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Structure (v.20, #3)

In This Issue (pp. vii-ix).
In This Issue (pp. vii-ix).
In This Issue (pp. vii-ix).

UBAP1: A New ESCRT Member Joins the cl_Ub by Natasha Pashkova; Robert C. Piper (pp. 383-385).
The ESCRTs play multiple roles within the cell, including degradation of ubiquitinated membrane proteins by sorting them into multivesicular bodies (MVBs). Two recent studies provide structural and functional insights into how the newly identified ESCRT-I component UBAP1 dedicates ESCRT-I function for sorting ubiquitinated proteins at the MVB (Agromayor et al., 2012 [this issue of Structure]; Stefani et al., 2011).

UBAP1: A New ESCRT Member Joins the cl_Ub by Natasha Pashkova; Robert C. Piper (pp. 383-385).
The ESCRTs play multiple roles within the cell, including degradation of ubiquitinated membrane proteins by sorting them into multivesicular bodies (MVBs). Two recent studies provide structural and functional insights into how the newly identified ESCRT-I component UBAP1 dedicates ESCRT-I function for sorting ubiquitinated proteins at the MVB (Agromayor et al., 2012 [this issue of Structure]; Stefani et al., 2011).

UBAP1: A New ESCRT Member Joins the cl_Ub by Natasha Pashkova; Robert C. Piper (pp. 383-385).
The ESCRTs play multiple roles within the cell, including degradation of ubiquitinated membrane proteins by sorting them into multivesicular bodies (MVBs). Two recent studies provide structural and functional insights into how the newly identified ESCRT-I component UBAP1 dedicates ESCRT-I function for sorting ubiquitinated proteins at the MVB (Agromayor et al., 2012 [this issue of Structure]; Stefani et al., 2011).

Ob-Stopping Obesity, Metabolic and Immune-Mediated Disorders by Giuseppe Matarese; Veronica De Rosa (pp. 385-387).
Despite its physiological importance, no part of human leptin receptor (ObR) has been structurally characterized before. In this issue of Structure, Carpenter et al. report the crystal structure of the leptin-binding domain of human ObR in complex with the Fab fragment of an ObR-blocking monoclonal antibody (9F8 mAb).

Ob-Stopping Obesity, Metabolic and Immune-Mediated Disorders by Giuseppe Matarese; Veronica De Rosa (pp. 385-387).
Despite its physiological importance, no part of human leptin receptor (ObR) has been structurally characterized before. In this issue of Structure, Carpenter et al. report the crystal structure of the leptin-binding domain of human ObR in complex with the Fab fragment of an ObR-blocking monoclonal antibody (9F8 mAb).

Ob-Stopping Obesity, Metabolic and Immune-Mediated Disorders by Giuseppe Matarese; Veronica De Rosa (pp. 385-387).
Despite its physiological importance, no part of human leptin receptor (ObR) has been structurally characterized before. In this issue of Structure, Carpenter et al. report the crystal structure of the leptin-binding domain of human ObR in complex with the Fab fragment of an ObR-blocking monoclonal antibody (9F8 mAb).

The 19S Cap Puzzle: A New Jigsaw Piece by Eva M. Huber; Michael Groll (pp. 387-388).
After elucidation of the atomic details of 20S proteasomes, current research focuses on the regulatory 19S particle. In this issue of Structure, He et al. present the crystal structure of Rpn2 and use electron microscopy to examine differences between Rpn2 and Rpn1.

The 19S Cap Puzzle: A New Jigsaw Piece by Eva M. Huber; Michael Groll (pp. 387-388).
After elucidation of the atomic details of 20S proteasomes, current research focuses on the regulatory 19S particle. In this issue of Structure, He et al. present the crystal structure of Rpn2 and use electron microscopy to examine differences between Rpn2 and Rpn1.

The 19S Cap Puzzle: A New Jigsaw Piece by Eva M. Huber; Michael Groll (pp. 387-388).
After elucidation of the atomic details of 20S proteasomes, current research focuses on the regulatory 19S particle. In this issue of Structure, He et al. present the crystal structure of Rpn2 and use electron microscopy to examine differences between Rpn2 and Rpn1.

Cas Protein Cmr2 Full of Surprises by Raven H. Huang (pp. 389-390).
The Cmr complex carries out target RNA degradation in organisms possessing the CRISPR-Cas system. In this issue of Structure, Cocozaki et al. present the crystal structure of Cmr2, providing insight into the architecture of the Cmr complex.

Cas Protein Cmr2 Full of Surprises by Raven H. Huang (pp. 389-390).
The Cmr complex carries out target RNA degradation in organisms possessing the CRISPR-Cas system. In this issue of Structure, Cocozaki et al. present the crystal structure of Cmr2, providing insight into the architecture of the Cmr complex.

Cas Protein Cmr2 Full of Surprises by Raven H. Huang (pp. 389-390).
The Cmr complex carries out target RNA degradation in organisms possessing the CRISPR-Cas system. In this issue of Structure, Cocozaki et al. present the crystal structure of Cmr2, providing insight into the architecture of the Cmr complex.

The Protein Data Bank at 40: Reflecting on the Past to Prepare for the Future by Helen M. Berman; Gerard J. Kleywegt; Haruki Nakamura; John L. Markley (pp. 391-396).
A symposium celebrating the 40th anniversary of the Protein Data Bank archive (PDB), organized by the Worldwide Protein Data Bank, was held at Cold Spring Harbor Laboratory (CSHL) October 28–30, 2011. PDB40's distinguished speakers highlighted four decades of innovation in structural biology, from the early era of structural determination to future directions for the field.

The Protein Data Bank at 40: Reflecting on the Past to Prepare for the Future by Helen M. Berman; Gerard J. Kleywegt; Haruki Nakamura; John L. Markley (pp. 391-396).
A symposium celebrating the 40th anniversary of the Protein Data Bank archive (PDB), organized by the Worldwide Protein Data Bank, was held at Cold Spring Harbor Laboratory (CSHL) October 28–30, 2011. PDB40's distinguished speakers highlighted four decades of innovation in structural biology, from the early era of structural determination to future directions for the field.

The Protein Data Bank at 40: Reflecting on the Past to Prepare for the Future by Helen M. Berman; Gerard J. Kleywegt; Haruki Nakamura; John L. Markley (pp. 391-396).
A symposium celebrating the 40th anniversary of the Protein Data Bank archive (PDB), organized by the Worldwide Protein Data Bank, was held at Cold Spring Harbor Laboratory (CSHL) October 28–30, 2011. PDB40's distinguished speakers highlighted four decades of innovation in structural biology, from the early era of structural determination to future directions for the field.

Structural and Functional Discussion of the Tetra-Trico-Peptide Repeat, a Protein Interaction Module by Natalie Zeytuni; Raz Zarivach (pp. 397-405).
Tetra-trico-peptide repeat (TPR) domains are found in numerous proteins, where they serve as interaction modules and multiprotein complex mediators. TPRs can be found in all kingdoms of life and regulate diverse biological processes, such as organelle targeting and protein import, vesicle fusion, and biomineralization. This review considers the structural features of TPR domains that permit the great ligand-binding diversity of this motif, given that TPR-interacting partners display variations in both sequence and secondary structure. In addition, tools for predicting TPR-interacting partners are discussed, as are the abilities of TPR domains to serve as protein-protein interaction scaffolds in biotechnology and therapeutics.

Structural and Functional Discussion of the Tetra-Trico-Peptide Repeat, a Protein Interaction Module by Natalie Zeytuni; Raz Zarivach (pp. 397-405).
Tetra-trico-peptide repeat (TPR) domains are found in numerous proteins, where they serve as interaction modules and multiprotein complex mediators. TPRs can be found in all kingdoms of life and regulate diverse biological processes, such as organelle targeting and protein import, vesicle fusion, and biomineralization. This review considers the structural features of TPR domains that permit the great ligand-binding diversity of this motif, given that TPR-interacting partners display variations in both sequence and secondary structure. In addition, tools for predicting TPR-interacting partners are discussed, as are the abilities of TPR domains to serve as protein-protein interaction scaffolds in biotechnology and therapeutics.

Structural and Functional Discussion of the Tetra-Trico-Peptide Repeat, a Protein Interaction Module by Natalie Zeytuni; Raz Zarivach (pp. 397-405).
Tetra-trico-peptide repeat (TPR) domains are found in numerous proteins, where they serve as interaction modules and multiprotein complex mediators. TPRs can be found in all kingdoms of life and regulate diverse biological processes, such as organelle targeting and protein import, vesicle fusion, and biomineralization. This review considers the structural features of TPR domains that permit the great ligand-binding diversity of this motif, given that TPR-interacting partners display variations in both sequence and secondary structure. In addition, tools for predicting TPR-interacting partners are discussed, as are the abilities of TPR domains to serve as protein-protein interaction scaffolds in biotechnology and therapeutics.

Improved Visualization of Vertebrate Nuclear Pore Complexes by Field Emission Scanning Electron Microscopy by Lihi Shaulov; Amnon Harel (pp. 407-413).
Field emission scanning electron microscopy (FESEM) can provide high-resolution three-dimensional surface imaging of many biological structures, including nuclear envelopes and nuclear pore complexes (NPCs). For this purpose, it is important to preserve NPCs as close as possible to their native morphology, embedded in undamaged nuclear membranes. We present optimized methodologies for FESEM imaging in a cell-free reconstitution system and for the direct visualization of mammalian cell nuclei. The use of anchored chromatin templates in the cell-free system is particularly advantageous for imaging fragile intermediates inhibited at early stages of assembly. Our new method for exposing the surface of mammalian nuclei results in an unprecedented quality of NPC images, avoiding detergent-induced and physical damage. These new methodologies pave the way for the combined use of FESEM imaging with biochemical and genetic manipulation, in cell-free systems and in mammalian cells.

Improved Visualization of Vertebrate Nuclear Pore Complexes by Field Emission Scanning Electron Microscopy by Lihi Shaulov; Amnon Harel (pp. 407-413).
Field emission scanning electron microscopy (FESEM) can provide high-resolution three-dimensional surface imaging of many biological structures, including nuclear envelopes and nuclear pore complexes (NPCs). For this purpose, it is important to preserve NPCs as close as possible to their native morphology, embedded in undamaged nuclear membranes. We present optimized methodologies for FESEM imaging in a cell-free reconstitution system and for the direct visualization of mammalian cell nuclei. The use of anchored chromatin templates in the cell-free system is particularly advantageous for imaging fragile intermediates inhibited at early stages of assembly. Our new method for exposing the surface of mammalian nuclei results in an unprecedented quality of NPC images, avoiding detergent-induced and physical damage. These new methodologies pave the way for the combined use of FESEM imaging with biochemical and genetic manipulation, in cell-free systems and in mammalian cells.

Improved Visualization of Vertebrate Nuclear Pore Complexes by Field Emission Scanning Electron Microscopy by Lihi Shaulov; Amnon Harel (pp. 407-413).
Field emission scanning electron microscopy (FESEM) can provide high-resolution three-dimensional surface imaging of many biological structures, including nuclear envelopes and nuclear pore complexes (NPCs). For this purpose, it is important to preserve NPCs as close as possible to their native morphology, embedded in undamaged nuclear membranes. We present optimized methodologies for FESEM imaging in a cell-free reconstitution system and for the direct visualization of mammalian cell nuclei. The use of anchored chromatin templates in the cell-free system is particularly advantageous for imaging fragile intermediates inhibited at early stages of assembly. Our new method for exposing the surface of mammalian nuclei results in an unprecedented quality of NPC images, avoiding detergent-induced and physical damage. These new methodologies pave the way for the combined use of FESEM imaging with biochemical and genetic manipulation, in cell-free systems and in mammalian cells.

The UBAP1 Subunit of ESCRT-I Interacts with Ubiquitin via a SOUBA Domain by Monica Agromayor; Nicolas Soler; Anna Caballe; Tonya Kueck; Stefan M. Freund; Mark D. Allen; Mark Bycroft; Olga Perisic; Yu Ye; Bethan McDonald; Hartmut Scheel; Kay Hofmann; Stuart J.D. Neil; Juan Martin-Serrano; Roger L. Williams (pp. 414-428).
The endosomal sorting complexes required for transport (ESCRTs) facilitate endosomal sorting of ubiquitinated cargo, MVB biogenesis, late stages of cytokinesis, and retroviral budding. Here we show that ubiquitin associated protein 1 (UBAP1), a subunit of human ESCRT-I, coassembles in a stable 1:1:1:1 complex with Vps23/TSG101, VPS28, and VPS37. The X-ray crystal structure of the C-terminal region of UBAP1 reveals a domain that we describe as a solenoid of overlapping UBAs (SOUBA). NMR analysis shows that each of the three rigidly arranged overlapping UBAs making up the SOUBA interact with ubiquitin. We demonstrate that UBAP1-containing ESCRT-I is essential for degradation of antiviral cell-surface proteins, such as tetherin (BST-2/CD317), by viral countermeasures, namely, the HIV-1 accessory protein Vpu and the Kaposi sarcoma-associated herpesvirus (KSHV) ubiquitin ligase K5.Display Omitted► ESCRT-I subunit UBAP1 is essential for degradation of antiviral protein tetherin ► UBAP1 has a domain consisting of a solenoid of overlapping UBAs (SOUBA) ► Each of the three UBAs in the SOUBA binds monoubiquitin

The UBAP1 Subunit of ESCRT-I Interacts with Ubiquitin via a SOUBA Domain by Monica Agromayor; Nicolas Soler; Anna Caballe; Tonya Kueck; Stefan M. Freund; Mark D. Allen; Mark Bycroft; Olga Perisic; Yu Ye; Bethan McDonald; Hartmut Scheel; Kay Hofmann; Stuart J.D. Neil; Juan Martin-Serrano; Roger L. Williams (pp. 414-428).
The endosomal sorting complexes required for transport (ESCRTs) facilitate endosomal sorting of ubiquitinated cargo, MVB biogenesis, late stages of cytokinesis, and retroviral budding. Here we show that ubiquitin associated protein 1 (UBAP1), a subunit of human ESCRT-I, coassembles in a stable 1:1:1:1 complex with Vps23/TSG101, VPS28, and VPS37. The X-ray crystal structure of the C-terminal region of UBAP1 reveals a domain that we describe as a solenoid of overlapping UBAs (SOUBA). NMR analysis shows that each of the three rigidly arranged overlapping UBAs making up the SOUBA interact with ubiquitin. We demonstrate that UBAP1-containing ESCRT-I is essential for degradation of antiviral cell-surface proteins, such as tetherin (BST-2/CD317), by viral countermeasures, namely, the HIV-1 accessory protein Vpu and the Kaposi sarcoma-associated herpesvirus (KSHV) ubiquitin ligase K5.Display Omitted► ESCRT-I subunit UBAP1 is essential for degradation of antiviral protein tetherin ► UBAP1 has a domain consisting of a solenoid of overlapping UBAs (SOUBA) ► Each of the three UBAs in the SOUBA binds monoubiquitin

The UBAP1 Subunit of ESCRT-I Interacts with Ubiquitin via a SOUBA Domain by Monica Agromayor; Nicolas Soler; Anna Caballe; Tonya Kueck; Stefan M. Freund; Mark D. Allen; Mark Bycroft; Olga Perisic; Yu Ye; Bethan McDonald; Hartmut Scheel; Kay Hofmann; Stuart J.D. Neil; Juan Martin-Serrano; Roger L. Williams (pp. 414-428).
The endosomal sorting complexes required for transport (ESCRTs) facilitate endosomal sorting of ubiquitinated cargo, MVB biogenesis, late stages of cytokinesis, and retroviral budding. Here we show that ubiquitin associated protein 1 (UBAP1), a subunit of human ESCRT-I, coassembles in a stable 1:1:1:1 complex with Vps23/TSG101, VPS28, and VPS37. The X-ray crystal structure of the C-terminal region of UBAP1 reveals a domain that we describe as a solenoid of overlapping UBAs (SOUBA). NMR analysis shows that each of the three rigidly arranged overlapping UBAs making up the SOUBA interact with ubiquitin. We demonstrate that UBAP1-containing ESCRT-I is essential for degradation of antiviral cell-surface proteins, such as tetherin (BST-2/CD317), by viral countermeasures, namely, the HIV-1 accessory protein Vpu and the Kaposi sarcoma-associated herpesvirus (KSHV) ubiquitin ligase K5.Display Omitted► ESCRT-I subunit UBAP1 is essential for degradation of antiviral protein tetherin ► UBAP1 has a domain consisting of a solenoid of overlapping UBAs (SOUBA) ► Each of the three UBAs in the SOUBA binds monoubiquitin

Moonlighting by Different Stressors: Crystal Structure of the Chaperone Species of a 2-Cys Peroxiredoxin by Fulvio Saccoccia; Patrizio Di Micco; Giovanna Boumis; Maurizio Brunori; Ilias Koutris; Adriana E. Miele; Veronica Morea; Palita Sriratana; David L. Williams; Andrea Bellelli; Francesco Angelucci (pp. 429-439).
2-Cys peroxiredoxins (Prxs) play two different roles depending on the physiological status of the cell. They are thioredoxin-dependent peroxidases under low oxidative stress and ATP-independent chaperones upon exposure to high peroxide concentrations. These alternative functions have been associated with changes in the oligomerization state from low-(LMW) to high-molecular-weight (HMW) species. Here we present the structures of Schistosoma mansoni PrxI in both states: the LMW decamer and the HMW 20-mer formed by two stacked decamers. The latter is the structure of a 2-Cys Prx chaperonic form. Comparison of the structures sheds light on the mechanism by which chemical stressors, such as high H2O2 concentration and acidic pH, are sensed and translated into a functional switch in this protein family. We also propose a model to account for the in vivo formation of long filaments of stacked Prx rings.Display Omitted► The Schistosoma mansoni peroxiredoxin I (SmPrxI) is a moonlighting protein ► SmPrxI switches from LMW peroxidase to HMW chaperone due to chemical stressors ► The three-dimensional structures of both LMW and HMW SmPrxI have been solved ► SmPrxI HMW is the structure of a Prx chaperone

Moonlighting by Different Stressors: Crystal Structure of the Chaperone Species of a 2-Cys Peroxiredoxin by Fulvio Saccoccia; Patrizio Di Micco; Giovanna Boumis; Maurizio Brunori; Ilias Koutris; Adriana E. Miele; Veronica Morea; Palita Sriratana; David L. Williams; Andrea Bellelli; Francesco Angelucci (pp. 429-439).
2-Cys peroxiredoxins (Prxs) play two different roles depending on the physiological status of the cell. They are thioredoxin-dependent peroxidases under low oxidative stress and ATP-independent chaperones upon exposure to high peroxide concentrations. These alternative functions have been associated with changes in the oligomerization state from low-(LMW) to high-molecular-weight (HMW) species. Here we present the structures of Schistosoma mansoni PrxI in both states: the LMW decamer and the HMW 20-mer formed by two stacked decamers. The latter is the structure of a 2-Cys Prx chaperonic form. Comparison of the structures sheds light on the mechanism by which chemical stressors, such as high H2O2 concentration and acidic pH, are sensed and translated into a functional switch in this protein family. We also propose a model to account for the in vivo formation of long filaments of stacked Prx rings.Display Omitted► The Schistosoma mansoni peroxiredoxin I (SmPrxI) is a moonlighting protein ► SmPrxI switches from LMW peroxidase to HMW chaperone due to chemical stressors ► The three-dimensional structures of both LMW and HMW SmPrxI have been solved ► SmPrxI HMW is the structure of a Prx chaperone

Moonlighting by Different Stressors: Crystal Structure of the Chaperone Species of a 2-Cys Peroxiredoxin by Fulvio Saccoccia; Patrizio Di Micco; Giovanna Boumis; Maurizio Brunori; Ilias Koutris; Adriana E. Miele; Veronica Morea; Palita Sriratana; David L. Williams; Andrea Bellelli; Francesco Angelucci (pp. 429-439).
2-Cys peroxiredoxins (Prxs) play two different roles depending on the physiological status of the cell. They are thioredoxin-dependent peroxidases under low oxidative stress and ATP-independent chaperones upon exposure to high peroxide concentrations. These alternative functions have been associated with changes in the oligomerization state from low-(LMW) to high-molecular-weight (HMW) species. Here we present the structures of Schistosoma mansoni PrxI in both states: the LMW decamer and the HMW 20-mer formed by two stacked decamers. The latter is the structure of a 2-Cys Prx chaperonic form. Comparison of the structures sheds light on the mechanism by which chemical stressors, such as high H2O2 concentration and acidic pH, are sensed and translated into a functional switch in this protein family. We also propose a model to account for the in vivo formation of long filaments of stacked Prx rings.Display Omitted► The Schistosoma mansoni peroxiredoxin I (SmPrxI) is a moonlighting protein ► SmPrxI switches from LMW peroxidase to HMW chaperone due to chemical stressors ► The three-dimensional structures of both LMW and HMW SmPrxI have been solved ► SmPrxI HMW is the structure of a Prx chaperone

Mechanistic Insights into the Activation of Rad51-Mediated Strand Exchange from the Structure of a Recombination Activator, the Swi5-Sfr1 Complex by Naoyuki Kuwabara; Yasuto Murayama; Hiroshi Hashimoto; Yuuichi Kokabu; Mitsunori Ikeguchi; Mamoru Sato; Kouta Mayanagi; Yasuhiro Tsutsui; Hiroshi Iwasaki; Toshiyuki Shimizu (pp. 440-449).
Rad51 forms a helical filament on single-stranded DNA and promotes strand exchange between two homologous DNA molecules during homologous recombination. The Swi5-Sfr1 complex interacts directly with Rad51 and stimulates strand exchange. Here we describe structural and functional aspects of the complex. Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex with a parallel coiled-coil heterodimer joined firmly together via two previously uncharacterized leucine-zipper motifs and a bundle. The resultant coiled coil is sharply kinked, generating an elongated crescent-shaped structure suitable for transient binding within the helical groove of the Rad51 filament. The N-terminal region of Sfr1, meanwhile, has an interface for binding of Rad51. Our data suggest that the snug fit resulting from the complementary geometry of the heterodimer activates the Rad51 filament and that the N-terminal domain of Sfr1 plays a role in the efficient recruitment of the Swi5-Sfr1 complex to the Rad51 filaments.► Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex ► The crystal structure of the Swi5-Sfr1 complex reveals an unusual kinked structure ► The molecular shape is suited to a snug fit into the groove of the Rad51 filament ► The N-terminal region of Sfr1 has an interface for binding of Rad51

Mechanistic Insights into the Activation of Rad51-Mediated Strand Exchange from the Structure of a Recombination Activator, the Swi5-Sfr1 Complex by Naoyuki Kuwabara; Yasuto Murayama; Hiroshi Hashimoto; Yuuichi Kokabu; Mitsunori Ikeguchi; Mamoru Sato; Kouta Mayanagi; Yasuhiro Tsutsui; Hiroshi Iwasaki; Toshiyuki Shimizu (pp. 440-449).
Rad51 forms a helical filament on single-stranded DNA and promotes strand exchange between two homologous DNA molecules during homologous recombination. The Swi5-Sfr1 complex interacts directly with Rad51 and stimulates strand exchange. Here we describe structural and functional aspects of the complex. Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex with a parallel coiled-coil heterodimer joined firmly together via two previously uncharacterized leucine-zipper motifs and a bundle. The resultant coiled coil is sharply kinked, generating an elongated crescent-shaped structure suitable for transient binding within the helical groove of the Rad51 filament. The N-terminal region of Sfr1, meanwhile, has an interface for binding of Rad51. Our data suggest that the snug fit resulting from the complementary geometry of the heterodimer activates the Rad51 filament and that the N-terminal domain of Sfr1 plays a role in the efficient recruitment of the Swi5-Sfr1 complex to the Rad51 filaments.► Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex ► The crystal structure of the Swi5-Sfr1 complex reveals an unusual kinked structure ► The molecular shape is suited to a snug fit into the groove of the Rad51 filament ► The N-terminal region of Sfr1 has an interface for binding of Rad51

Mechanistic Insights into the Activation of Rad51-Mediated Strand Exchange from the Structure of a Recombination Activator, the Swi5-Sfr1 Complex by Naoyuki Kuwabara; Yasuto Murayama; Hiroshi Hashimoto; Yuuichi Kokabu; Mitsunori Ikeguchi; Mamoru Sato; Kouta Mayanagi; Yasuhiro Tsutsui; Hiroshi Iwasaki; Toshiyuki Shimizu (pp. 440-449).
Rad51 forms a helical filament on single-stranded DNA and promotes strand exchange between two homologous DNA molecules during homologous recombination. The Swi5-Sfr1 complex interacts directly with Rad51 and stimulates strand exchange. Here we describe structural and functional aspects of the complex. Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex with a parallel coiled-coil heterodimer joined firmly together via two previously uncharacterized leucine-zipper motifs and a bundle. The resultant coiled coil is sharply kinked, generating an elongated crescent-shaped structure suitable for transient binding within the helical groove of the Rad51 filament. The N-terminal region of Sfr1, meanwhile, has an interface for binding of Rad51. Our data suggest that the snug fit resulting from the complementary geometry of the heterodimer activates the Rad51 filament and that the N-terminal domain of Sfr1 plays a role in the efficient recruitment of the Swi5-Sfr1 complex to the Rad51 filaments.► Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex ► The crystal structure of the Swi5-Sfr1 complex reveals an unusual kinked structure ► The molecular shape is suited to a snug fit into the groove of the Rad51 filament ► The N-terminal region of Sfr1 has an interface for binding of Rad51

Constructing and Validating Initial Cα Models from Subnanometer Resolution Density Maps with Pathwalking by Mariah R. Baker; Ian Rees; Steven J. Ludtke; Wah Chiu; Matthew L. Baker (pp. 450-463).
A significant number of macromolecular structures solved by electron cryo-microscopy and X-ray crystallography obtain resolutions of 3.5–6Å, at which direct atomistic interpretation is difficult. To address this, we developed pathwalking, a semi-automated protocol to enumerate reasonable Cα models from near-atomic resolution density maps without a structural template or sequence-structure correspondence. Pathwalking uses an approach derived from the Traveling Salesman Problem to rapidly generate an ensemble of initial models for individual proteins, which can later be optimized to produce full atomic models. Pathwalking can also be used to validate and identify potential structural ambiguities in models generated from near-atomic resolution density maps. In this work, examples from the EMDB and PDB are used to assess the broad applicability and accuracy of our method. With the growing number of near-atomic resolution density maps from cryo-EM and X-ray crystallograph y, pathwalking can become an important tool in modeling protein structures.► Pathwalking uses Traveling Salesman Problem in protein folding ► Rapid creation of first-approach Cα-models directly from density maps ► Pathwalking generates models without requiring a sequence or template ► Pathwalking can be used for de novo model validation

Constructing and Validating Initial Cα Models from Subnanometer Resolution Density Maps with Pathwalking by Mariah R. Baker; Ian Rees; Steven J. Ludtke; Wah Chiu; Matthew L. Baker (pp. 450-463).
A significant number of macromolecular structures solved by electron cryo-microscopy and X-ray crystallography obtain resolutions of 3.5–6Å, at which direct atomistic interpretation is difficult. To address this, we developed pathwalking, a semi-automated protocol to enumerate reasonable Cα models from near-atomic resolution density maps without a structural template or sequence-structure correspondence. Pathwalking uses an approach derived from the Traveling Salesman Problem to rapidly generate an ensemble of initial models for individual proteins, which can later be optimized to produce full atomic models. Pathwalking can also be used to validate and identify potential structural ambiguities in models generated from near-atomic resolution density maps. In this work, examples from the EMDB and PDB are used to assess the broad applicability and accuracy of our method. With the growing number of near-atomic resolution density maps from cryo-EM and X-ray crystallograph y, pathwalking can become an important tool in modeling protein structures.► Pathwalking uses Traveling Salesman Problem in protein folding ► Rapid creation of first-approach Cα-models directly from density maps ► Pathwalking generates models without requiring a sequence or template ► Pathwalking can be used for de novo model validation

Constructing and Validating Initial Cα Models from Subnanometer Resolution Density Maps with Pathwalking by Mariah R. Baker; Ian Rees; Steven J. Ludtke; Wah Chiu; Matthew L. Baker (pp. 450-463).
A significant number of macromolecular structures solved by electron cryo-microscopy and X-ray crystallography obtain resolutions of 3.5–6Å, at which direct atomistic interpretation is difficult. To address this, we developed pathwalking, a semi-automated protocol to enumerate reasonable Cα models from near-atomic resolution density maps without a structural template or sequence-structure correspondence. Pathwalking uses an approach derived from the Traveling Salesman Problem to rapidly generate an ensemble of initial models for individual proteins, which can later be optimized to produce full atomic models. Pathwalking can also be used to validate and identify potential structural ambiguities in models generated from near-atomic resolution density maps. In this work, examples from the EMDB and PDB are used to assess the broad applicability and accuracy of our method. With the growing number of near-atomic resolution density maps from cryo-EM and X-ray crystallograph y, pathwalking can become an important tool in modeling protein structures.► Pathwalking uses Traveling Salesman Problem in protein folding ► Rapid creation of first-approach Cα-models directly from density maps ► Pathwalking generates models without requiring a sequence or template ► Pathwalking can be used for de novo model validation

EM-Fold: De Novo Atomic-Detail Protein Structure Determination from Medium-Resolution Density Maps by Steffen Lindert; Nathan Alexander; Nils Wötzel; Mert Karakaş; Phoebe L. Stewart; Jens Meiler (pp. 464-478).
Electron density maps of membrane proteins or large macromolecular complexes are frequently only determined at medium resolution between 4 Å and 10 Å, either by cryo-electron microscopy or X-ray crystallography. In these density maps, the general arrangement of secondary structure elements (SSEs) is revealed, whereas their directionality and connectivity remain elusive. We demonstrate that the topology of proteins with up to 250 amino acids can be determined from such density maps when combined with a computational protein folding protocol. Furthermore, we accurately reconstruct atomic detail in loop regions and amino acid side chains not visible in the experimental data. The EM-Fold algorithm assembles the SSEs de novo before atomic detail is added using Rosetta. In a benchmark of 27 proteins, the protocol consistently and reproducibly achieves models with root mean square deviation values <3 Å.► EM-Fold yields atomic detail not present in the medium resolution density map ► EM-Fold consistently finds the correct topology from medium-resolution density maps

EM-Fold: De Novo Atomic-Detail Protein Structure Determination from Medium-Resolution Density Maps by Steffen Lindert; Nathan Alexander; Nils Wötzel; Mert Karakaş; Phoebe L. Stewart; Jens Meiler (pp. 464-478).
Electron density maps of membrane proteins or large macromolecular complexes are frequently only determined at medium resolution between 4 Å and 10 Å, either by cryo-electron microscopy or X-ray crystallography. In these density maps, the general arrangement of secondary structure elements (SSEs) is revealed, whereas their directionality and connectivity remain elusive. We demonstrate that the topology of proteins with up to 250 amino acids can be determined from such density maps when combined with a computational protein folding protocol. Furthermore, we accurately reconstruct atomic detail in loop regions and amino acid side chains not visible in the experimental data. The EM-Fold algorithm assembles the SSEs de novo before atomic detail is added using Rosetta. In a benchmark of 27 proteins, the protocol consistently and reproducibly achieves models with root mean square deviation values <3 Å.► EM-Fold yields atomic detail not present in the medium resolution density map ► EM-Fold consistently finds the correct topology from medium-resolution density maps

EM-Fold: De Novo Atomic-Detail Protein Structure Determination from Medium-Resolution Density Maps by Steffen Lindert; Nathan Alexander; Nils Wötzel; Mert Karakaş; Phoebe L. Stewart; Jens Meiler (pp. 464-478).
Electron density maps of membrane proteins or large macromolecular complexes are frequently only determined at medium resolution between 4 Å and 10 Å, either by cryo-electron microscopy or X-ray crystallography. In these density maps, the general arrangement of secondary structure elements (SSEs) is revealed, whereas their directionality and connectivity remain elusive. We demonstrate that the topology of proteins with up to 250 amino acids can be determined from such density maps when combined with a computational protein folding protocol. Furthermore, we accurately reconstruct atomic detail in loop regions and amino acid side chains not visible in the experimental data. The EM-Fold algorithm assembles the SSEs de novo before atomic detail is added using Rosetta. In a benchmark of 27 proteins, the protocol consistently and reproducibly achieves models with root mean square deviation values <3 Å.► EM-Fold yields atomic detail not present in the medium resolution density map ► EM-Fold consistently finds the correct topology from medium-resolution density maps

Variable Lymphocyte Receptor Recognition of the Immunodominant Glycoprotein of Bacillus anthracis Spores by Robert N. Kirchdoerfer; Brantley R. Herrin; Byung Woo Han; Charles L. Turnbough Jr.; Max D. Cooper; Ian A. Wilson (pp. 479-486).
Variable lymphocyte receptors (VLRs) are the adaptive immune receptors of jawless fish, which evolved adaptive immunity independent of other vertebrates. In lieu of the immunoglobulin fold-based T and B cell receptors, lymphocyte-like cells of jawless fish express VLRs (VLRA, VLRB, or VLRC) composed of leucine-rich repeats and are similar to toll-like receptors (TLRs) in structure, but antibodies (VLRB) and T cell receptors (VLRA and VLRC) in function. Here, we present the structural and biochemical characterization of VLR4, a VLRB, in complex with BclA, the immunodominant glycoprotein of Bacillus anthracis spores. Using a combination of crystallography, mutagenesis, and binding studies, we delineate the mode of antigen recognition and binding between VLR4 and BclA, examine commonalities in VLRB recognition of antigens, and demonstrate the potential of VLR4 as a diagnostic tool for the identification of B. anthracis spores.Display Omitted► VLRBs use their C-terminal LRRs and the LRRCT-loop to interact with antigen ► Sequence-related VLRBs exhibit differential recognition of their BclA epitopes ► VLR4 binds a conserved protein epitope, yet is specific for B. anthracis spores

Variable Lymphocyte Receptor Recognition of the Immunodominant Glycoprotein of Bacillus anthracis Spores by Robert N. Kirchdoerfer; Brantley R. Herrin; Byung Woo Han; Charles L. Turnbough Jr.; Max D. Cooper; Ian A. Wilson (pp. 479-486).
Variable lymphocyte receptors (VLRs) are the adaptive immune receptors of jawless fish, which evolved adaptive immunity independent of other vertebrates. In lieu of the immunoglobulin fold-based T and B cell receptors, lymphocyte-like cells of jawless fish express VLRs (VLRA, VLRB, or VLRC) composed of leucine-rich repeats and are similar to toll-like receptors (TLRs) in structure, but antibodies (VLRB) and T cell receptors (VLRA and VLRC) in function. Here, we present the structural and biochemical characterization of VLR4, a VLRB, in complex with BclA, the immunodominant glycoprotein of Bacillus anthracis spores. Using a combination of crystallography, mutagenesis, and binding studies, we delineate the mode of antigen recognition and binding between VLR4 and BclA, examine commonalities in VLRB recognition of antigens, and demonstrate the potential of VLR4 as a diagnostic tool for the identification of B. anthracis spores.Display Omitted► VLRBs use their C-terminal LRRs and the LRRCT-loop to interact with antigen ► Sequence-related VLRBs exhibit differential recognition of their BclA epitopes ► VLR4 binds a conserved protein epitope, yet is specific for B. anthracis spores

Variable Lymphocyte Receptor Recognition of the Immunodominant Glycoprotein of Bacillus anthracis Spores by Robert N. Kirchdoerfer; Brantley R. Herrin; Byung Woo Han; Charles L. Turnbough Jr.; Max D. Cooper; Ian A. Wilson (pp. 479-486).
Variable lymphocyte receptors (VLRs) are the adaptive immune receptors of jawless fish, which evolved adaptive immunity independent of other vertebrates. In lieu of the immunoglobulin fold-based T and B cell receptors, lymphocyte-like cells of jawless fish express VLRs (VLRA, VLRB, or VLRC) composed of leucine-rich repeats and are similar to toll-like receptors (TLRs) in structure, but antibodies (VLRB) and T cell receptors (VLRA and VLRC) in function. Here, we present the structural and biochemical characterization of VLR4, a VLRB, in complex with BclA, the immunodominant glycoprotein of Bacillus anthracis spores. Using a combination of crystallography, mutagenesis, and binding studies, we delineate the mode of antigen recognition and binding between VLR4 and BclA, examine commonalities in VLRB recognition of antigens, and demonstrate the potential of VLR4 as a diagnostic tool for the identification of B. anthracis spores.Display Omitted► VLRBs use their C-terminal LRRs and the LRRCT-loop to interact with antigen ► Sequence-related VLRBs exhibit differential recognition of their BclA epitopes ► VLR4 binds a conserved protein epitope, yet is specific for B. anthracis spores

Structure of the Human Obesity Receptor Leptin-Binding Domain Reveals the Mechanism of Leptin Antagonism by a Monoclonal Antibody by Byron Carpenter; Glyn R. Hemsworth; Zida Wu; Mabrouka Maamra; Christian J. Strasburger; Richard J. Ross; Peter J. Artymiuk (pp. 487-497).
Leptin regulates energy homeostasis, fertility, and the immune system, making it an important drug target. However, due to a complete lack of structural data for the obesity receptor (ObR), leptin's mechanism of receptor activation remains poorly understood. We have crystallized the Fab fragment of a leptin-blocking monoclonal antibody (9F8), both in its uncomplexed state and bound to the leptin-binding domain (LBD) of human ObR. We describe the structure of the LBD-9F8 Fab complex and the conformational changes in 9F8 associated with LBD binding. A molecular model of the putative leptin-LBD complex reveals that 9F8 Fab blocks leptin binding through only a small (10%) overlap in their binding sites, and that leptin binding is likely to involve an induced fit mechanism. This crystal structure of the leptin-binding domain of the obesity receptor will facilitate the design of therapeutics to modulate leptin signaling.► Structure of the leptin binding domain of the human obesity receptor (ObR) ► Structure of a leptin blocking antibody was solved in both free and ObR-bound forms ► A model is proposed for leptin binding to the leptin-binding domain (LBD) of ObR ► 9F8 Fab blocks leptin binding to ObR through a small overlap in their epitopes

Structure of the Human Obesity Receptor Leptin-Binding Domain Reveals the Mechanism of Leptin Antagonism by a Monoclonal Antibody by Byron Carpenter; Glyn R. Hemsworth; Zida Wu; Mabrouka Maamra; Christian J. Strasburger; Richard J. Ross; Peter J. Artymiuk (pp. 487-497).
Leptin regulates energy homeostasis, fertility, and the immune system, making it an important drug target. However, due to a complete lack of structural data for the obesity receptor (ObR), leptin's mechanism of receptor activation remains poorly understood. We have crystallized the Fab fragment of a leptin-blocking monoclonal antibody (9F8), both in its uncomplexed state and bound to the leptin-binding domain (LBD) of human ObR. We describe the structure of the LBD-9F8 Fab complex and the conformational changes in 9F8 associated with LBD binding. A molecular model of the putative leptin-LBD complex reveals that 9F8 Fab blocks leptin binding through only a small (10%) overlap in their binding sites, and that leptin binding is likely to involve an induced fit mechanism. This crystal structure of the leptin-binding domain of the obesity receptor will facilitate the design of therapeutics to modulate leptin signaling.► Structure of the leptin binding domain of the human obesity receptor (ObR) ► Structure of a leptin blocking antibody was solved in both free and ObR-bound forms ► A model is proposed for leptin binding to the leptin-binding domain (LBD) of ObR ► 9F8 Fab blocks leptin binding to ObR through a small overlap in their epitopes

Structure of the Human Obesity Receptor Leptin-Binding Domain Reveals the Mechanism of Leptin Antagonism by a Monoclonal Antibody by Byron Carpenter; Glyn R. Hemsworth; Zida Wu; Mabrouka Maamra; Christian J. Strasburger; Richard J. Ross; Peter J. Artymiuk (pp. 487-497).
Leptin regulates energy homeostasis, fertility, and the immune system, making it an important drug target. However, due to a complete lack of structural data for the obesity receptor (ObR), leptin's mechanism of receptor activation remains poorly understood. We have crystallized the Fab fragment of a leptin-blocking monoclonal antibody (9F8), both in its uncomplexed state and bound to the leptin-binding domain (LBD) of human ObR. We describe the structure of the LBD-9F8 Fab complex and the conformational changes in 9F8 associated with LBD binding. A molecular model of the putative leptin-LBD complex reveals that 9F8 Fab blocks leptin binding through only a small (10%) overlap in their binding sites, and that leptin binding is likely to involve an induced fit mechanism. This crystal structure of the leptin-binding domain of the obesity receptor will facilitate the design of therapeutics to modulate leptin signaling.► Structure of the leptin binding domain of the human obesity receptor (ObR) ► Structure of a leptin blocking antibody was solved in both free and ObR-bound forms ► A model is proposed for leptin binding to the leptin-binding domain (LBD) of ObR ► 9F8 Fab blocks leptin binding to ObR through a small overlap in their epitopes

Capsomer Dynamics and Stabilization in the T = 12 Marine Bacteriophage SIO-2 and Its Procapsid Studied by CryoEM by Gabriel C. Lander; Anne-Claire Baudoux; Farooq Azam; Clinton S. Potter; Bridget Carragher; John E. Johnson (pp. 498-503).
We report the subnanometer cryo-electron microscopy (cryoEM) reconstruction of a marine siphovirus, the Vibrio phage SIO-2. This phage is lytic for related Vibrio species with significant ecological importance, including the broadly antagonistic bacterium Vibrio sp. SWAT3. The three-dimensional structure of the 800 Å SIO-2, icosahedrally averaged head of the tailed particle revealed a T = 12 quasi-symmetry not previously described in a bacteriophage. Two morphologically distinct types of auxiliary proteins were also identified; one species bound to the surface of hexamers, and the other bound to pentamers. The secondary structure, evident in the electron density, shows that the major capsid protein has the HK97-like fold. The three-dimensional structure of the procapsid form, also presented here, has no “decoration” proteins and reveals a capsomer organization due to the constraints of the T = 12 symmetry.► The SIO2 dsDNA bacteriophage displays T = 12 quasi-symmetry ► This places icosahedral symmetry constraints on subunit hexamers ► The restrictions are conspicuous in the procapsid where “hexamers” are skewed ► The balance of energetics required by stages of assembly and symmetry are discussed

Capsomer Dynamics and Stabilization in the T = 12 Marine Bacteriophage SIO-2 and Its Procapsid Studied by CryoEM by Gabriel C. Lander; Anne-Claire Baudoux; Farooq Azam; Clinton S. Potter; Bridget Carragher; John E. Johnson (pp. 498-503).
We report the subnanometer cryo-electron microscopy (cryoEM) reconstruction of a marine siphovirus, the Vibrio phage SIO-2. This phage is lytic for related Vibrio species with significant ecological importance, including the broadly antagonistic bacterium Vibrio sp. SWAT3. The three-dimensional structure of the 800 Å SIO-2, icosahedrally averaged head of the tailed particle revealed a T = 12 quasi-symmetry not previously described in a bacteriophage. Two morphologically distinct types of auxiliary proteins were also identified; one species bound to the surface of hexamers, and the other bound to pentamers. The secondary structure, evident in the electron density, shows that the major capsid protein has the HK97-like fold. The three-dimensional structure of the procapsid form, also presented here, has no “decoration” proteins and reveals a capsomer organization due to the constraints of the T = 12 symmetry.► The SIO2 dsDNA bacteriophage displays T = 12 quasi-symmetry ► This places icosahedral symmetry constraints on subunit hexamers ► The restrictions are conspicuous in the procapsid where “hexamers” are skewed ► The balance of energetics required by stages of assembly and symmetry are discussed

Capsomer Dynamics and Stabilization in the T = 12 Marine Bacteriophage SIO-2 and Its Procapsid Studied by CryoEM by Gabriel C. Lander; Anne-Claire Baudoux; Farooq Azam; Clinton S. Potter; Bridget Carragher; John E. Johnson (pp. 498-503).
We report the subnanometer cryo-electron microscopy (cryoEM) reconstruction of a marine siphovirus, the Vibrio phage SIO-2. This phage is lytic for related Vibrio species with significant ecological importance, including the broadly antagonistic bacterium Vibrio sp. SWAT3. The three-dimensional structure of the 800 Å SIO-2, icosahedrally averaged head of the tailed particle revealed a T = 12 quasi-symmetry not previously described in a bacteriophage. Two morphologically distinct types of auxiliary proteins were also identified; one species bound to the surface of hexamers, and the other bound to pentamers. The secondary structure, evident in the electron density, shows that the major capsid protein has the HK97-like fold. The three-dimensional structure of the procapsid form, also presented here, has no “decoration” proteins and reveals a capsomer organization due to the constraints of the T = 12 symmetry.► The SIO2 dsDNA bacteriophage displays T = 12 quasi-symmetry ► This places icosahedral symmetry constraints on subunit hexamers ► The restrictions are conspicuous in the procapsid where “hexamers” are skewed ► The balance of energetics required by stages of assembly and symmetry are discussed

Structural Dynamics Associated with Intermediate Formation in an Archetypal Conformational Disease by Mun Peak Nyon; Lakshmi Segu; Lisa D. Cabrita; Géraldine R. Lévy; John Kirkpatrick; Benoit D. Roussel; Anathe O.M. Patschull; Tracey E. Barrett; Ugo I. Ekeowa; Richard Kerr; Christopher A. Waudby; Noor Kalsheker; Marian Hill; Konstantinos Thalassinos; David A. Lomas; John Christodoulou; Bibek Gooptu (pp. 504-512).
In conformational diseases, native protein conformers convert to pathological intermediates that polymerize. Structural characterization of these key intermediates is challenging. They are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. We have characterized a forme fruste deficiency variant of α1-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo. Lys154Asn α1-antitrypsin populates an intermediate ensemble along the polymerization pathway at physiological temperatures. Nuclear magnetic resonance spectroscopy was used to report the structural and dynamic changes associated with this. Our data highlight an interaction network likely to regulate conformational change and do not support the recent contention that the disease-relevant intermediate is substantially unfolded. Conformational disease intermediates may best be defined using powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions.► An α1-antitrypsin deficiency mutant is a forme fruste model for the Z variant ► NMR spectroscopic and ion-mobility mass spectrometric characterization of the mutant ► Residue-specific discrimination of disease-relevant and denaturant-induced ensembles ► A “clasp” motif caps a network of stabilizing interactions in α1-antitrypsin

Structural Dynamics Associated with Intermediate Formation in an Archetypal Conformational Disease by Mun Peak Nyon; Lakshmi Segu; Lisa D. Cabrita; Géraldine R. Lévy; John Kirkpatrick; Benoit D. Roussel; Anathe O.M. Patschull; Tracey E. Barrett; Ugo I. Ekeowa; Richard Kerr; Christopher A. Waudby; Noor Kalsheker; Marian Hill; Konstantinos Thalassinos; David A. Lomas; John Christodoulou; Bibek Gooptu (pp. 504-512).
In conformational diseases, native protein conformers convert to pathological intermediates that polymerize. Structural characterization of these key intermediates is challenging. They are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. We have characterized a forme fruste deficiency variant of α1-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo. Lys154Asn α1-antitrypsin populates an intermediate ensemble along the polymerization pathway at physiological temperatures. Nuclear magnetic resonance spectroscopy was used to report the structural and dynamic changes associated with this. Our data highlight an interaction network likely to regulate conformational change and do not support the recent contention that the disease-relevant intermediate is substantially unfolded. Conformational disease intermediates may best be defined using powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions.► An α1-antitrypsin deficiency mutant is a forme fruste model for the Z variant ► NMR spectroscopic and ion-mobility mass spectrometric characterization of the mutant ► Residue-specific discrimination of disease-relevant and denaturant-induced ensembles ► A “clasp” motif caps a network of stabilizing interactions in α1-antitrypsin

Structural Dynamics Associated with Intermediate Formation in an Archetypal Conformational Disease by Mun Peak Nyon; Lakshmi Segu; Lisa D. Cabrita; Géraldine R. Lévy; John Kirkpatrick; Benoit D. Roussel; Anathe O.M. Patschull; Tracey E. Barrett; Ugo I. Ekeowa; Richard Kerr; Christopher A. Waudby; Noor Kalsheker; Marian Hill; Konstantinos Thalassinos; David A. Lomas; John Christodoulou; Bibek Gooptu (pp. 504-512).
In conformational diseases, native protein conformers convert to pathological intermediates that polymerize. Structural characterization of these key intermediates is challenging. They are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. We have characterized a forme fruste deficiency variant of α1-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo. Lys154Asn α1-antitrypsin populates an intermediate ensemble along the polymerization pathway at physiological temperatures. Nuclear magnetic resonance spectroscopy was used to report the structural and dynamic changes associated with this. Our data highlight an interaction network likely to regulate conformational change and do not support the recent contention that the disease-relevant intermediate is substantially unfolded. Conformational disease intermediates may best be defined using powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions.► An α1-antitrypsin deficiency mutant is a forme fruste model for the Z variant ► NMR spectroscopic and ion-mobility mass spectrometric characterization of the mutant ► Residue-specific discrimination of disease-relevant and denaturant-induced ensembles ► A “clasp” motif caps a network of stabilizing interactions in α1-antitrypsin

The Structure of the 26S Proteasome Subunit Rpn2 Reveals Its PC Repeat Domain as a Closed Toroid of Two Concentric α-Helical Rings by Jun He; Kiran Kulkarni; Paula C.A. da Fonseca; Dasha Krutauz; Michael H. Glickman; David Barford; Edward P. Morris (pp. 513-521).
The 26S proteasome proteolyses ubiquitylated proteins and is assembled from a 20S proteolytic core and two 19S regulatory particles (19S-RP). The 19S-RP scaffolding subunits Rpn1 and Rpn2 function to engage ubiquitin receptors. Rpn1 and Rpn2 are characterized by eleven tandem copies of a 35–40 amino acid repeat motif termed the proteasome/cyclosome (PC) repeat. Here, we reveal that the eleven PC repeats of Rpn2 form a closed toroidal structure incorporating two concentric rings of α helices encircling two axial α helices. A rod-like N-terminal domain consisting of 17 stacked α helices and a globular C-terminal domain emerge from one face of the toroid. Rpn13, an ubiquitin receptor, binds to the C-terminal 20 residues of Rpn2. Rpn1 adopts a similar conformation to Rpn2 but differs in the orientation of its rod-like N-terminal domain. These findings have implications for understanding how 19S-RPs recognize, unfold, and deliver ubiquitylated substrates to the 20S core.► The crystal structure of Rpn2, a key subunit of the 26S proteasome, has a distinctive fold ► Rpn2 has a toroidal PC, extended N-terminal, and globular C-terminal domain ► The Rpn2 PC domain is formed from eleven PC repeats and two axial α-helices ► Electron microscopy shows that Rpn1 adopts a similar structure to Rpn2

The Structure of the 26S Proteasome Subunit Rpn2 Reveals Its PC Repeat Domain as a Closed Toroid of Two Concentric α-Helical Rings by Jun He; Kiran Kulkarni; Paula C.A. da Fonseca; Dasha Krutauz; Michael H. Glickman; David Barford; Edward P. Morris (pp. 513-521).
The 26S proteasome proteolyses ubiquitylated proteins and is assembled from a 20S proteolytic core and two 19S regulatory particles (19S-RP). The 19S-RP scaffolding subunits Rpn1 and Rpn2 function to engage ubiquitin receptors. Rpn1 and Rpn2 are characterized by eleven tandem copies of a 35–40 amino acid repeat motif termed the proteasome/cyclosome (PC) repeat. Here, we reveal that the eleven PC repeats of Rpn2 form a closed toroidal structure incorporating two concentric rings of α helices encircling two axial α helices. A rod-like N-terminal domain consisting of 17 stacked α helices and a globular C-terminal domain emerge from one face of the toroid. Rpn13, an ubiquitin receptor, binds to the C-terminal 20 residues of Rpn2. Rpn1 adopts a similar conformation to Rpn2 but differs in the orientation of its rod-like N-terminal domain. These findings have implications for understanding how 19S-RPs recognize, unfold, and deliver ubiquitylated substrates to the 20S core.► The crystal structure of Rpn2, a key subunit of the 26S proteasome, has a distinctive fold ► Rpn2 has a toroidal PC, extended N-terminal, and globular C-terminal domain ► The Rpn2 PC domain is formed from eleven PC repeats and two axial α-helices ► Electron microscopy shows that Rpn1 adopts a similar structure to Rpn2

The Structure of the 26S Proteasome Subunit Rpn2 Reveals Its PC Repeat Domain as a Closed Toroid of Two Concentric α-Helical Rings by Jun He; Kiran Kulkarni; Paula C.A. da Fonseca; Dasha Krutauz; Michael H. Glickman; David Barford; Edward P. Morris (pp. 513-521).
The 26S proteasome proteolyses ubiquitylated proteins and is assembled from a 20S proteolytic core and two 19S regulatory particles (19S-RP). The 19S-RP scaffolding subunits Rpn1 and Rpn2 function to engage ubiquitin receptors. Rpn1 and Rpn2 are characterized by eleven tandem copies of a 35–40 amino acid repeat motif termed the proteasome/cyclosome (PC) repeat. Here, we reveal that the eleven PC repeats of Rpn2 form a closed toroidal structure incorporating two concentric rings of α helices encircling two axial α helices. A rod-like N-terminal domain consisting of 17 stacked α helices and a globular C-terminal domain emerge from one face of the toroid. Rpn13, an ubiquitin receptor, binds to the C-terminal 20 residues of Rpn2. Rpn1 adopts a similar conformation to Rpn2 but differs in the orientation of its rod-like N-terminal domain. These findings have implications for understanding how 19S-RPs recognize, unfold, and deliver ubiquitylated substrates to the 20S core.► The crystal structure of Rpn2, a key subunit of the 26S proteasome, has a distinctive fold ► Rpn2 has a toroidal PC, extended N-terminal, and globular C-terminal domain ► The Rpn2 PC domain is formed from eleven PC repeats and two axial α-helices ► Electron microscopy shows that Rpn1 adopts a similar structure to Rpn2

Recognition Pliability Is Coupled to Structural Heterogeneity: A Calmodulin Intrinsically Disordered Binding Region Complex by Malini Nagulapalli; Giacomo Parigi; Jing Yuan; Joerg Gsponer; George Deraos; Vladimir V. Bamm; George Harauz; John Matsoukas; Maurits R.R. de Planque; Ioannis P. Gerothanassis; M. Madan Babu; Claudio Luchinat; Andreas G. Tzakos (pp. 522-533).
Protein interactions within regulatory networks should adapt in a spatiotemporal-dependent dynamic environment, in order to process and respond to diverse and versatile cellular signals. However, the principles governing recognition pliability in protein complexes are not well understood. We have investigated a region of the intrinsically disordered protein myelin basic protein (MBP145–165) that interacts with calmodulin, but that also promiscuously binds other biomolecules (membranes, modifying enzymes). To characterize this interaction, we implemented an NMR spectroscopic approach that calculates, for each conformation of the complex, the maximum occurrence based on recorded pseudocontact shifts and residual dipolar couplings. We found that the MBP145–165-calmodulin interaction is characterized by structural heterogeneity. Quantitative comparative analysis indicated that distinct conformational landscapes of structural heterogeneity are sampled for different calmodulin-target complexes. Such structural heterogeneity in protein complexes could potentially explain the way that transient and promiscuous protein interactions are optimized and tuned in complex regulatory networks.Display Omitted► A disordered noncanonical CaM-binding region is located at the C terminus of MBP ► This region binds to semi-open CaM with an affinity similar to full-length MBP ► CaM adopts a large ensemble of conformations when in complex with the MBP peptide ► Distinct spatial heterogeneity is sampled for different CaM-target complexes

Recognition Pliability Is Coupled to Structural Heterogeneity: A Calmodulin Intrinsically Disordered Binding Region Complex by Malini Nagulapalli; Giacomo Parigi; Jing Yuan; Joerg Gsponer; George Deraos; Vladimir V. Bamm; George Harauz; John Matsoukas; Maurits R.R. de Planque; Ioannis P. Gerothanassis; M. Madan Babu; Claudio Luchinat; Andreas G. Tzakos (pp. 522-533).
Protein interactions within regulatory networks should adapt in a spatiotemporal-dependent dynamic environment, in order to process and respond to diverse and versatile cellular signals. However, the principles governing recognition pliability in protein complexes are not well understood. We have investigated a region of the intrinsically disordered protein myelin basic protein (MBP145–165) that interacts with calmodulin, but that also promiscuously binds other biomolecules (membranes, modifying enzymes). To characterize this interaction, we implemented an NMR spectroscopic approach that calculates, for each conformation of the complex, the maximum occurrence based on recorded pseudocontact shifts and residual dipolar couplings. We found that the MBP145–165-calmodulin interaction is characterized by structural heterogeneity. Quantitative comparative analysis indicated that distinct conformational landscapes of structural heterogeneity are sampled for different calmodulin-target complexes. Such structural heterogeneity in protein complexes could potentially explain the way that transient and promiscuous protein interactions are optimized and tuned in complex regulatory networks.Display Omitted► A disordered noncanonical CaM-binding region is located at the C terminus of MBP ► This region binds to semi-open CaM with an affinity similar to full-length MBP ► CaM adopts a large ensemble of conformations when in complex with the MBP peptide ► Distinct spatial heterogeneity is sampled for different CaM-target complexes

Recognition Pliability Is Coupled to Structural Heterogeneity: A Calmodulin Intrinsically Disordered Binding Region Complex by Malini Nagulapalli; Giacomo Parigi; Jing Yuan; Joerg Gsponer; George Deraos; Vladimir V. Bamm; George Harauz; John Matsoukas; Maurits R.R. de Planque; Ioannis P. Gerothanassis; M. Madan Babu; Claudio Luchinat; Andreas G. Tzakos (pp. 522-533).
Protein interactions within regulatory networks should adapt in a spatiotemporal-dependent dynamic environment, in order to process and respond to diverse and versatile cellular signals. However, the principles governing recognition pliability in protein complexes are not well understood. We have investigated a region of the intrinsically disordered protein myelin basic protein (MBP145–165) that interacts with calmodulin, but that also promiscuously binds other biomolecules (membranes, modifying enzymes). To characterize this interaction, we implemented an NMR spectroscopic approach that calculates, for each conformation of the complex, the maximum occurrence based on recorded pseudocontact shifts and residual dipolar couplings. We found that the MBP145–165-calmodulin interaction is characterized by structural heterogeneity. Quantitative comparative analysis indicated that distinct conformational landscapes of structural heterogeneity are sampled for different calmodulin-target complexes. Such structural heterogeneity in protein complexes could potentially explain the way that transient and promiscuous protein interactions are optimized and tuned in complex regulatory networks.Display Omitted► A disordered noncanonical CaM-binding region is located at the C terminus of MBP ► This region binds to semi-open CaM with an affinity similar to full-length MBP ► CaM adopts a large ensemble of conformations when in complex with the MBP peptide ► Distinct spatial heterogeneity is sampled for different CaM-target complexes

Cdc6-Induced Conformational Changes in ORC Bound to Origin DNA Revealed by Cryo-Electron Microscopy by Jingchuan Sun; Hironori Kawakami; Juergen Zech; Christian Speck; Bruce Stillman; Huilin Li (pp. 534-544).
The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here, we report a single-particle cryo-EM-derived structure of the supramolecular assembly comprising Saccharomyces cerevisiae ORC, the replication initiation factor Cdc6, and double-stranded ARS1 origin DNA in the presence of ATPγS. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, in particular reorienting the Orc1 N-terminal BAH domain. Segmentation of the 3D map of ORC-Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 protein suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus, ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends.Display Omitted► We have determined the cryo-EM structures of ORC, ORC-DNA, and ORC-Cdc6-DNA ► We show that ORC is arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2 ► DNA and Cdc6 binding causes large conformational changes in ORC ► Origin DNA is proposed to bind to the interior surface of the crescent-shaped ORC

Cdc6-Induced Conformational Changes in ORC Bound to Origin DNA Revealed by Cryo-Electron Microscopy by Jingchuan Sun; Hironori Kawakami; Juergen Zech; Christian Speck; Bruce Stillman; Huilin Li (pp. 534-544).
The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here, we report a single-particle cryo-EM-derived structure of the supramolecular assembly comprising Saccharomyces cerevisiae ORC, the replication initiation factor Cdc6, and double-stranded ARS1 origin DNA in the presence of ATPγS. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, in particular reorienting the Orc1 N-terminal BAH domain. Segmentation of the 3D map of ORC-Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 protein suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus, ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends.Display Omitted► We have determined the cryo-EM structures of ORC, ORC-DNA, and ORC-Cdc6-DNA ► We show that ORC is arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2 ► DNA and Cdc6 binding causes large conformational changes in ORC ► Origin DNA is proposed to bind to the interior surface of the crescent-shaped ORC

Cdc6-Induced Conformational Changes in ORC Bound to Origin DNA Revealed by Cryo-Electron Microscopy by Jingchuan Sun; Hironori Kawakami; Juergen Zech; Christian Speck; Bruce Stillman; Huilin Li (pp. 534-544).
The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here, we report a single-particle cryo-EM-derived structure of the supramolecular assembly comprising Saccharomyces cerevisiae ORC, the replication initiation factor Cdc6, and double-stranded ARS1 origin DNA in the presence of ATPγS. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, in particular reorienting the Orc1 N-terminal BAH domain. Segmentation of the 3D map of ORC-Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 protein suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus, ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends.Display Omitted► We have determined the cryo-EM structures of ORC, ORC-DNA, and ORC-Cdc6-DNA ► We show that ORC is arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2 ► DNA and Cdc6 binding causes large conformational changes in ORC ► Origin DNA is proposed to bind to the interior surface of the crescent-shaped ORC

Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex by Alexis I. Cocozaki; Nancy F. Ramia; Yaming Shao; Caryn R. Hale; Rebecca M. Terns; Michael P. Terns; Hong Li (pp. 545-553).
Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3Å crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two α-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.► Crystal structure of the largest subunit of the CRISPR mediated RNA cleavage (CMR) complex ► The CMR complex contains an adenylyl cyclase-like subunit ► The adenylyl cyclase-like subunit binds ATP and divalent metals but is not the site of RNA cleavage ► The HD domain of CMR complex is not required for RNA cleavage activity

Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex by Alexis I. Cocozaki; Nancy F. Ramia; Yaming Shao; Caryn R. Hale; Rebecca M. Terns; Michael P. Terns; Hong Li (pp. 545-553).
Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3Å crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two α-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.► Crystal structure of the largest subunit of the CRISPR mediated RNA cleavage (CMR) complex ► The CMR complex contains an adenylyl cyclase-like subunit ► The adenylyl cyclase-like subunit binds ATP and divalent metals but is not the site of RNA cleavage ► The HD domain of CMR complex is not required for RNA cleavage activity

Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex by Alexis I. Cocozaki; Nancy F. Ramia; Yaming Shao; Caryn R. Hale; Rebecca M. Terns; Michael P. Terns; Hong Li (pp. 545-553).
Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3Å crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two α-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.► Crystal structure of the largest subunit of the CRISPR mediated RNA cleavage (CMR) complex ► The CMR complex contains an adenylyl cyclase-like subunit ► The adenylyl cyclase-like subunit binds ATP and divalent metals but is not the site of RNA cleavage ► The HD domain of CMR complex is not required for RNA cleavage activity

Architecture of a Dodecameric Bacterial Replicative Helicase by Meike Stelter; Irina Gutsche; Ulrike Kapp; Alexandre Bazin; Goran Bajic; Gaël Goret; Marc Jamin; Joanna Timmins; Laurent Terradot (pp. 554-564).
Hexameric DnaB helicases are often loaded at DNA replication forks by interacting with the initiator protein DnaA and/or a helicase loader (DnaC in Escherichia coli). These loaders are not universally required, and DnaB from Helicobacter pylori was found to bypass DnaC when expressed in E. coli cells. The crystal structure of Helicobacter pylori DnaB C-terminal domain (HpDnaB-CTD) reveals a large two-helix insertion (named HPI) in the ATPase domain that protrudes away from the RecA fold. Biophysical characterization and electron microscopy (EM) analysis of the full-length protein show that HpDnaB forms head-to-head double hexamers remarkably similar to helicases found in some eukaryotes, archaea, and viruses. The docking of the HpDnaB-CTD structure into EM reconstruction of HpDnaB provides a model that shows how hexamerization of the CTD is facilitated by HPI-HPI interactions. The HpDnaB double-hexamer architecture supports an alternative strategy to load bacterial helicases onto forks in the absence of helicase loaders.Display Omitted► Crystal structure of the ATPase domain of DnaB from Helicobacter pylori ► MALLS analysis shows that HpDnaB forms dodecamers ► EM reconstruction shows that HpDnaB forms head-to-head double hexamers

Architecture of a Dodecameric Bacterial Replicative Helicase by Meike Stelter; Irina Gutsche; Ulrike Kapp; Alexandre Bazin; Goran Bajic; Gaël Goret; Marc Jamin; Joanna Timmins; Laurent Terradot (pp. 554-564).
Hexameric DnaB helicases are often loaded at DNA replication forks by interacting with the initiator protein DnaA and/or a helicase loader (DnaC in Escherichia coli). These loaders are not universally required, and DnaB from Helicobacter pylori was found to bypass DnaC when expressed in E. coli cells. The crystal structure of Helicobacter pylori DnaB C-terminal domain (HpDnaB-CTD) reveals a large two-helix insertion (named HPI) in the ATPase domain that protrudes away from the RecA fold. Biophysical characterization and electron microscopy (EM) analysis of the full-length protein show that HpDnaB forms head-to-head double hexamers remarkably similar to helicases found in some eukaryotes, archaea, and viruses. The docking of the HpDnaB-CTD structure into EM reconstruction of HpDnaB provides a model that shows how hexamerization of the CTD is facilitated by HPI-HPI interactions. The HpDnaB double-hexamer architecture supports an alternative strategy to load bacterial helicases onto forks in the absence of helicase loaders.Display Omitted► Crystal structure of the ATPase domain of DnaB from Helicobacter pylori ► MALLS analysis shows that HpDnaB forms dodecamers ► EM reconstruction shows that HpDnaB forms head-to-head double hexamers

Architecture of a Dodecameric Bacterial Replicative Helicase by Meike Stelter; Irina Gutsche; Ulrike Kapp; Alexandre Bazin; Goran Bajic; Gaël Goret; Marc Jamin; Joanna Timmins; Laurent Terradot (pp. 554-564).
Hexameric DnaB helicases are often loaded at DNA replication forks by interacting with the initiator protein DnaA and/or a helicase loader (DnaC in Escherichia coli). These loaders are not universally required, and DnaB from Helicobacter pylori was found to bypass DnaC when expressed in E. coli cells. The crystal structure of Helicobacter pylori DnaB C-terminal domain (HpDnaB-CTD) reveals a large two-helix insertion (named HPI) in the ATPase domain that protrudes away from the RecA fold. Biophysical characterization and electron microscopy (EM) analysis of the full-length protein show that HpDnaB forms head-to-head double hexamers remarkably similar to helicases found in some eukaryotes, archaea, and viruses. The docking of the HpDnaB-CTD structure into EM reconstruction of HpDnaB provides a model that shows how hexamerization of the CTD is facilitated by HPI-HPI interactions. The HpDnaB double-hexamer architecture supports an alternative strategy to load bacterial helicases onto forks in the absence of helicase loaders.Display Omitted► Crystal structure of the ATPase domain of DnaB from Helicobacter pylori ► MALLS analysis shows that HpDnaB forms dodecamers ► EM reconstruction shows that HpDnaB forms head-to-head double hexamers
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