY: dynamic 0.9943983 complex_assembly cleaner0 2023-06-29T12:05:48Z GO: Spy:Im7 protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.99670714 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.9471477 protein_state cleaner0 2023-06-29T12:12:48Z DUMMY: bound to protein_type MESH: cleaner0 2023-06-29T10:01:04Z chaperones RESULTS title_1 4089 RESULTS RESULTS title_2 4097 Crystallizing the Spy:Im7 complex 0.97671187 experimental_method cleaner0 2023-06-29T12:00:41Z MESH: Crystallizing 0.990619 complex_assembly cleaner0 2023-06-29T12:05:55Z GO: Spy:Im7 RESULTS paragraph 4131 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. 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. We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals. Subsequent crystal washing and dissolution experiments confirmed the presence of the substrates in the co-crystals (Supplementary Fig. 2). The crystals diffracted to ~1.8 Å resolution. 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. 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. Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a). We hypothesized that the fragmented density was due to multiple, partially occupied conformations of the substrate bound within the crystal. Such residual density is typically not considered usable by traditional X-ray crystallography methods. Thus, we developed a new approach to interpret the chaperone-bound substrate in multiple conformations. 0.96328425 evidence cleaner0 2023-06-29T09:51:24Z DUMMY: crystals 0.9971807 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.5414726 experimental_method cleaner0 2023-06-29T10:01:47Z MESH: crystallization conditions 0.9924412 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.98916316 evidence cleaner0 2023-06-29T09:51:24Z DUMMY: crystals 0.8674296 protein_state cleaner0 2023-06-29T12:12:53Z DUMMY: presence of 0.6865464 protein_state cleaner0 2023-06-29T12:12:56Z DUMMY: absence 0.59963125 experimental_method cleaner0 2023-06-29T12:00:53Z MESH: screened 0.8132744 experimental_method cleaner0 2023-06-29T10:01:47Z MESH: crystallization conditions 0.99615026 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.47748753 protein_state cleaner0 2023-06-29T12:12:58Z DUMMY: unfolded 0.88614196 taxonomy_domain cleaner0 2023-06-29T09:50:27Z DUMMY: bovine 0.9140386 chemical cleaner0 2023-06-29T12:05:02Z CHEBI: α-casein 0.9974244 protein_state cleaner0 2023-06-29T12:13:00Z DUMMY: wild-type 0.99624205 protein_state cleaner0 2023-06-29T12:13:02Z DUMMY: WT 0.9939699 species cleaner0 2023-06-29T09:50:35Z MESH: E. coli 0.9985669 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.99383175 protein_state cleaner0 2023-06-29T12:13:05Z DUMMY: unfolded 0.9981888 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 mutant MESH: cleaner0 2023-06-29T10:03:28Z L18A mutant MESH: cleaner0 2023-06-29T10:03:39Z L19A mutant MESH: cleaner0 2023-06-29T10:03:48Z L37A 0.9933337 structure_element cleaner0 2023-06-29T12:18:23Z SO: N-terminal half 0.99848396 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.9150071 mutant cleaner0 2023-06-29T09:56:05Z MESH: Im76-45 structure_element SO: cleaner0 2023-06-29T10:14:38Z Spy-binding portion 0.9982128 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.98329085 experimental_method cleaner0 2023-06-29T12:00:58Z MESH: co-crystallized 0.71110064 protein_state cleaner0 2023-06-29T12:13:09Z DUMMY: with 0.9975012 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.99698585 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.8080074 protein_state cleaner0 2023-06-29T12:13:12Z DUMMY: alone 0.94558287 evidence cleaner0 2023-06-29T09:51:24Z DUMMY: crystals 0.8897016 experimental_method cleaner0 2023-06-29T12:01:05Z MESH: crystal washing and dissolution 0.8521347 experimental_method cleaner0 2023-06-29T12:01:09Z MESH: co-crystals 0.9779488 evidence cleaner0 2023-06-29T09:51:23Z DUMMY: crystals complex_assembly GO: cleaner0 2023-06-29T09:55:24Z Spy:Im76-45 0.9705391 chemical cleaner0 2023-06-29T10:05:46Z CHEBI: selenomethionine 0.98479927 evidence cleaner0 2023-06-29T09:51:24Z DUMMY: crystals 0.9901266 experimental_method cleaner0 2023-06-29T10:04:01Z MESH: single-wavelength anomalous diffraction 0.957922 experimental_method cleaner0 2023-06-29T10:04:05Z MESH: SAD 0.99743783 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.99567246 evidence cleaner0 2023-06-29T09:50:58Z DUMMY: electron density structure_element SO: cleaner0 2023-06-29T10:14:56Z minimal binding portion 0.99820125 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 mutant MESH: cleaner0 2023-06-29T09:56:05Z Im76-45 0.9937921 evidence cleaner0 2023-06-29T09:50:59Z DUMMY: electron density 0.88117105 evidence cleaner0 2023-06-29T11:54:22Z DUMMY: density 0.6665854 evidence cleaner0 2023-06-29T09:51:13Z DUMMY: crystal 0.9937469 experimental_method cleaner0 2023-06-29T09:58:23Z MESH: X-ray crystallography 0.99554175 protein_state cleaner0 2023-06-29T12:13:15Z DUMMY: chaperone-bound RESULTS title_2 5875 READ: a strategy to visualize heterogeneous and dynamic biomolecules experimental_method MESH: cleaner0 2023-06-29T09:51:47Z READ RESULTS paragraph 5944 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. 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. (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. (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. (5) Finally, we validated the ensemble using multiple statistical tests. 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. These two forms of data are therefore complementary: by labeling individual residues, one can locate them to specific points in space. The electron density then allowed us to connect the labeled residues of the substrate by confining the protein chain within regions of detectable density. In this way, the two forms of data together were able to describe multiple conformations of the substrate within the crystal. 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. However, we believe that READ will prove generally applicable to visualizing heterogeneous and dynamic complexes that have previously escaped detailed structural analysis. 0.9949292 evidence cleaner0 2023-06-29T11:54:25Z DUMMY: structure protein PR: cleaner0 2023-06-29T09:57:25Z Spy 0.5212672 experimental_method cleaner0 2023-06-29T09:51:47Z MESH: READ 0.9872874 experimental_method cleaner0 2023-06-29T09:52:08Z MESH: Residual Electron and Anomalous Density protein PR: cleaner0 2023-06-29T09:57:25Z Spy 0.92912316 evidence cleaner0 2023-06-29T11:54:31Z DUMMY: co-crystal 0.9963336 evidence cleaner0 2023-06-29T11:54:37Z DUMMY: structure 0.6307563 protein_state cleaner0 2023-06-29T12:13:19Z DUMMY: folded 0.572075 structure_element cleaner0 2023-06-29T12:18:35Z SO: domain 0.9985532 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9903908 evidence cleaner0 2023-06-29T09:52:41Z DUMMY: residual electron density 0.97443974 protein_state cleaner0 2023-06-29T12:13:22Z DUMMY: dynamic 0.93532926 protein_state cleaner0 2023-06-29T12:13:25Z DUMMY: flexible 0.99564326 chemical cleaner0 2023-06-29T12:05:09Z CHEBI: iodine 0.9940871 evidence cleaner0 2023-06-29T09:51:30Z DUMMY: anomalous density 0.98526114 experimental_method cleaner0 2023-06-29T09:52:19Z MESH: molecular dynamics 0.9845114 experimental_method cleaner0 2023-06-29T09:52:24Z MESH: MD 0.76252395 experimental_method cleaner0 2023-06-29T10:19:31Z MESH: simulations 0.991301 protein_state cleaner0 2023-06-29T12:13:29Z DUMMY: dynamic 0.9059882 experimental_method cleaner0 2023-06-29T12:01:15Z MESH: sample-and-select algorithm evidence DUMMY: cleaner0 2023-06-29T11:55:18Z residual and anomalous density 0.97203445 protein_state cleaner0 2023-06-29T12:13:31Z DUMMY: flexible 0.9963092 chemical cleaner0 2023-06-29T12:05:09Z CHEBI: iodine 0.9915202 evidence cleaner0 2023-06-29T09:52:42Z DUMMY: residual electron density 0.76906615 protein_state cleaner0 2023-06-29T12:13:33Z DUMMY: flexible 0.99548495 evidence cleaner0 2023-06-29T09:50:59Z DUMMY: electron density 0.91406024 evidence cleaner0 2023-06-29T11:55:28Z DUMMY: density 0.99207217 evidence cleaner0 2023-06-29T09:51:14Z DUMMY: crystal 0.92841744 experimental_method cleaner0 2023-06-29T09:51:47Z MESH: READ structure_element SO: cleaner0 2023-06-29T10:16:14Z Spy-binding domain 0.9985055 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 mutant MESH: cleaner0 2023-06-29T09:56:05Z Im76-45 0.9920535 protein_state cleaner0 2023-06-29T12:13:35Z DUMMY: bound to 0.99868625 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.87583953 experimental_method cleaner0 2023-06-29T09:51:47Z MESH: READ RESULTS title_2 7950 Collecting READ data for the Spy:Im76-45 complex experimental_method MESH: cleaner0 2023-06-29T09:51:47Z READ 0.9432124 complex_assembly cleaner0 2023-06-29T09:55:24Z GO: Spy:Im76-45 RESULTS paragraph 7999 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). Moreover, binding experiments indicated that Im76-45 comprises the entire Spy-binding region. To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe). 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. 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). 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. Consistent with the fragmented density, however, we observed multiple iodine positions for seven of the eight substituted residues. Together, these results indicated that the Im7 substrate binds Spy in multiple conformations. 0.6954702 experimental_method cleaner0 2023-06-29T12:01:19Z MESH: READ technique 0.90497404 protein_type cleaner0 2023-06-29T09:57:11Z MESH: chaperone 0.9988024 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.95279837 mutant cleaner0 2023-06-29T09:56:05Z MESH: Im76-45 0.9865262 experimental_method cleaner0 2023-06-29T12:01:23Z MESH: NMR 0.92164403 mutant cleaner0 2023-06-29T09:56:05Z MESH: Im76-45 0.9502464 protein_state cleaner0 2023-06-29T12:13:41Z DUMMY: unfolded 0.5735778 protein_state cleaner0 2023-06-29T12:13:43Z DUMMY: folded 0.9982692 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.9560363 experimental_method cleaner0 2023-06-29T12:01:26Z MESH: binding experiments mutant MESH: cleaner0 2023-06-29T09:56:05Z Im76-45 0.9808938 site cleaner0 2023-06-29T12:07:12Z SO: Spy-binding region 0.99265575 chemical cleaner0 2023-06-29T12:05:09Z CHEBI: iodine 0.99013966 experimental_method cleaner0 2023-06-29T12:01:31Z MESH: replaced mutant MESH: cleaner0 2023-06-29T09:56:05Z Im76-45 0.9939632 chemical cleaner0 2023-06-29T12:05:22Z CHEBI: 4-iodophenylalanine 0.99561006 chemical cleaner0 2023-06-29T10:18:17Z CHEBI: pI-Phe 0.82237387 evidence cleaner0 2023-06-29T09:53:13Z DUMMY: anomalous scattering mutant MESH: cleaner0 2023-06-29T09:56:05Z Im76-45 0.9948049 evidence cleaner0 2023-06-29T09:51:14Z DUMMY: crystal 0.9945221 experimental_method cleaner0 2023-06-29T12:01:36Z MESH: co-crystallized 0.9980811 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9786275 mutant cleaner0 2023-06-29T09:56:05Z MESH: Im76-45 0.91171074 chemical cleaner0 2023-06-29T10:18:17Z CHEBI: pI-Phe 0.44063374 experimental_method cleaner0 2023-06-29T12:01:40Z MESH: substitution 0.6336847 experimental_method cleaner0 2023-06-29T12:01:44Z MESH: collected 0.70439875 evidence cleaner0 2023-06-29T09:53:27Z DUMMY: anomalous data 0.99490327 complex_assembly cleaner0 2023-06-29T09:55:24Z GO: Spy:Im76-45 0.99621254 evidence cleaner0 2023-06-29T09:53:23Z DUMMY: electron density map 0.81312895 evidence cleaner0 2023-06-29T09:53:18Z DUMMY: anomalous signals 0.96556896 site cleaner0 2023-06-29T10:23:09Z SO: cradle 0.99820566 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.43931144 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.99788827 site cleaner0 2023-06-29T12:07:15Z SO: substrate binding site 0.8927793 evidence cleaner0 2023-06-29T11:55:34Z DUMMY: density 0.52766275 chemical cleaner0 2023-06-29T09:53:41Z CHEBI: iodine 0.9940791 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.99801326 protein cleaner0 2023-06-29T09:57:25Z PR: Spy RESULTS title_2 9410 READ sample-and-select procedure experimental_method MESH: cleaner0 2023-06-29T09:51:47Z READ 0.7578166 experimental_method cleaner0 2023-06-29T12:01:48Z MESH: sample-and-select RESULTS paragraph 9443 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. 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. This procedure iteratively constructs structural ensembles and then compares them to the experimental data. During each round of the selection, a genetic algorithm alters the ensemble and its agreement to the experimental data is re-evaluated. If successful, the selection identifies the smallest group of specific conformations that best fits the residual electron density and anomalous signals. The READ sample-and-select algorithm is diagrammed in Fig. 2. 0.99216205 mutant cleaner0 2023-06-29T09:55:51Z MESH: Im76-45 0.99342895 protein_state cleaner0 2023-06-29T12:13:45Z DUMMY: bound to 0.9970419 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9833829 evidence cleaner0 2023-06-29T09:52:42Z DUMMY: residual electron density 0.93260133 evidence cleaner0 2023-06-29T09:53:19Z DUMMY: anomalous signals chemical CHEBI: cleaner0 2023-06-29T10:18:17Z pI-Phe complex_assembly GO: cleaner0 2023-06-29T09:55:24Z Spy:Im76-45 protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.928301 experimental_method cleaner0 2023-06-29T12:01:52Z MESH: sample-and-select 0.9818716 experimental_method cleaner0 2023-06-29T09:59:39Z MESH: NMR spectroscopy 0.8969105 experimental_method cleaner0 2023-06-29T12:02:02Z MESH: genetic algorithm 0.8385226 evidence cleaner0 2023-06-29T09:52:42Z DUMMY: residual electron density 0.7360922 evidence cleaner0 2023-06-29T09:53:19Z DUMMY: anomalous signals experimental_method MESH: cleaner0 2023-06-29T09:51:47Z READ 0.93616086 experimental_method cleaner0 2023-06-29T12:02:05Z MESH: sample-and-select algorithm RESULTS paragraph 10274 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). 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). 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). Im76-45 binds and unbinds to Spy throughout the simulations. This strategy allows a wide range of substrate conformations to interact with the chaperone. From the MD simulations, we extracted ~10,000 diverse Spy:Im76-45 complexes to be used by the ensuing selection. Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate. 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. protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.88118553 experimental_method cleaner0 2023-06-29T12:02:08Z MESH: coarse-grained MD simulations 0.6608005 experimental_method cleaner0 2023-06-29T12:02:11Z MESH: pseudo-crystal environment 0.9891684 experimental_method cleaner0 2023-06-29T12:02:13Z MESH: coarse-grained simulations 0.9837824 complex_assembly cleaner0 2023-06-29T12:06:04Z GO: Spy-Im76-45 0.9687606 experimental_method cleaner0 2023-06-29T12:02:17Z MESH: binding simulations protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.9812668 mutant cleaner0 2023-06-29T09:56:05Z MESH: Im76-45 0.99846727 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9011532 experimental_method cleaner0 2023-06-29T10:19:30Z MESH: simulations protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.9936941 experimental_method cleaner0 2023-06-29T09:52:24Z MESH: MD 0.97938156 experimental_method cleaner0 2023-06-29T10:19:31Z MESH: simulations 0.99521255 complex_assembly cleaner0 2023-06-29T09:55:24Z GO: Spy:Im76-45 0.99863607 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9957451 oligomeric_state cleaner0 2023-06-29T12:10:19Z DUMMY: dimer 0.9443531 protein_state cleaner0 2023-06-29T12:13:49Z DUMMY: bound to 0.9847695 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9121185 evidence cleaner0 2023-06-29T11:56:15Z DUMMY: residual electron and anomalous crystallographic data RESULTS paragraph 11368 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. Simultaneously, it uses the residual electron density to help choose ensembles. 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. To accomplish this task, we generated a compressed version of the experimental 2mFo−DFc electron density map for use in the selection. This process provided us with a target map that the ensuing selection tried to recapitulate. 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. 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). 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. 0.5931571 evidence cleaner0 2023-06-29T09:53:14Z DUMMY: anomalous scattering chemical CHEBI: cleaner0 2023-06-29T10:18:17Z pI-Phe 0.9956863 chemical cleaner0 2023-06-29T12:05:09Z CHEBI: iodine 0.9964419 chemical cleaner0 2023-06-29T12:05:09Z CHEBI: iodine evidence DUMMY: cleaner0 2023-06-29T09:53:19Z anomalous signals evidence DUMMY: cleaner0 2023-06-29T09:52:42Z residual electron density 0.9872704 experimental_method cleaner0 2023-06-29T12:02:22Z MESH: electron density selection 0.98625016 evidence cleaner0 2023-06-29T09:50:59Z DUMMY: electron density evidence DUMMY: cleaner0 2023-06-29T10:20:35Z 2mFo−DFc electron density map 0.9862191 evidence cleaner0 2023-06-29T11:56:29Z DUMMY: map 0.9359912 evidence cleaner0 2023-06-29T11:56:31Z DUMMY: map 0.9649724 evidence cleaner0 2023-06-29T11:56:36Z DUMMY: density 0.98811775 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9733921 complex_assembly cleaner0 2023-06-29T09:55:24Z GO: Spy:Im76-45 0.9643926 experimental_method cleaner0 2023-06-29T09:52:24Z MESH: MD experimental_method MESH: cleaner0 2023-06-29T10:19:31Z simulations 0.54676366 experimental_method cleaner0 2023-06-29T12:02:51Z MESH: coarse-grained 0.5649066 experimental_method cleaner0 2023-06-29T09:52:24Z MESH: MD 0.99560875 evidence cleaner0 2023-06-29T09:50:59Z DUMMY: electron density 0.9872942 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9960478 evidence cleaner0 2023-06-29T09:53:23Z DUMMY: electron density map 0.9960303 evidence cleaner0 2023-06-29T10:21:36Z DUMMY: electron density maps 0.9947624 evidence cleaner0 2023-06-29T09:53:23Z DUMMY: electron density map RESULTS paragraph 12790 This approach allowed us to simultaneously use both the iodine anomalous signals and the residual electron density in the selection procedure. The selection resulted in small ensembles from the MD pool that best fit the READ data (Fig. 1c,d). 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). Combined, these validation tests confirmed that the selection procedure and selected six-member ensemble recapitulate the experimental data. 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. 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). 0.98970246 chemical cleaner0 2023-06-29T12:05:09Z CHEBI: iodine 0.83912575 evidence cleaner0 2023-06-29T09:53:19Z DUMMY: anomalous signals evidence DUMMY: cleaner0 2023-06-29T09:52:42Z residual electron density 0.49437568 experimental_method cleaner0 2023-06-29T09:52:24Z MESH: MD experimental_method MESH: cleaner0 2023-06-29T09:51:47Z READ 0.99502945 complex_assembly cleaner0 2023-06-29T09:55:24Z GO: Spy:Im76-45 0.99570507 evidence cleaner0 2023-06-29T11:56:51Z DUMMY: RFree experimental_method MESH: cleaner0 2023-06-29T10:22:23Z 10-fold cross-validation test 0.9134777 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.98730475 protein_state cleaner0 2023-06-29T12:14:07Z DUMMY: bound to 0.4701963 protein_type cleaner0 2023-06-29T09:57:11Z MESH: chaperone 0.99870205 protein cleaner0 2023-06-29T09:57:25Z PR: Spy RESULTS title_2 13698 Folding and interactions of Im7 while bound to Spy 0.99858224 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.9928488 protein_state cleaner0 2023-06-29T12:14:14Z DUMMY: bound to 0.98990685 protein cleaner0 2023-06-29T09:57:25Z PR: Spy RESULTS paragraph 13749 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. 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. 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. We found these conformations to be highly heterogeneous and to include unfolded, partially folded, and native-like states (Fig. 3). 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). experimental_method MESH: cleaner0 2023-06-29T09:51:47Z READ protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.9957247 evidence cleaner0 2023-06-29T11:57:00Z DUMMY: structures 0.99441284 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9951389 protein_state cleaner0 2023-06-29T12:14:10Z DUMMY: bound to 0.98687464 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.99474734 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.99560237 protein_state cleaner0 2023-06-29T12:14:18Z DUMMY: bound 0.9946273 protein_state cleaner0 2023-06-29T12:14:21Z DUMMY: unfolded 0.8663503 protein_state cleaner0 2023-06-29T12:14:23Z DUMMY: partially folded 0.9229308 protein_state cleaner0 2023-06-29T12:14:24Z DUMMY: native-like 0.9941633 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.8029603 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.92596716 site cleaner0 2023-06-29T10:23:09Z SO: cradle 0.93264157 site cleaner0 2023-06-29T10:23:09Z SO: cradle RESULTS paragraph 14640 We constructed a contact map of the complex, which shows the frequency of interactions for chaperone-substrate residue pairs (Fig. 4). 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). The Spy-contacting residues comprise a mixture of charged, polar, and hydrophobic residues. 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. This mixture suggests the importance of both electrostatic and hydrophobic components in binding the Im76-45 ensemble. With respect to the substrate, we observed that nearly every residue in Im76-45 is in contact with Spy (Fig. 4a). However, we did notice that despite this uniformity, regions of Im76-45 preferentially interact with different regions in Spy (Fig. 4b). 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. 0.99227893 evidence cleaner0 2023-06-29T11:57:04Z DUMMY: contact map protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.8847004 site cleaner0 2023-06-29T12:07:23Z SO: interaction sites 0.9974826 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.99912685 residue_name_number cleaner0 2023-06-29T12:10:42Z DUMMY: Arg122 0.99911207 residue_name_number cleaner0 2023-06-29T12:10:44Z DUMMY: Thr124 0.99908876 residue_name_number cleaner0 2023-06-29T12:10:47Z DUMMY: Phe29 protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.99911886 residue_name_number cleaner0 2023-06-29T12:10:49Z DUMMY: Arg61 0.9991086 residue_name_number cleaner0 2023-06-29T12:10:51Z DUMMY: Arg43 0.9991142 residue_name_number cleaner0 2023-06-29T12:10:54Z DUMMY: Lys47 0.99912184 residue_name_number cleaner0 2023-06-29T12:10:56Z DUMMY: His96 0.99911183 residue_name_number cleaner0 2023-06-29T12:10:59Z DUMMY: Met46 0.88955724 site cleaner0 2023-06-29T12:08:02Z SO: Spy-contacting residues 0.99504805 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.99063617 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.7740819 site cleaner0 2023-06-29T10:23:09Z SO: cradle 0.5764325 site cleaner0 2023-06-29T10:23:09Z SO: cradle 0.98738986 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9951992 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9969289 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9949441 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.997964 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.98398495 structure_element cleaner0 2023-06-29T12:18:39Z SO: N-terminal half 0.99496967 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.8808872 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.70677155 site cleaner0 2023-06-29T10:23:09Z SO: cradle 0.97302437 structure_element cleaner0 2023-06-29T12:18:42Z SO: C-terminal half 0.9961086 protein cleaner0 2023-06-29T09:57:25Z PR: Spy site SO: cleaner0 2023-06-29T12:08:44Z concave surface RESULTS paragraph 15807 Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly. 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). 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. The diversity of conformations and binding sites observed here emphasizes the dynamic and heterogeneous nature of the chaperone-substrate ensemble. 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. 0.9955931 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9971462 protein_state cleaner0 2023-06-29T12:14:29Z DUMMY: unfolded 0.99407166 protein_state cleaner0 2023-06-29T12:14:32Z DUMMY: native 0.99749655 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9956022 protein_state cleaner0 2023-06-29T12:14:35Z DUMMY: least-folded 0.9922649 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9970746 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9937761 protein_state cleaner0 2023-06-29T12:14:38Z DUMMY: most-folded 0.99426556 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.99790394 site cleaner0 2023-06-29T12:08:19Z SO: interaction sites 0.9976718 site cleaner0 2023-06-29T12:08:22Z SO: binding sites protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.99526626 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.981724 protein_state cleaner0 2023-06-29T12:14:41Z DUMMY: bound to protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone RESULTS title_2 16694 Spy changes conformation upon substrate binding 0.9074146 protein cleaner0 2023-06-29T09:57:25Z PR: Spy RESULTS paragraph 16742 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). Upon substrate binding, the Spy dimer twists 9° about its center relative to its apo form. This twist yields asymmetry and results in substantially different interaction patterns in the two Spy monomers (Fig. 4b). It is possible that this twist serves to increase heterogeneity in Spy by providing more binding poses. Additionally, we observed that the linker region (residues 47–57) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate. This increased disorder might explain how Spy is able to recognize and bind different substrates and/or differing conformations of the same substrate. Importantly, we observed the same structural changes in Spy regardless of which of the four substrates was bound (Fig. 5b, Table 1). 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. 0.99717087 evidence cleaner0 2023-06-29T11:57:09Z DUMMY: structure 0.9987859 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.99704045 protein_state cleaner0 2023-06-29T12:14:44Z DUMMY: substrate-bound 0.9966618 protein_state cleaner0 2023-06-29T12:14:47Z DUMMY: apo 0.9987929 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9964521 oligomeric_state cleaner0 2023-06-29T12:10:23Z DUMMY: dimer 0.99871683 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9962998 oligomeric_state cleaner0 2023-06-29T12:10:26Z DUMMY: dimer 0.9975248 protein_state cleaner0 2023-06-29T12:14:49Z DUMMY: apo 0.99865746 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.99570364 oligomeric_state cleaner0 2023-06-29T12:10:28Z DUMMY: monomers 0.99836415 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.99685514 structure_element cleaner0 2023-06-29T12:18:49Z SO: linker region 0.97453624 residue_range cleaner0 2023-06-29T12:11:54Z DUMMY: 47–57 0.9987594 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.8976868 protein_state cleaner0 2023-06-29T12:14:53Z DUMMY: disordered 0.99830097 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.99859184 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.99523723 evidence cleaner0 2023-06-29T11:57:12Z DUMMY: RMSD 0.95354253 protein_state cleaner0 2023-06-29T12:14:55Z DUMMY: well-folded 0.9984742 protein cleaner0 2023-06-29T09:57:25Z PR: Spy protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.78872776 experimental_method cleaner0 2023-06-29T12:02:59Z MESH: competition experiments protein PR: cleaner0 2023-06-29T09:57:25Z Spy 0.9981789 site cleaner0 2023-06-29T12:08:29Z SO: Spy binding site DISCUSS title_1 18074 DISCUSSION DISCUSS paragraph 18085 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. As a substrate, we used Im76-45, the chaperone-interacting portion of the protein-folding model protein Im7. In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states. The ensemble provides an unprecedented description of the conformations that a substrate assumes while exploring its chaperone-associated folding landscape. This substrate-chaperone ensemble helps accomplish the longstanding goal of obtaining a detailed view of how a chaperone aids protein folding. protein_type MESH: cleaner0 2023-06-29T10:01:05Z chaperones 0.87664104 experimental_method cleaner0 2023-06-29T09:51:47Z MESH: READ 0.9228798 evidence cleaner0 2023-06-29T11:57:16Z DUMMY: conformational ensemble 0.88223135 protein_type cleaner0 2023-06-29T09:57:11Z MESH: chaperone 0.998847 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9225119 protein_state cleaner0 2023-06-29T12:15:00Z DUMMY: bound to substrate 0.914196 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.7164347 structure_element cleaner0 2023-06-29T12:18:53Z SO: chaperone-interacting portion 0.9980854 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.9964735 protein_state cleaner0 2023-06-29T12:15:03Z DUMMY: chaperone-bound 0.8293746 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.99512637 protein_state cleaner0 2023-06-29T12:15:05Z DUMMY: unfolded 0.96388763 protein_state cleaner0 2023-06-29T12:15:08Z DUMMY: folded 0.9537819 protein_state cleaner0 2023-06-29T12:15:10Z DUMMY: native protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone DISCUSS paragraph 18808 We recently showed that Im7 can fold while remaining continuously bound to Spy. The high-resolution ensemble obtained here now provides insight into exactly how this occurs. 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. 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. This model is consistent with previous studies postulating that the flexible binding of chaperones allows for substrate protein folding. 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. 0.99826306 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.9076788 protein_state cleaner0 2023-06-29T12:15:13Z DUMMY: continuously bound to 0.99854445 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9263354 evidence cleaner0 2023-06-29T11:57:54Z DUMMY: ensemble 0.996702 evidence cleaner0 2023-06-29T11:57:58Z DUMMY: structures 0.79658026 evidence cleaner0 2023-06-29T11:58:00Z DUMMY: ensemble protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.71427774 site cleaner0 2023-06-29T12:08:44Z SO: concave surface 0.9980413 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.61538374 evidence cleaner0 2023-06-29T11:58:03Z DUMMY: ensemble 0.9962405 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.96033084 site cleaner0 2023-06-29T12:08:54Z SO: amphipathic surface 0.98483825 protein_state cleaner0 2023-06-29T12:15:15Z DUMMY: bound to protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone protein_type MESH: cleaner0 2023-06-29T10:01:05Z chaperones 0.7650008 site cleaner0 2023-06-29T12:08:44Z SO: concave surface 0.9985177 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9986675 protein cleaner0 2023-06-29T09:57:25Z PR: Spy protein_type MESH: cleaner0 2023-06-29T10:01:05Z chaperones DISCUSS paragraph 19698 In contrast to Spy’s binding hotspots, Im76-45 displays substantially less specificity in its binding sites. Nearly all Im76-45 residues come in contact with Spy. Unfolded substrate conformers interact with Spy through both hydrophobic and hydrophilic interactions, whereas the binding of native-like states is mainly hydrophilic. 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. 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. Notably, the most frequent contacts between Spy and Im76-45 are charge-charge interactions. 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. Residues Asp32 and Asp35 are close to each other in the folded state of Im7. This proximity likely causes electrostatic repulsion that destabilizes Im7’s native state. Interaction with Spy’s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation. 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. 0.97845733 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.997659 site cleaner0 2023-06-29T12:08:58Z SO: binding hotspots mutant MESH: cleaner0 2023-06-29T09:56:06Z Im76-45 0.9979149 site cleaner0 2023-06-29T12:09:01Z SO: binding sites mutant MESH: cleaner0 2023-06-29T09:56:06Z Im76-45 0.99680966 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.99753666 protein_state cleaner0 2023-06-29T12:15:19Z DUMMY: Unfolded 0.99649507 protein cleaner0 2023-06-29T09:57:25Z PR: Spy bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:54Z hydrophobic and hydrophilic interactions 0.98912925 protein_state cleaner0 2023-06-29T12:15:21Z DUMMY: native-like 0.98963326 protein_state cleaner0 2023-06-29T12:15:35Z DUMMY: ATP-independent protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.9956173 protein_state cleaner0 2023-06-29T12:15:38Z DUMMY: unfolded bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:54Z hydrophobic interactions 0.7040446 site cleaner0 2023-06-29T12:09:05Z SO: hydrophobic regions protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.9965184 protein cleaner0 2023-06-29T09:57:25Z PR: Spy mutant MESH: cleaner0 2023-06-29T09:56:06Z Im76-45 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:54Z charge-charge interactions 0.99789584 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.9990938 residue_name_number cleaner0 2023-06-29T12:11:05Z DUMMY: Glu21 0.9990645 residue_name_number cleaner0 2023-06-29T12:11:08Z DUMMY: Asp32 0.9990693 residue_name_number cleaner0 2023-06-29T12:11:10Z DUMMY: Asp35 0.9972257 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.99615 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.9320808 site cleaner0 2023-06-29T10:23:09Z SO: cradle 0.9975612 protein_state cleaner0 2023-06-29T12:15:42Z DUMMY: unfolded 0.993737 protein_state cleaner0 2023-06-29T12:15:44Z DUMMY: native-like 0.9990429 residue_name_number cleaner0 2023-06-29T12:11:12Z DUMMY: Asp32 0.9990433 residue_name_number cleaner0 2023-06-29T12:11:16Z DUMMY: Asp35 0.9975516 protein_state cleaner0 2023-06-29T12:15:46Z DUMMY: folded 0.9979709 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.99788946 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.82028353 protein_state cleaner0 2023-06-29T12:15:48Z DUMMY: native 0.99727935 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.999037 residue_name_number cleaner0 2023-06-29T12:11:19Z DUMMY: Asp32 0.99904805 residue_name_number cleaner0 2023-06-29T12:11:21Z DUMMY: Asp35 0.7348036 protein_state cleaner0 2023-06-29T12:15:55Z DUMMY: helical conformation bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:54Z hydrophobic interactions 0.9968719 protein cleaner0 2023-06-29T09:57:25Z PR: Spy protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.7177229 protein_state cleaner0 2023-06-29T12:15:57Z DUMMY: folded DISCUSS paragraph 21394 Recently, we employed a genetic selection system to improve the chaperone activity of Spy. This selection resulted in “Super Spy” variants that were more effective at both preventing aggregation and promoting protein folding. In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function. 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. 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). 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. This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a). This expansion would increase the structural plasticity for substrate binding. By sampling multiple conformations, this linker region may allow diverse substrate conformations to be accommodated. 0.95396966 experimental_method cleaner0 2023-06-29T12:03:16Z MESH: genetic selection system protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.9985115 protein cleaner0 2023-06-29T09:57:25Z PR: Spy 0.27333757 protein cleaner0 2023-06-29T09:57:25Z PR: Spy protein_state DUMMY: cleaner0 2023-06-29T11:52:25Z variants 0.9951461 protein_state cleaner0 2023-06-29T12:16:04Z DUMMY: bound mutant MESH: cleaner0 2023-06-29T09:56:06Z Im76-45 0.9421 evidence cleaner0 2023-06-29T11:58:11Z DUMMY: ensemble protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.31445134 protein cleaner0 2023-06-29T09:57:26Z PR: Spy protein_state DUMMY: cleaner0 2023-06-29T11:52:24Z variants 0.9420128 protein_state cleaner0 2023-06-29T12:16:08Z DUMMY: bound 0.53971404 protein cleaner0 2023-06-29T09:57:02Z PR: Im7 0.99709225 protein_state cleaner0 2023-06-29T12:16:10Z DUMMY: WT 0.9983999 protein cleaner0 2023-06-29T09:57:26Z PR: Spy protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.9924574 experimental_method cleaner0 2023-06-29T12:03:23Z MESH: H/D exchange 0.76765364 evidence cleaner0 2023-06-29T12:03:40Z DUMMY: ensemble protein PR: cleaner0 2023-06-29T09:57:26Z Spy protein_state DUMMY: cleaner0 2023-06-29T11:52:57Z mutations 0.9960479 mutant cleaner0 2023-06-29T12:09:16Z MESH: H96L 0.996639 mutant cleaner0 2023-06-29T12:09:19Z MESH: Q100L 0.931379 site cleaner0 2023-06-29T11:51:25Z SO: chaperone contact surface 0.6865122 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9421161 evidence cleaner0 2023-06-29T11:58:15Z DUMMY: co-structure 0.99530506 mutant cleaner0 2023-06-29T12:09:22Z MESH: L32P 0.99812347 protein cleaner0 2023-06-29T09:57:26Z PR: Spy structure_element SO: cleaner0 2023-06-29T12:19:27Z N-terminal helix 0.82643086 protein_state cleaner0 2023-06-29T12:16:13Z DUMMY: disordered 0.994686 structure_element cleaner0 2023-06-29T12:19:03Z SO: linker protein PR: cleaner0 2023-06-29T09:57:26Z Spy 0.91852707 evidence cleaner0 2023-06-29T11:58:18Z DUMMY: structures 0.99480677 structure_element cleaner0 2023-06-29T12:19:05Z SO: linker region 0.99762577 protein_state cleaner0 2023-06-29T12:16:15Z DUMMY: apo 0.9925647 structure_element cleaner0 2023-06-29T12:19:08Z SO: linker region DISCUSS paragraph 22639 Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding. This residue does not directly contact Im76-45 in our READ-derived ensemble. Instead, when Spy is bound to substrate, F115 engages in close CH⋯π hydrogen bonds with Tyr104 (Fig. 6b). This interaction presumably reduces the mobility of the C-terminal helix. The F115I/L substitutions would replace these hydrogen bonds with hydrophobic interactions that have little angular dependence. 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. Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions. 0.5755169 protein cleaner0 2023-06-29T09:57:26Z PR: Spy protein_state DUMMY: cleaner0 2023-06-29T11:52:58Z mutations 0.9984133 mutant cleaner0 2023-06-29T12:09:24Z MESH: F115I 0.99841094 mutant cleaner0 2023-06-29T12:09:27Z MESH: F115L 0.95090145 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 experimental_method MESH: cleaner0 2023-06-29T09:51:47Z READ 0.9659466 evidence cleaner0 2023-06-29T11:58:21Z DUMMY: ensemble 0.99572515 protein cleaner0 2023-06-29T09:57:26Z PR: Spy 0.9169791 protein_state cleaner0 2023-06-29T12:16:20Z DUMMY: bound to 0.999062 residue_name_number cleaner0 2023-06-29T12:11:27Z DUMMY: F115 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:54Z hydrogen bonds 0.9991068 residue_name_number cleaner0 2023-06-29T12:11:30Z DUMMY: Tyr104 0.9650637 structure_element cleaner0 2023-06-29T12:19:12Z SO: C-terminal helix 0.99830073 mutant cleaner0 2023-06-29T12:09:31Z MESH: F115I 0.95239127 mutant cleaner0 2023-06-29T12:09:35Z MESH: L bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:54Z hydrogen bonds bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:54Z hydrophobic interactions 0.8771667 protein_state cleaner0 2023-06-29T12:16:23Z DUMMY: flexible 0.98513585 structure_element cleaner0 2023-06-29T12:19:32Z SO: linker 0.9851317 evidence cleaner0 2023-06-29T11:58:24Z DUMMY: ensemble 0.28606677 protein cleaner0 2023-06-29T09:57:26Z PR: Spy protein_state DUMMY: cleaner0 2023-06-29T11:52:25Z variants protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone DISCUSS paragraph 23493 Despite extensive studies, exactly how complex chaperone machines help proteins fold remains controversial. 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. We speculate that many other chaperones could utilize a similar strategy. ATP and co-chaperone dependencies may have emerged later through evolution to better modulate and control chaperone action. protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.9588935 protein_type cleaner0 2023-06-29T09:57:11Z MESH: chaperone 0.9988973 protein cleaner0 2023-06-29T09:57:26Z PR: Spy protein_type MESH: cleaner0 2023-06-29T10:01:05Z chaperones 0.9926553 chemical cleaner0 2023-06-29T12:05:31Z CHEBI: ATP protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone DISCUSS paragraph 24027 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. Heterogeneous dynamic complexes or disordered regions of single proteins, once considered solely approachable by NMR spectroscopy, can now be visualized through X-ray crystallography. Consequently, this technique could enable structural characterization of many important dynamic and heterogeneous biomolecular systems. protein_type MESH: cleaner0 2023-06-29T09:57:11Z chaperone 0.9962828 experimental_method cleaner0 2023-06-29T09:58:23Z MESH: X-ray crystallography 0.9899347 experimental_method cleaner0 2023-06-29T12:03:45Z MESH: computational modeling 0.93940705 protein_state cleaner0 2023-06-29T12:16:27Z DUMMY: disordered 0.98850334 experimental_method cleaner0 2023-06-29T09:59:39Z MESH: NMR spectroscopy 0.9961585 experimental_method cleaner0 2023-06-29T09:58:23Z MESH: X-ray crystallography METHODS title_1 24557 ONLINE METHODS METHODS paragraph 24572 For computational methods, including simulations of Spy-substrate interactions, binning the residual Im7 electron density, ensemble selection, validation tests, and contact map generation, please see Supplementary Note 1. METHODS title_2 24794 Spy truncation mutants’ construction and in vitro and in vivo activity measurements METHODS paragraph 24880 To facilitate crystallization, we used Spy 29-124, a truncated Spy version that removes the unstructured N- and C-terminal tails (full length Spy is 138 amino acids). To determine if these alterations impact Spy’s chaperone activity in vitro, we performed in vitro chaperone activity assays and found that they had no significant effect; these deletions also had only a minor effect on Spy’s ability to stabilize Im7 in vivo (Supplementary Fig. 1). The in vitro activity of Spy 29-124 was assessed using the aldolase refolding assay as previously described. Briefly, in the denaturing step, 100 μM aldolase was denatured in buffer containing 6.6 M GdmCl, 40 mM HEPES pH 7.5, and 50 mM NaCl overnight at 22 °C (room temperature). In the refolding step, denatured aldolase was diluted to 3 μM in refolding buffer (40 mM HEPES, 150 mM NaCl, 5 mM DTT pH 7.5) in the presence of 6 μM WT Spy or Spy 29-124 (Spy:aldolase = 2:1). As a control, an identical experiment without Spy added was also performed. The refolding temperature was 37 °C with continuous shaking. The refolding status was monitored at different time points (1 min, 4 min, 10 min, and 20 min) and tested by diluting the refolding sample by 15-fold into the reaction buffer (0.15 mM NADH, 2 mM F1,6-DP, 1.8 U/ml GDH/TPI, 40 mM HEPES, and 150 mM NaCl pH 7.5) at 28 °C. The absorbance was monitored for 1.5 min at 340 nm. The percentage refolding was calculated and averaged over three repeats. METHODS paragraph 26343 To determine the in vivo activity of the Spy mutants, the quantity of the unstable Im7 variant L53A I54A expressed in the periplasm was compared during Spy variant co-expression as previously described. Plasmid Spy (pTrc-spy) was used as the template for the construction of the variant plasmids of Spy for in vivo chaperone activity measurement (Supplementary Table 3). To use the native signal sequence of spy for the periplasmic export of the Spy variants, an NheI site was first introduced between the signal sequence and the mature protein coding region of Spy. The vector was then digested with NheI and BamHI, purified, and ligated with the linear fragments corresponding to truncated sequences (21–130, 24–130, 27–130, 30–130, and 33–130) of Spy. METHODS paragraph 27108 Cells containing a strain that expressed the unstable Im7 mutant IL53A I54A (pCDFTrc-ssIm7L53A I54A) were transformed with plasmids that expressed either WT or one of the five truncated Spy mutants and grown to mid-log phase in LB medium at 37 °C. Im7 L53A I54A and Spy expression were induced with various concentrations of IPTG for 2 h to compare the in vivo chaperone activity of WT Spy and the truncated Spy mutants at similar expression levels. Periplasmic fractions were prepared as previously described and were separated on 16% Tricine gel (Life Technologies Inc.). The bands corresponding to Spy and the C-terminal His-tagged Im7 were either directly visualized on Coomassie stained gels or determined by western blot using anti-His antibody (Abcam ab1187; validation provided on manufacturer’s website). METHODS title_2 27925 Protein expression and purification METHODS paragraph 27961 The gene for spy 29-124 was amplified from plasmid pET28sumo-spy with primer 1 (5′-CGC GGG ATC CTT CAA AGA CCT GAA CCT GAC CG-3′) and primer 2 (5′-CGC GCT CGA GTT ATG TCA GAC GCT TCT CAA AAT TAG C-3′), and was cloned into pET28sumo via BamHI and XhoI sites. The H96L variant was made by Phusion site-directed mutagenesis (New England Biolabs). WT and H96L Spy 29-124 were expressed and purified as described previously with the exception that Ni-HisTrap columns (GE Healthcare) were utilized instead of the Ni-NTA beads and mini-chromatography column. ULP1 cleavage occurred following elution from the Ni-HisTrap column overnight at 4 °C while dialyzing to 40 mM Tris, 300 mM NaCl, pH 8.0. After dialysis, Spy was passed over the HisTrap column to remove the cleaved SUMO tag (20 mM imidazole was left over from the dialysis). Cleavage of the SUMO tag leaves a single serine in position 28 of Spy. The flow-through was then concentrated and diluted 5 times with 20 mM Tris, pH 8 for further purification on a HiTrap Q column. Spy has an isoelectric point of 9.5 and therefore was collected in the flow-through. The flow-through containing Spy was concentrated and diluted 5-fold with 50 mM sodium phosphate at pH 6.5 and passed over a HiTrap SP column. Spy was then eluted with a gradient from 0 M to 1 M NaCl. Re-buffering to the final reaction buffer was accomplished by gel filtration, passing the pooled and concentrated fractions containing Spy over a HiLoad 75 column in 40 mM HEPES, 100 mM NaCl, pH 7.5. Fractions containing Spy were then concentrated, frozen in liquid nitrogen, and stored at −80 °C. WT Im7, Im7 L18A L19A L37A H40W, and Im7 L18A L19A L37A were purified by the same protocol as Spy, but without the SP column step. In addition to WT Im7 and these various Im7 mutants, co-crystallization experiments extensively utilized Im76-45, a minimal Spy-binding segment that encompasses the first two helices of Im7 and contains 46% of the total Im7 sequence. It displays partial helicity when free in solution (Supplementary Fig. 3). The 6-45 portion of Im7 (H2N-SISDYTEAEFVQLLKEIEKENVAATDDVLD VLLEHFVKIT-OH), 4-iodophenlyalanine variants, and a peptide corresponding to a portion of bovine alpha casein S1 148-177 (Ac-ELFRQFYQLDAYPSGAWYYVPLGTQYTDAP-amide) were obtained from New England Peptide at ≥ 95% purity. Anomalous signals for residues E12, E14, L19, and E21 substitutions were determined using a peptide containing Im7 6–26, which was also obtained from New England Peptide at ≥ 95% purity. METHODS title_2 30494 Protein crystallization METHODS paragraph 30518 Co-crystals of WT Spy 29-124 and Spy H96L 29-124 in complex with Im7 variants and casein were grown by vapor diffusion. 25–130 mg/ml dimer Spy was incubated with various Im7 or casein substrates at concentrations ranging from equimolar to three-fold excess substrate in 22%–33% PEG 3000, 0.88–1.0 M imidazole pH 8.0, and 40–310 mM zinc acetate at 20 °C. Crystals were flash frozen in liquid nitrogen using 35% PEG 3000 as a cryo-protectant. It is worthwhile to note that the flash freezing could somewhat bias the conformations observed in the crystal structure. However, we chose to freeze the crystal to provide us with the maximum capability to identify and interpret the iodine anomalous signals. METHODS title_2 31228 Assessing presence of substrate in crystals METHODS paragraph 31272 Crystals were washed by sequential transfer between three to six 2 μl drops of mother liquor, incubating in each wash solution for 2–10 s in an effort to remove all surface bound and precipitated substrate protein before being dissolved for visualization by SDS-PAGE. Before loading, samples were boiled for 10 min in reducing loading buffer, and then loaded onto 16% Tricine gels. Wash samples and dissolved crystal samples were analyzed by Lumetein staining (Biotium) and Flamingo staining (Bio-Rad) per manufacturer’s instructions, and imaged using a FluorChem M Imager (ProteinSimple). METHODS title_2 31867 X-ray crystallography METHODS paragraph 31889 Data were collected at the LS-CAT beamlines at the Advanced Photon Source at 100 K. SeMet and native Spy:Im76-45 crystals were collected at 12.7 keV and 9.7 keV, respectively. Spy:Casein 148-177, and Spy H96L:WT Im7 crystals were collected also collected at 12.7 keV. Data integration and scaling were performed with iMosflm and AIMLESS, respectively. As molecular replacement attempts using the previously published apo Spy structures (PDB IDs: 3O39 and 3OEO) were unsuccessful, the Spy:Im76-45 complex was solved using Se-SAD phasing with SeMet-Spy, followed by density modification and initial model building by AutoSol in Phenix. The initial model was completed and refined using the native Spy:Im76-45 complex data. The rest of the structures were built using the native Spy:Im76-45 structure as a molecular replacement search model. Refinements, including TLS refinement, were performed using COOT and Phenix. All refined structures were validated using the Molprobity server, with Clashscores ranking better than the 90th percentile for all structures. Structural figures were rendered using PyMOL and UCSF Chimera, and movies generated using UCSF Chimera. Several partially occupied zinc atoms were observed in the crystal structure. Although some of these zinc atoms could also potentially modelled as water molecules, doing so resulted in an increase in the RFree. Additionally, a section of density near His A96 that is potentially partially occupied by a combination of water, Spy linker region, and possibly zinc, was modelled as containing water molecules. Spy H96L:Im76-45 was employed for iodine anomalous scattering experiments due to increased robustness and reproducibility of the crystals. METHODS paragraph 33599 The expected anomalous scatterers in the structures were S in methionine residues of Spy, Zn from the crystallization buffer, and I in the single pI-Phe residue of each synthetic Im76-45 peptide. Each I site is expected to be partially occupied as Im76-45 had diffuse density corresponding to multiple, partially occupied conformers; the Zn sites also may be partially occupied. To identify I, S, and Zn atomic positions using anomalous scattering, datasets were collected at 6.5 keV and 14.0 keV at 100 K using the ID-D beamline at LS-CAT. Anomalous difference maps for initial anomalous signal screening were calculated with phases from a molecular replacement search using the native Spy:Im76-45 (with no Im76-45 built in) complex as the search model. METHODS paragraph 34354 Anomalous difference maps calculated with the 14.0 keV data were used as controls to distinguish iodine from zinc atoms, as the iodine and zinc anomalous scattering factors are comparable at 14.0 keV, whereas at 6.5 keV, f″ is ~9-fold greater for iodine than for zinc. Anomalous differences were also collected and analyzed for a crystal of WT Spy 29-124:Im76-45 containing no iodine. The resulting anomalous difference map was inspected for peaks corresponding to sulfur, which were then excluded when selecting iodine peaks. Also, peaks that overlapped with Spy in the crystal lattice were excluded from analysis. METHODS paragraph 34972 As an initial screen for placing iodine atoms in the 6.5 keV anomalous difference maps, the median methionine sulfur signal was used as a cutoff for each individual map to control for varying data quality between crystals. Then, all anomalous atoms were refined in Phenix using anomalous group refinement. Refined B-factor of placed iodine ions was then used to estimate the positional fluctuation of the anomalous signals. This positional fluctuation was used as estimated error in the ensuing selection. A summary of all the anomalous signal heights (Supplementary Table 1) and anomalous difference maps (Supplemental Dataset 1) are displayed at varying contour levels for maximum clarity of iodine and methionine peak heights. METHODS title_2 35702 Substrate binding to Spy METHODS paragraph 35727 The dissociation constant of Im76-45 was determined via a fluorescence-based competition experiment with Im7 L18A L19A L37A H40W, and its ability to compete with casein 148-177 for Spy binding was tested. Im7 L18A L19A L37A H40W was chosen for competition experiments due to its tight binding (Supplementary Fig. 8) and substantial fluorescence change upon binding. This mutant binds to Spy tighter than Im7 L18A L19A L37A. 10 μM Spy 29-124 dimer was mixed with 10 μM Im7 L18A L19A L37A H40W or casein 148-177 to form a 1:1 complex in a buffer containing 40 mM HEPES pH 7.5 and 100 mM NaCl at 22 °C. Complex formation was monitored with a QuantaMaster 400 (Photon Technology International) using the tryptophan fluorescence of Im7 L18A L19A L37A H40W. Naturally tryptophan-free Im76-45 was then titrated into the complex to compete with Im7 L18A L19A L37A H40W for Spy binding. The observed fluorescence intensity at 350 nm was plotted as a function of the logarithm of the Im76-45 or casein 148-177 concentration. The data were fit for a one-site-binding competition model (OriginLab 9.1): where A1 and A2 are the maximum and minimum asymptotes, respectively, and x is the concentration of Im76-45. x0 is the apparent KD for Im76-45 based on its ability to compete with Im7 L18A L19A L37A H40W. Using the KD of Im7 L18A L19A L37A H40W binding to Spy 29-124, we then calculated the KD for Im76-45 binding to Spy 29-124 using the Cheng-Prusoff equation: where L is the concentration of Im7 L18A L19A L37A H40W and KD is the dissociation constant for Im7 L18A L19A L37A H40W binding to Spy. Due to interaction between higher oligomer states of Im76-45 and casein 148-177 (Supplementary Fig. 8), the competition curve was unable to be fit for casein 148-177 competing with Im76-45. METHODS paragraph 37513 The stoichiometry of binding of casein 148-177 and Spy was determined by tryptophan fluorescence of the casein upon Spy 29-124 addition. Increasing concentrations of Spy 29-124 were titrated to 20 μM of casein 148-177 in 40 mM HEPES (pH 7.5), 100 mM NaCl, at 22 °C. Complex formation was monitored with a QuantaMaster 400 (Photon Technology International) using the tryptophan fluorescence of casein 148-177. The observed fluorescence intensity at 339 nm was plotted as a function of the Spy 29-124 dimer concentration and fit with a quadratic equation using Origin 9.1 (OriginLab). METHODS paragraph 38098 To determine the dissociation constant, increasing concentrations of Spy 29-124 were titrated to 0.25 μM of casein in 40 mM HEPES (pH 7.5), 100 mM NaCl, at 22 °C. Complex formation was monitored with a QuantaMaster 400 (Photon Technology International) using the tryptophan fluorescence of casein 148-177. The observed fluorescence intensity at 339 nm was corrected for dilution due to the titration and then plotted as a function of the Spy 29-124 dimer concentration. The data were fit using a square hyperbola function in Origin 9.1 (OriginLab): where F is the recorded fluorescence signal, Fmax is the maximum fluorescence reached upon saturation of the complex, L is the concentration of free Spy in solution, KD is the dissociation constant, and C is a parameter for the offset. The calculated KD is an average of three independent repetitions. The measured dissociation constants for the different substrates ranged from 0.1 to 1 μM. METHODS title_2 39044 Isothermal titration calorimetry (ITC) METHODS paragraph 39083 Spy 29-124 and Im7-L18A L19A L37A H40W were dialyzed overnight against 40 mM HEPES, 100 mM NaCl, pH 7.5. 165 μM Spy dimer was loaded into a syringe and titrated into a cell containing 15 μM Im7 L18A L19 AL37A H40W at 25 °C in an iTC200 (Malvern Instruments) with an injection interval of 120 s and an initial delay time of 60 s. The solution was stirred at 1000 rpm, and the reference power was set to 6 μcal s−1 in high feedback mode. Data analysis was conducted using a plugin for Origin 7 (OriginLab), the software provided by the manufacturer. METHODS title_2 39637 Analytical ultracentrifugation METHODS paragraph 39668 Sedimentation velocity experiments for the Im76-45 and the bovine α-S1-casein peptide were performed using a Beckman Proteome Lab XL-I analytical ultracentrifuge (Beckman Coulter). Both peptides were first dialyzed against 40 mM HEPES, 100 mM NaCl, pH 7.5, then diluted to a concentration of 10 μM using the dialysis buffer. Samples were loaded into cells containing standard sector shaped 2-channel Epon centerpieces with 1.2 cm path-length (Beckman Coulter) and equilibrated to 22 °C for at least 1 h prior to sedimentation. All samples were spun at 48,000 rpm in a Beckman AN-50 Ti rotor, and the sedimentation of the protein was monitored continuously using interference optics, since the Im76-45 does not absorb strongly at 280 nm. Data analysis was conducted with SEDFIT (version 14.1), using the continuous c(s) distribution model. The confidence level for the maximum entropy (ME) regularization was set to 0.95. Buffer density and viscosity were calculated using SEDNTERP (http://sednterp.unh.edu/). SUPPL title_1 40682 Supplementary Material SUPPL footnote 40705 ACCESSION CODES SUPPL footnote 40721 Structures and datasets in this work have been deposited in the PDB under the IDs 5INA, 5IOG, 5IOE, and 5IOA. SUPPL footnote 40831 AUTHOR CONTRIBUTIONS SUPPL footnote 40852 Overall concept was conceived by S.H. and J.B. Experiments were designed by S.H., S.Q., J.B., R.T., H.B., and P.K. Experiments were performed by S.H., S.Q., P.K., R.M., and L.W. Analysis and computational modeling was designed by C.B., L.S., P.A., L.A., H.B., and S.H. Computational analysis was carried out by Q.X., S.H., L.S., L.A., P.A., P.K., and R.M. The manuscript was written primarily by S.H. and J.B., with assistance from L.S., L.A. and all other authors. 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(a) 2mFo−DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 σ. This is the residual density that is used in the READ selection. (b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence. Multiple iodine positions were detected for most residues. 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). The agreement from back-calculating a subset of data excluded from the selection procedure is shown by the red curve (cross-validation). The cost function, χ2, decreases as the agreement to the experimental data increases and is defined in the Online Methods. evidence DUMMY: cleaner0 2023-06-29T11:46:25Z 2mFo−DFc omit map 0.9824786 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.87016684 structure_element cleaner0 2023-06-29T12:19:36Z SO: flexible linker 0.7679258 evidence cleaner0 2023-06-29T09:50:59Z DUMMY: electron density 0.5860164 evidence cleaner0 2023-06-29T11:58:30Z DUMMY: density experimental_method MESH: cleaner0 2023-06-29T09:51:48Z READ chemical CHEBI: cleaner0 2023-06-29T12:05:10Z iodine evidence DUMMY: cleaner0 2023-06-29T09:53:19Z anomalous signals chemical CHEBI: cleaner0 2023-06-29T10:18:17Z pI-Phe 0.8892446 experimental_method cleaner0 2023-06-29T12:03:53Z MESH: substitutions chemical CHEBI: cleaner0 2023-06-29T12:05:10Z iodine 0.9806015 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9915528 evidence cleaner0 2023-06-29T09:50:59Z DUMMY: electron density evidence DUMMY: cleaner0 2023-06-29T11:59:03Z anomalous iodine signals 0.50510055 experimental_method cleaner0 2023-06-29T09:52:24Z MESH: MD 0.8905258 evidence cleaner0 2023-06-29T11:59:11Z DUMMY: cost function 0.970643 evidence cleaner0 2023-06-29T11:59:15Z DUMMY: χ2 nihms785109f2.jpg F2 FIG fig_caption 46008 Flowchart of the READ sample-and-select process. experimental_method MESH: cleaner0 2023-06-29T09:51:48Z READ 0.827946 experimental_method cleaner0 2023-06-29T12:04:08Z MESH: sample-and-select nihms785109f3.jpg F3 FIG fig_caption 46057 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. Spy is depicted as a gray surface and the Im76-45 conformer is shown as orange balls. 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. Dashed lines connect non-contiguous segments of the Im76-45 substrate. Residues of the Spy flexible linker region that fit the residual electron density are shown as larger gray spheres. 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. complex_assembly GO: cleaner0 2023-06-29T09:55:24Z Spy:Im76-45 0.9943816 evidence cleaner0 2023-06-29T11:59:19Z DUMMY: RMSD 0.57364076 protein_state cleaner0 2023-06-29T12:16:32Z DUMMY: native 0.9697895 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 experimental_method MESH: cleaner0 2023-06-29T09:51:48Z READ 0.99273866 protein cleaner0 2023-06-29T09:57:26Z PR: Spy 0.73834866 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 experimental_method MESH: cleaner0 2023-06-29T09:51:48Z READ evidence DUMMY: cleaner0 2023-06-29T09:52:42Z residual electron density 0.9689309 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9660642 protein cleaner0 2023-06-29T09:57:26Z PR: Spy 0.7810079 structure_element cleaner0 2023-06-29T12:19:40Z SO: linker region evidence DUMMY: cleaner0 2023-06-29T09:52:42Z residual electron density 0.99446154 evidence cleaner0 2023-06-29T11:59:23Z DUMMY: RMSD 0.7643502 protein_state cleaner0 2023-06-29T12:16:36Z DUMMY: native 0.9704957 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.95991987 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.963916 protein cleaner0 2023-06-29T09:57:26Z PR: Spy nihms785109f4.jpg F4 FIG fig_caption 46956 Contact maps of Spy:Im76-45 complex. (a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures. For clarity, Im76-45 is represented with a single conformation. The frequency plotted is calculated as the average contact frequency from Spy to every residue of Im76-45 and vice-versa. 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. Contacts to the two Spy monomers are depicted separately. Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps. 0.99464214 evidence cleaner0 2023-06-29T11:59:28Z DUMMY: Contact maps 0.9935497 complex_assembly cleaner0 2023-06-29T09:55:24Z GO: Spy:Im76-45 0.9907131 complex_assembly cleaner0 2023-06-29T09:55:24Z GO: Spy:Im76-45 0.994877 evidence cleaner0 2023-06-29T11:59:31Z DUMMY: contact map 0.99700975 protein_state cleaner0 2023-06-29T12:16:41Z DUMMY: bound 0.99881434 protein cleaner0 2023-06-29T09:57:26Z PR: Spy 0.9960406 oligomeric_state cleaner0 2023-06-29T12:10:33Z DUMMY: dimer 0.9831422 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9948394 evidence cleaner0 2023-06-29T11:59:34Z DUMMY: structures 0.98120135 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.990936 evidence cleaner0 2023-06-29T11:59:37Z DUMMY: contact frequency 0.99847955 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.99095154 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9880468 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9985318 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.9932604 evidence cleaner0 2023-06-29T11:59:41Z DUMMY: contact map 0.9946891 evidence cleaner0 2023-06-29T11:59:44Z DUMMY: contact maps 0.97827303 complex_assembly cleaner0 2023-06-29T09:55:24Z GO: Spy:Im76-45 0.9984988 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.99316674 oligomeric_state cleaner0 2023-06-29T12:10:36Z DUMMY: monomers 0.78499204 protein_state cleaner0 2023-06-29T12:16:43Z DUMMY: flexible 0.9960276 structure_element cleaner0 2023-06-29T12:19:44Z SO: linker region 0.99871933 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.9779859 residue_range cleaner0 2023-06-29T12:11:59Z DUMMY: 47–57 0.99175453 evidence cleaner0 2023-06-29T11:59:47Z DUMMY: contact maps nihms785109f5.jpg F5 FIG fig_caption 47612 Spy conformation changes upon substrate binding. (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). Error bars depict standard deviations of n=3 technical replicates. 0.9942 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.99299896 experimental_method cleaner0 2023-06-29T12:04:13Z MESH: Overlay 0.99786407 protein_state cleaner0 2023-06-29T12:16:48Z DUMMY: apo 0.99821895 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.99747664 protein_state cleaner0 2023-06-29T12:16:50Z DUMMY: bound 0.9980399 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.98867375 experimental_method cleaner0 2023-06-29T12:04:15Z MESH: Overlay 0.99659353 protein_state cleaner0 2023-06-29T12:16:53Z DUMMY: WT 0.9979012 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.988338 protein_state cleaner0 2023-06-29T12:16:55Z DUMMY: bound to 0.63883996 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9976901 mutant cleaner0 2023-06-29T12:09:41Z MESH: H96L 0.99283606 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.94561666 protein_state cleaner0 2023-06-29T12:16:57Z DUMMY: bound to 0.996292 protein cleaner0 2023-06-29T09:57:03Z PR: Im7 mutant MESH: cleaner0 2023-06-29T11:48:36Z L18A mutant MESH: cleaner0 2023-06-29T11:49:02Z L19 A mutant MESH: cleaner0 2023-06-29T11:49:14Z L13A 0.9974648 mutant cleaner0 2023-06-29T12:09:50Z MESH: H96L 0.99459994 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.9761287 protein_state cleaner0 2023-06-29T12:17:00Z DUMMY: bound to 0.99709237 protein_state cleaner0 2023-06-29T12:17:03Z DUMMY: WT 0.99832183 protein cleaner0 2023-06-29T09:57:03Z PR: Im7 0.9970504 protein_state cleaner0 2023-06-29T12:17:05Z DUMMY: WT 0.99810314 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.9752623 protein_state cleaner0 2023-06-29T12:17:08Z DUMMY: bound to 0.99773103 chemical cleaner0 2023-06-29T12:05:38Z CHEBI: casein 0.9873357 experimental_method cleaner0 2023-06-29T12:04:19Z MESH: Competition assay 0.6766577 mutant cleaner0 2023-06-29T09:56:06Z MESH: Im76-45 0.9897697 protein cleaner0 2023-06-29T09:57:03Z PR: Im7 mutant MESH: cleaner0 2023-06-29T11:49:29Z L18A mutant MESH: cleaner0 2023-06-29T11:49:38Z L19A mutant MESH: cleaner0 2023-06-29T11:49:46Z L37A mutant MESH: cleaner0 2023-06-29T11:49:57Z H40W 0.9944518 site cleaner0 2023-06-29T12:09:10Z SO: binding site 0.99822253 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.98650044 experimental_method cleaner0 2023-06-29T12:04:22Z MESH: substrate competition assays nihms785109f6.jpg F6 FIG fig_caption 48143 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). (b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility. The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region. 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. 0.59827995 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.9926208 structure_element cleaner0 2023-06-29T12:19:50Z SO: linker region 0.19467086 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.80332744 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.99444485 structure_element cleaner0 2023-06-29T12:19:52Z SO: linker region 0.99736905 protein_state cleaner0 2023-06-29T12:17:14Z DUMMY: apo 0.9945273 protein_state cleaner0 2023-06-29T12:17:17Z DUMMY: bound 0.9987766 residue_name_number cleaner0 2023-06-29T12:11:36Z DUMMY: F115 0.99887234 residue_name_number cleaner0 2023-06-29T12:11:39Z DUMMY: L32 0.99102753 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.99522245 structure_element cleaner0 2023-06-29T12:19:55Z SO: linker region 0.57751876 site cleaner0 2023-06-29T10:23:09Z SO: cradle protein PR: cleaner0 2023-06-29T09:57:27Z Spy 0.8755758 structure_element cleaner0 2023-06-29T12:19:57Z SO: linker region 0.21077903 protein cleaner0 2023-06-29T09:57:27Z PR: Spy 0.99865603 mutant cleaner0 2023-06-29T12:09:59Z MESH: F115L 0.9986588 mutant cleaner0 2023-06-29T12:10:02Z MESH: F115I 0.9984819 mutant cleaner0 2023-06-29T12:10:05Z MESH: L32P 0.9920492 structure_element cleaner0 2023-06-29T12:19:59Z SO: linker region 0.99873227 residue_name_number cleaner0 2023-06-29T12:11:42Z DUMMY: L32 0.9988135 residue_name_number cleaner0 2023-06-29T12:11:43Z DUMMY: F115 0.9988507 residue_name_number cleaner0 2023-06-29T12:11:46Z DUMMY: Y104 protein PR: cleaner0 2023-06-29T09:57:27Z Spy protein_state DUMMY: cleaner0 2023-06-29T11:52:58Z mutations bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:54Z hydrogen bonds T1.xml T1 TABLE table_caption 48804 Crystallography Statistics T1.xml T1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="void" rules="none"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1"/><th valign="top" align="left" rowspan="1" colspan="1">SeMet Spy:Im7_6-45</th><th valign="top" align="left" rowspan="1" colspan="1">Spy:Im7_6-45</th><th valign="top" align="left" rowspan="1" colspan="1">Spy:Casein 148-177, substrate not modeled</th><th valign="top" align="left" rowspan="1" colspan="1">Spy H96L:Im7 L18A L19A L37A, substrate not modeled</th><th valign="top" align="left" rowspan="1" colspan="1">Spy H96L:WT Im7, substrate not modeled</th></tr></thead><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1">PDB ID</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">5INA</td><td align="left" valign="top" rowspan="1" colspan="1">5IOG</td><td align="left" valign="top" rowspan="1" colspan="1">5IOE</td><td align="left" valign="top" rowspan="1" colspan="1">5IOA</td></tr><tr><td colspan="6" align="left" valign="top" rowspan="1"><bold>Data collection</bold></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Space group</td><td align="left" valign="top" rowspan="1" colspan="1">P4₁22</td><td align="left" valign="top" rowspan="1" colspan="1">P4₁22</td><td align="left" valign="top" rowspan="1" colspan="1">P4₁22</td><td align="left" valign="top" rowspan="1" colspan="1">P4₁22</td><td align="left" valign="top" rowspan="1" colspan="1">P4₁22</td></tr><tr><td colspan="6" align="left" valign="top" rowspan="1">Cell dimensions</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td align="left" valign="top" rowspan="1" colspan="1">42.9, 42.9, 259.3</td><td align="left" valign="top" rowspan="1" colspan="1">42.9, 42.9, 260.2</td><td align="left" valign="top" rowspan="1" colspan="1">43.0, 43.0, 258.2</td><td align="left" valign="top" rowspan="1" colspan="1">43.1, 43.1, 258.7</td><td align="left" valign="top" rowspan="1" colspan="1">43.1, 43.14, 260.2</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> <italic>α</italic>, <italic>β</italic>, <italic>γ</italic> (°)</td><td align="left" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="left" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="left" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="left" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="left" valign="top" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="left" valign="top" rowspan="1" colspan="1">64.82–2.44(2.53–2.44)</td><td align="left" valign="top" rowspan="1" colspan="1">30.50–1.79(1.83–1.79)</td><td align="left" valign="top" rowspan="1" colspan="1">36.88–1.77 (1.80–1.77)</td><td align="left" valign="top" rowspan="1" colspan="1">30.48–1.87(1.91–1.87)</td><td align="left" valign="top" rowspan="1" colspan="1">33.21–1.87(1.91–1.87)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><italic>R</italic>_merge (%)</td><td align="left" valign="top" rowspan="1" colspan="1">10.6(36)</td><td align="left" valign="top" rowspan="1" colspan="1">8.2(108)</td><td align="left" valign="top" rowspan="1" colspan="1">6.2(134)</td><td align="left" valign="top" rowspan="1" colspan="1">8.4(152)</td><td align="left" valign="top" rowspan="1" colspan="1">9.6(249)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><italic>I</italic>/σ(<italic>I)</italic></td><td align="left" valign="top" rowspan="1" colspan="1">15.1(6.8)</td><td align="left" valign="top" rowspan="1" colspan="1">7.0(1.1)</td><td align="left" valign="top" rowspan="1" colspan="1">15.3(1.6)</td><td align="left" valign="top" rowspan="1" colspan="1">13.8(1.8)</td><td align="left" valign="top" rowspan="1" colspan="1">13.2(1.3)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Completeness (%)</td><td align="left" valign="top" rowspan="1" colspan="1">100(100)</td><td align="left" valign="top" rowspan="1" colspan="1">94.0(90.1)</td><td align="left" valign="top" rowspan="1" colspan="1">99.9(99.5)</td><td align="left" valign="top" rowspan="1" colspan="1">100(100)</td><td align="left" valign="top" rowspan="1" colspan="1">96.8(93.1)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Redundancy</td><td align="left" valign="top" rowspan="1" colspan="1">15.6(15.6)</td><td align="left" valign="top" rowspan="1" colspan="1">4.3(4.2)</td><td align="left" valign="top" rowspan="1" colspan="1">8.7(8.2)</td><td align="left" valign="top" rowspan="1" colspan="1">9.6(9.4)</td><td align="left" valign="top" rowspan="1" colspan="1">8.2(8.2)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">CC1/2</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">0.998(0.689)</td><td align="left" valign="top" rowspan="1" colspan="1">0.999(0.745)</td><td align="left" valign="top" rowspan="1" colspan="1">0.999(0.676)</td><td align="left" valign="top" rowspan="1" colspan="1">0.998(0.606)</td></tr><tr><td colspan="6" align="left" valign="top" rowspan="1"><bold>Refinement</bold></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">1.79</td><td align="left" valign="top" rowspan="1" colspan="1">1.77</td><td align="left" valign="top" rowspan="1" colspan="1">1.87</td><td align="left" valign="top" rowspan="1" colspan="1">1.87</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">No. of Reflections</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">22583</td><td align="left" valign="top" rowspan="1" colspan="1">25052</td><td align="left" valign="top" rowspan="1" colspan="1">21505</td><td align="left" valign="top" rowspan="1" colspan="1">20838</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><italic>R</italic>_work/<italic>R</italic>_free</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">0.22/0.23</td><td align="left" valign="top" rowspan="1" colspan="1">0.21/0.24</td><td align="left" valign="top" rowspan="1" colspan="1">0.22/0.24</td><td align="left" valign="top" rowspan="1" colspan="1">0.21/0.25</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">No. of Atoms</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">1765</td><td align="left" valign="top" rowspan="1" colspan="1">1669</td><td align="left" valign="top" rowspan="1" colspan="1">1715</td><td align="left" valign="top" rowspan="1" colspan="1">1653</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Protein</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">1586</td><td align="left" valign="top" rowspan="1" colspan="1">1493</td><td align="left" valign="top" rowspan="1" colspan="1">1541</td><td align="left" valign="top" rowspan="1" colspan="1">1444</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Ligand/ion</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">30</td><td align="left" valign="top" rowspan="1" colspan="1">56</td><td align="left" valign="top" rowspan="1" colspan="1">60</td><td align="left" valign="top" rowspan="1" colspan="1">30</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Water</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">149</td><td align="left" valign="top" rowspan="1" colspan="1">120</td><td align="left" valign="top" rowspan="1" colspan="1">114</td><td align="left" valign="top" rowspan="1" colspan="1">179</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">B-factors</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">49.4</td><td align="left" valign="top" rowspan="1" colspan="1">48.5</td><td align="left" valign="top" rowspan="1" colspan="1">47.4</td><td align="left" valign="top" rowspan="1" colspan="1">39.2</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Protein</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">49.0</td><td align="left" valign="top" rowspan="1" colspan="1">47.5</td><td align="left" valign="top" rowspan="1" colspan="1">46.3</td><td align="left" valign="top" rowspan="1" colspan="1">38.3</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Ligand/ion</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">48.6</td><td align="left" valign="top" rowspan="1" colspan="1">65.9</td><td align="left" valign="top" rowspan="1" colspan="1">80.4</td><td align="left" valign="top" rowspan="1" colspan="1">62.9</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Water</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">54.2</td><td align="left" valign="top" rowspan="1" colspan="1">51.9</td><td align="left" valign="top" rowspan="1" colspan="1">44.5</td><td align="left" valign="top" rowspan="1" colspan="1">42.1</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">r.m.s. Deviations</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Bond lengths (Å)</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">0.013</td><td align="left" valign="top" rowspan="1" colspan="1">0.013</td><td align="left" valign="top" rowspan="1" colspan="1">0.013</td><td align="left" valign="top" rowspan="1" colspan="1">0.014</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Bond angles (º)</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">1.24</td><td align="left" valign="top" rowspan="1" colspan="1">1.30</td><td align="left" valign="top" rowspan="1" colspan="1">1.24</td><td align="left" valign="top" rowspan="1" colspan="1">1.39</td></tr></tbody></table> 48831 SeMet Spy:Im76-45 Spy:Im76-45 Spy:Casein 148-177, substrate not modeled Spy H96L:Im7 L18A L19A L37A, substrate not modeled Spy H96L:WT Im7, substrate not modeled PDB ID 5INA 5IOG 5IOE 5IOA Data collection Space group P4122 P4122 P4122 P4122 P4122 Cell dimensions  a, b, c (Å) 42.9, 42.9, 259.3 42.9, 42.9, 260.2 43.0, 43.0, 258.2 43.1, 43.1, 258.7 43.1, 43.14, 260.2  α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å) 64.82–2.44(2.53–2.44) 30.50–1.79(1.83–1.79) 36.88–1.77 (1.80–1.77) 30.48–1.87(1.91–1.87) 33.21–1.87(1.91–1.87) Rmerge (%) 10.6(36) 8.2(108) 6.2(134) 8.4(152) 9.6(249) I/σ(I) 15.1(6.8) 7.0(1.1) 15.3(1.6) 13.8(1.8) 13.2(1.3) Completeness (%) 100(100) 94.0(90.1) 99.9(99.5) 100(100) 96.8(93.1) Redundancy 15.6(15.6) 4.3(4.2) 8.7(8.2) 9.6(9.4) 8.2(8.2) CC1/2 0.998(0.689) 0.999(0.745) 0.999(0.676) 0.998(0.606) Refinement Resolution (Å) 1.79 1.77 1.87 1.87 No. of Reflections 22583 25052 21505 20838 Rwork/Rfree 0.22/0.23 0.21/0.24 0.22/0.24 0.21/0.25 No. of Atoms 1765 1669 1715 1653  Protein 1586 1493 1541 1444  Ligand/ion 30 56 60 30  Water 149 120 114 179 B-factors 49.4 48.5 47.4 39.2  Protein 49.0 47.5 46.3 38.3  Ligand/ion 48.6 65.9 80.4 62.9  Water 54.2 51.9 44.5 42.1 r.m.s. Deviations  Bond lengths (Å) 0.013 0.013 0.013 0.014  Bond angles (º) 1.24 1.30 1.24 1.39