Single Isomorphous Replacement Research Articles

Single-support serial isomorphous replacement phasing.

The use of single isomorphous replacement (SIR) has become less widespread due to difficulties in sample preparation and the identification of isomorphous native and derivative data sets. Non-isomorphism becomes even more problematic in serial experiments, because it adds natural inter-crystal non-isomorphism to heavy-atom-soaking-induced non-isomorphism. Here, a method that can successfully address these issues (and indeed can benefit from differences in heavy-atom occupancy) and additionally significantly simplifies the SIR experiment is presented. A single heavy-atom soak into a microcrystalline slurry is performed, followed by automated serial data collection of partial data sets. This produces a set of data collections with a gradient of heavy-atom occupancies, which are reflected in differential merging statistics. These differences can be exploited by an optimized genetic algorithm to segregate the pool of data sets into `native' and `derivative' groups, which can then be used to successfully determine phases experimentally by SIR.

Building and destroying synaptic bridges: How do Hevin/Sparcl1, SPARC, and MDGAs modify trans-synaptic neurexin-neuroligin interactions?

Astrocyte-secreted Hevin induces synapse formation by bridging the trans-synaptic organizers, neuroligins and neurexins. In this issue of Structure, Fan etal. (2021) elucidate the structural basis of Hevin's interaction with these molecules and reveal the competitive interplay between Hevin, SPARC, and MDGAs.

SHELIXIR: automation of experimental phasing procedures using SHELXC/D/E

The program SHELIXIR represents a simple and efficient tool for routine phase-problem solution using data for experimental phasing by the single-wavelength anomalous dispersion, multiwavelength anomalous dispersion, single isomorphous replacement with anomalous scattering and radiation-damage-induced phasing methods. As indicated in its name, all calculation procedures are performed with the SHELXC/D/E program package. SHELIXIR provides screening for alternative space groups, optimal solvent content, and high- and low-resolution limits. The procedures of SHELXE are parallelized to minimize the computational time. The automation and parallelization of such procedures are suitable for phasing at synchrotron beamlines directly or for finding the optimal parameters for further data processing. A simple graphical interface is designed to make use easier and to increase efficiency during beam time.

Interplay between hevin, SPARC, and MDGAs: Modulators of neurexin-neuroligin transsynaptic bridges

•An elaborate protein network regulates neurexin-neuroligin transsynaptic bridges•The hevin FS domain binds both neurexin and neuroligin via a direct interaction•Antagonist SPARC also binds neurexin/neuroligin•Hevin and MDGAs, which exert opposite actions, compete for binding to neuroligin Hevin is secreted by astrocytes and its synaptogenic effects are antagonized by the related protein, SPARC. Hevin stabilizes neurexin-neuroligin transsynaptic bridges in vivo. A third protein, membrane-tethered MDGA, blocks these bridges. Here, we reveal the molecular underpinnings of a regulatory network formed by this trio of proteins. The hevin FS-EC structure differs from SPARC, in that the EC domain appears rearranged around a conserved core. The FS domain is structurally conserved and it houses nanomolar affinity binding sites for neurexin and neuroligin. SPARC also binds neurexin and neuroligin, competing with hevin, so its antagonist action is rooted in its shortened N-terminal region. Strikingly, the hevin FS domain competes with MDGA for an overlapping binding site on neuroligin, while the hevin EC domain binds the extracellular matrix protein collagen (like SPARC), so that this trio of proteins can regulate neurexin-neuroligin transsynaptic bridges and also extracellular matrix interactions, impacting synapse formation and ultimately neural circuits. Hevin is secreted by astrocytes and its synaptogenic effects are antagonized by the related protein, SPARC. Hevin stabilizes neurexin-neuroligin transsynaptic bridges in vivo. A third protein, membrane-tethered MDGA, blocks these bridges. Here, we reveal the molecular underpinnings of a regulatory network formed by this trio of proteins. The hevin FS-EC structure differs from SPARC, in that the EC domain appears rearranged around a conserved core. The FS domain is structurally conserved and it houses nanomolar affinity binding sites for neurexin and neuroligin. SPARC also binds neurexin and neuroligin, competing with hevin, so its antagonist action is rooted in its shortened N-terminal region. Strikingly, the hevin FS domain competes with MDGA for an overlapping binding site on neuroligin, while the hevin EC domain binds the extracellular matrix protein collagen (like SPARC), so that this trio of proteins can regulate neurexin-neuroligin transsynaptic bridges and also extracellular matrix interactions, impacting synapse formation and ultimately neural circuits. Hevin (high endothelial venule protein), also known as SPARCL1 (SPARC-like 1, SC1, secreted protein acidic and rich in cysteine-like 1), is a secreted matricellular protein that promotes the formation and maintenance of neural circuits in mammalian brain by altering synaptic connections between neurons as well as impacting the position of neurons as they migrate (Allen and Eroglu, 2017Allen N.J. Eroglu C. Cell biology of astrocyte-synapse interactions.Neuron. 2017; 96: 697-708Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar; Ferrer-Ferrer and Dityatev, 2018Ferrer-Ferrer M. Dityatev A. Shaping synapses by the neural extracellular matrix.Front. Neuroanat. 2018; 12: 40Crossref PubMed Scopus (69) Google Scholar; Jones and Bouvier, 2014Jones E.V. Bouvier D.S. Astrocyte-secreted matricellular proteins in CNS remodelling during development and disease.Neural Plast. 2014; 2014: 321209Crossref PubMed Scopus (106) Google Scholar; Yuzaki, 2018Yuzaki M. Two classes of secreted synaptic organizers in the central nervous system.Annu. Rev. Physiol. 2018; 80: 243-262Crossref PubMed Scopus (50) Google Scholar). Hevin is composed of an N-terminal thread-like, flexible acidic region and a C-terminal globular region containing a follistatin-like (FS) domain and an extracellular calcium-binding (EC) domain (Hambrock et al., 2003Hambrock H.O. Nitsche D.P. Hansen U. Bruckner P. Paulsson M. Maurer P. Hartmann U. SC1/hevin. An extracellular calcium-modulated protein that binds collagen I.J. Biol. Chem. 2003; 278: 11351-11358Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The FS-EC tandem has ∼61% sequence identity to the matricellular protein SPARC (secreted protein acidic and rich in cysteine), a much smaller protein with a very short N-terminal region that is not conserved in hevin (Bradshaw, 2012Bradshaw A.D. Diverse biological functions of the SPARC family of proteins.Int. J. Biochem. Cell Biol. 2012; 44: 480-488Crossref PubMed Scopus (158) Google Scholar; Girard and Springer, 1995Girard J.P. Springer T.A. Cloning from purified high endothelial venule cells of hevin, a close relative of the antiadhesive extracellular matrix protein SPARC.Immunity. 1995; 2: 113-123Abstract Full Text PDF PubMed Scopus (143) Google Scholar; Murphy-Ullrich and Sage, 2014Murphy-Ullrich J.E. Sage E.H. Revisiting the matricellular concept.Matrix Biol. 2014; 37: 1-14Crossref PubMed Scopus (239) Google Scholar). Proteins that induce and/or maintain synapses are an important focal point of study because they are increasingly linked to neuropsychiatric disorders (Südhof, 2017Südhof T.C. Synaptic neurexin complexes: a molecular code for the logic of neural circuits.Cell. 2017; 171: 745-769Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar), but for many, including hevin, relatively little is known about their molecular mechanisms. Hevin and SPARC have both similar as well as opposing biological functions in the brain. Hevin stimulates excitatory synapse formation (Gan and Südhof, 2019Gan K.J. Südhof T.C. Specific factors in blood from young but not old mice directly promote synapse formation and NMDA-receptor recruitment.Proc. Natl. Acad. Sci. U S A. 2019; 116: 12524-12533Crossref PubMed Scopus (39) Google Scholar; Gan and Südhof, 2020Gan K.J. Südhof T.C. SPARCL1 promotes excitatory but not inhibitory synapse formation and function independent of neurexins and neuroligins.J. Neurosci. 2020; 40: 8088-8102Crossref PubMed Scopus (16) Google Scholar; Kucukdereli et al., 2011Kucukdereli H. Allen N.J. Lee A.T. Feng A. Ozlu M.I. Conatser L.M. Chakraborty C. Workman G. Weaver M. Sage E.H. et al.Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC.Proc. Natl. Acad. Sci. U S A. 2011; 108: E440-E449Crossref PubMed Scopus (329) Google Scholar). Its action is important to generate refined neural circuits; for instance, hevin secreted by astrocytes promotes and stabilizes thalamocortical excitatory synapses in the developing mouse visual cortex, strengthening the connections between the two brain regions, at the cost of the intracortical excitatory connections, which are eliminated (Kucukdereli et al., 2011Kucukdereli H. Allen N.J. Lee A.T. Feng A. Ozlu M.I. Conatser L.M. Chakraborty C. Workman G. Weaver M. Sage E.H. et al.Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC.Proc. Natl. Acad. Sci. U S A. 2011; 108: E440-E449Crossref PubMed Scopus (329) Google Scholar; Risher et al., 2014Risher W.C. Patel S. Kim I.H. Uezu A. Bhagat S. Wilton D.K. Pilaz L.-J. Singh Alvarado J. Calhan O.Y. Silver D.L. et al.Astrocytes refine cortical connectivity at dendritic spines.Elife. 2014; 3: e04047Crossref Scopus (87) Google Scholar; Singh et al., 2016Singh S.K. Stogsdill J.A. Pulimood N.S. Dingsdale H. Kim Y.H. Pilaz L.-J. Kim I.H. Manhaes A.C. Rodrigues W.S. Pamukcu A. et al.Astrocytes assemble thalamocortical synapses by bridging NRX1α and NL1 via hevin.Cell. 2016; 164: 183-196Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). SPARC also affects synapse formation, albeit differently than hevin (Bradshaw, 2012Bradshaw A.D. Diverse biological functions of the SPARC family of proteins.Int. J. Biochem. Cell Biol. 2012; 44: 480-488Crossref PubMed Scopus (158) Google Scholar; Murphy-Ullrich and Sage, 2014Murphy-Ullrich J.E. Sage E.H. Revisiting the matricellular concept.Matrix Biol. 2014; 37: 1-14Crossref PubMed Scopus (239) Google Scholar). SPARC lacks the ability to induce synaptogenesis and it blocks hevin action (Kucukdereli et al., 2011Kucukdereli H. Allen N.J. Lee A.T. Feng A. Ozlu M.I. Conatser L.M. Chakraborty C. Workman G. Weaver M. Sage E.H. et al.Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC.Proc. Natl. Acad. Sci. U S A. 2011; 108: E440-E449Crossref PubMed Scopus (329) Google Scholar). Interestingly, a fragment of hevin, SLF (SPARC-like fragment), that encompasses the FS and EC domains just like SPARC, is also not synaptogenic and likewise blocks hevin-induced synapse formation (Kucukdereli et al., 2011Kucukdereli H. Allen N.J. Lee A.T. Feng A. Ozlu M.I. Conatser L.M. Chakraborty C. Workman G. Weaver M. Sage E.H. et al.Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC.Proc. Natl. Acad. Sci. U S A. 2011; 108: E440-E449Crossref PubMed Scopus (329) Google Scholar). SPARC triggers a cell-autonomous program of synapse elimination in cholinergic neurons (López-Murcia et al., 2015López-Murcia F.J. Terni B. Llobet A. SPARC triggers a cell-autonomous program of synapse elimination.Proc. Natl. Acad. Sci. U S A. 2015; 112: 13366-13371Crossref PubMed Scopus (31) Google Scholar), although in single-cell cholinergic neuron microcultures it increases the formation of autapses (i.e., synapses from one neuron onto itself) (Albrecht et al., 2012Albrecht D. López-Murcia F.J. Pérez-González A.P. Lichtner G. Solsona C. Llobet A. SPARC prevents maturation of cholinergic presynaptic terminals.Mol. Cell. Neurosci. 2012; 49: 364-374Crossref PubMed Scopus (26) Google Scholar). SPARC also regulates the efficiency of synaptic communication in developing hippocampus by controlling the number of AMPA-receptor subunits at synapses via a β3-integrin-mediated mechanism, while hevin does not (Jones et al., 2011Jones E.V. Bernardinelli Y. Tse Y.C. Chierzi S. Wong T.P. Murai K.K. Astrocytes control glutamate receptor levels at developing synapses through SPARC-beta-integrin interactions.J. Neurosci. 2011; 31: 4154-4165Crossref PubMed Scopus (85) Google Scholar). However, both hevin and SPARC alter neurite outgrowth (thus influencing neuron morphology), as well as neuronal migration and connectivities during postnatal development (López-Murcia et al., 2015López-Murcia F.J. Terni B. Llobet A. SPARC triggers a cell-autonomous program of synapse elimination.Proc. Natl. Acad. Sci. U S A. 2015; 112: 13366-13371Crossref PubMed Scopus (31) Google Scholar; Vincent et al., 2008Vincent A.J. Lau P.W. Roskams A.J. SPARC is expressed by macroglia and microglia in the developing and mature nervous system.Dev. Dyn. 2008; 237: 1449-1462Crossref PubMed Scopus (53) Google Scholar), perhaps via interactions with the extracellular matrix. SPARC has a well-described role in extracellular matrix assembly through its ability to bind collagen, which has been elucidated by structural studies. Whether hevin binds collagen as well is unclear (Bradshaw, 2012Bradshaw A.D. Diverse biological functions of the SPARC family of proteins.Int. J. Biochem. Cell Biol. 2012; 44: 480-488Crossref PubMed Scopus (158) Google Scholar; Hohenester et al., 2008Hohenester E. Sasaki T. Giudici C. Farndale R.W. Bächinger H.P. Structural basis of sequence-specific collagen recognition by SPARC.Proc. Natl. Acad. Sci. U S A. 2008; 105: 18273-18277Crossref PubMed Scopus (104) Google Scholar; Murphy-Ullrich and Sage, 2014Murphy-Ullrich J.E. Sage E.H. Revisiting the matricellular concept.Matrix Biol. 2014; 37: 1-14Crossref PubMed Scopus (239) Google Scholar). In the brain, hevin and SPARC show characteristic expression patterns that are temporally and spatially regulated, placing these molecules in highly strategic positions to modulate neural circuit formation and maintenance during development and beyond. Throughout postnatal development, hevin is abundantly expressed in astrocytes and in subsets of projection neurons, escalating to high levels during a peak period of synaptic remodeling (Lively and Brown, 2008aLively S. Brown I.R. Localization of the extracellular matrix protein SC1 coincides with synaptogenesis during rat postnatal development.Neurochem. Res. 2008; 33: 1692-1700Crossref PubMed Scopus (21) Google Scholar; Lloyd-Burton and Roskams, 2012Lloyd-Burton S. Roskams A.J. SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS.J. Comp. Neurol. 2012; 520: 2575-2590Crossref PubMed Scopus (16) Google Scholar; Mendis et al., 1996Mendis D.B. Shahin S. Gurd J.W. Brown I.R. SC1, a SPARC-related glycoprotein, exhibits features of an ECM component in the developing and adult brain.Brain Res. 1996; 713: 53-63Crossref PubMed Scopus (37) Google Scholar; Risher et al., 2014Risher W.C. Patel S. Kim I.H. Uezu A. Bhagat S. Wilton D.K. Pilaz L.-J. Singh Alvarado J. Calhan O.Y. Silver D.L. et al.Astrocytes refine cortical connectivity at dendritic spines.Elife. 2014; 3: e04047Crossref Scopus (87) Google Scholar). However, hevin is also strongly expressed in many regions in adult brain in most astrocytes, and also in select populations of inhibitory and excitatory neurons (Hashimoto et al., 2016Hashimoto N. Sato T. Yajima T. Fujita M. Sato A. Shimizu Y. Shimada Y. Shoji N. Sasano T. Ichikawa H. SPARCL1-containing neurons in the human brainstem and sensory ganglion.Somatosens. Mot. Res. 2016; 33: 112-117Crossref PubMed Scopus (8) Google Scholar; Lively et al., 2007Lively S. Ringuette M.J. Brown I.R. Localization of the extracellular matrix protein SC1 to synapses in the adult rat brain.Neurochem. Res. 2007; 32: 65-71Crossref PubMed Scopus (31) Google Scholar; Lloyd-Burton and Roskams, 2012Lloyd-Burton S. Roskams A.J. SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS.J. Comp. Neurol. 2012; 520: 2575-2590Crossref PubMed Scopus (16) Google Scholar; Mendis et al., 1996Mendis D.B. Shahin S. Gurd J.W. Brown I.R. SC1, a SPARC-related glycoprotein, exhibits features of an ECM component in the developing and adult brain.Brain Res. 1996; 713: 53-63Crossref PubMed Scopus (37) Google Scholar; Mongrédien et al., 2019Mongrédien R. Erdozain A.M. Dumas S. Cutando L. Del Moral A.N. Puighermanal E. Rezai Amin S. Giros B. Valjent E. Meana J.J. et al.Cartography of hevin-expressing cells in the adult brain reveals prominent expression in astrocytes and parvalbumin neurons.Brain Struct. Funct. 2019; 224: 1219-1244Crossref PubMed Scopus (6) Google Scholar; Risher et al., 2014Risher W.C. Patel S. Kim I.H. Uezu A. Bhagat S. Wilton D.K. Pilaz L.-J. Singh Alvarado J. Calhan O.Y. Silver D.L. et al.Astrocytes refine cortical connectivity at dendritic spines.Elife. 2014; 3: e04047Crossref Scopus (87) Google Scholar). Like hevin, SPARC is broadly expressed during development in glial cells and radial glia (progenitor cells that additionally function as guide cells along which neurons migrate) (Vincent et al., 2008Vincent A.J. Lau P.W. Roskams A.J. SPARC is expressed by macroglia and microglia in the developing and mature nervous system.Dev. Dyn. 2008; 237: 1449-1462Crossref PubMed Scopus (53) Google Scholar), but in adult CNS, SPARC is expressed only in very limited regions by astrocytes and microglia but not neurons (Lloyd-Burton and Roskams, 2012Lloyd-Burton S. Roskams A.J. SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS.J. Comp. Neurol. 2012; 520: 2575-2590Crossref PubMed Scopus (16) Google Scholar; Mendis et al., 1995Mendis D.B. Malaval L. Brown I.R. SPARC, an extracellular matrix glycoprotein containing the follistatin module, is expressed by astrocytes in synaptic enriched regions of the adult brain.Brain Res. 1995; 676: 69-79Crossref PubMed Scopus (46) Google Scholar; Mongrédien et al., 2019Mongrédien R. Erdozain A.M. Dumas S. Cutando L. Del Moral A.N. Puighermanal E. Rezai Amin S. Giros B. Valjent E. Meana J.J. et al.Cartography of hevin-expressing cells in the adult brain reveals prominent expression in astrocytes and parvalbumin neurons.Brain Struct. Funct. 2019; 224: 1219-1244Crossref PubMed Scopus (6) Google Scholar; Vincent et al., 2008Vincent A.J. Lau P.W. Roskams A.J. SPARC is expressed by macroglia and microglia in the developing and mature nervous system.Dev. Dyn. 2008; 237: 1449-1462Crossref PubMed Scopus (53) Google Scholar). Given the high sequence identity, the striking biological differences between hevin and SPARC remain puzzling, especially as they also seem to share certain functions, as described above, and this dichotomy is important given the characteristic temporal and spatial expression patterns of hevin and SPARC. The molecular mechanisms of hevin and SPARC actions in the brain are unclear. In vivo, hevin appears to promote the interaction between two families of synaptic organizers, the post-synaptic neuroligins and the pre-synaptic neurexins, and it is proposed to join them together in transsynaptic bridges that span and stabilize synaptic junctions (Singh et al., 2016Singh S.K. Stogsdill J.A. Pulimood N.S. Dingsdale H. Kim Y.H. Pilaz L.-J. Kim I.H. Manhaes A.C. Rodrigues W.S. Pamukcu A. et al.Astrocytes assemble thalamocortical synapses by bridging NRX1α and NL1 via hevin.Cell. 2016; 164: 183-196Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Neurexin-neuroligin complexes are among the most extensively studied transsynaptic bridges and are particularly notable because of their diverse composition and their links to neuropsychiatric disorders (Rudenko, 2019Rudenko G. Neurexins—versatile molecular platforms in the synaptic cleft.Curr. Opin. Struct. Biol. 2019; 54: 112-121Crossref PubMed Scopus (15) Google Scholar; Südhof, 2017Südhof T.C. Synaptic neurexin complexes: a molecular code for the logic of neural circuits.Cell. 2017; 171: 745-769Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). In humans, five neuroligin (NLGN) genes encode proteins containing a membrane-tethered, dimeric ectodomain with a cholinesterase-like fold, which is variably diversified through alternative splicing at two splice insert sites, site A (SSA) and site B (SSB) (Bemben et al., 2015Bemben M.A. Shipman S.L. Nicoll R.A. Roche K.W. The cellular and molecular landscape of neuroligins.Trends Neurosci. 2015; 38: 496-505Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar; Südhof, 2017Südhof T.C. Synaptic neurexin complexes: a molecular code for the logic of neural circuits.Cell. 2017; 171: 745-769Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Similarly, three neurexin (NRXN) genes generate membrane-tethered proteins with highly variable extracellular regions. α-Neurexins contain 6 LNS (laminin G, neurexin, sex-hormone binding globule) domains and 3 EGF (epidermal growth factor)-like repeats, while β-neurexins are composed of only a single LNS domain. Neurexins are also extensively diversified at alternative splice sites (SS1–6) (Südhof, 2017Südhof T.C. Synaptic neurexin complexes: a molecular code for the logic of neural circuits.Cell. 2017; 171: 745-769Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Contrasting hevin as a promoter of neurexin-neuroligin transsynaptic bridges, the MDGA (MAM domain-containing glycosylphosphatidylinositol anchor protein) family of synaptic organizers (composed of six immunoglobulin [Ig] domains, one fibronectin III, and one MAM [meprin, A5 protein, PTPμ] domain tethered to the post-synaptic membrane) destabilizes neurexin-neuroligin transsynaptic bridges. They do so by binding to neuroligin and blocking the recruitment of neurexin (Elegheert et al., 2017Elegheert J. Cvetkovska V. Clayton A.J. Heroven C. Vennekens K.M. Smukowski S.N. Regan M.C. Jia W. Smith A.C. Furukawa H. et al.Structural mechanism for modulation of synaptic neuroligin-neurexin signaling by MDGA proteins.Neuron. 2017; 95: 896-913.e10Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar; Gangwar et al., 2017Gangwar S.P. Zhong X. Seshadrinathan S. Chen H. Machius M. Rudenko G. Molecular mechanism of MDGA1: regulation of neuroligin 2:neurexin trans-synaptic bridges.Neuron. 2017; 94: 1132-1141.e4Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar; Kim et al., 2017Kim J.A. Kim D. Won S.Y. Han K.A. Park D. Cho E. Yun N. An H.J. Um J.W. Kim E. et al.Structural insights into modulation of neurexin-neuroligin trans-synaptic adhesion by MDGA1/neuroligin-2 complex.Neuron. 2017; 94: 1121-1131.e6Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). But it is not known if, and how, MDGAs, hevin, and SPARC interplay. Furthermore, recent studies have drawn into question whether hevin interacts with the neurexin-neuroligin transsynaptic bridge directly, or even at all, and whether hevin's synaptogenic effect involves these transsynaptic bridges (Elegheert et al., 2017Elegheert J. Cvetkovska V. Clayton A.J. Heroven C. Vennekens K.M. Smukowski S.N. Regan M.C. Jia W. Smith A.C. Furukawa H. et al.Structural mechanism for modulation of synaptic neuroligin-neurexin signaling by MDGA proteins.Neuron. 2017; 95: 896-913.e10Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar; Gan and Südhof, 2020Gan K.J. Südhof T.C. SPARCL1 promotes excitatory but not inhibitory synapse formation and function independent of neurexins and neuroligins.J. Neurosci. 2020; 40: 8088-8102Crossref PubMed Scopus (16) Google Scholar). For SPARC, it is also not known if it binds neurexin and/or neuroligin. So, while hevin and SPARC are strategically positioned to impact neural circuitries in developing and mature brain, whether they carry out synergistic, opposing, or indeed independent functions, is not clear, in part due to the lack of structure-function relationships. Here, we establish interplay between the trio of proteins, hevin, SPARC, and MDGA, and elucidate the molecular mechanism. First, we show that the hevin FS-EC tandem has striking structural differences compared with SPARC, predominantly in the EC domain. We show that hevin and SPARC are both able to bind to neuroligins and neurexins, suggesting that they can act in concert to regulate neurexin-neuroligin transsynaptic bridges. We further show that the hevin FS domain, which is structurally conserved in SPARC, is sufficient to bind both neuroligin and neurexin with nanomolar affinity. Importantly, we find that hevin and MDGAs occupy overlapping binding sites on neuroligin, explaining how they compete with each other for neuroligin binding. Finally, we show that hevin, like SPARC, binds to collagen in solution, but we do not observe the same collagen-binding site as in the SPARC structure. Collectively, our results establish that hevin and SPARC can take part in a complex symphony of competing protein interactions, vying for synaptic organizers as well as interacting with extracellular matrix components. Key structural features provide rationales for both the shared and fundamentally different biological roles of hevin and SPARC in the brain. Hevin FS-EC and fragments were produced using baculovirus-mediated overexpression (Figures 1A and S1A). Because attempts to solve the hevin FS-EC X-ray crystal structure by molecular replacement using SPARC as a search model (PDB: 1BMO and 2V53) were unsuccessful, the Ca2+ ions in the EF-hands were substituted with holmium and the hevin crystal structure determined through single isomorphous replacement with anomalous scattering phasing (Table 1). The final model of hevin FS-EC was refined using data to 2.27 Å resolution (Rwork = 18.9%; Rfree = 24.1%) (Table 1). The hevin FS domain (V430-C509) is composed of an EGF-like repeat (V430-Q457) containing two disulfide bonds as well as a Kazal domain (D458-K509) containing a mixed α/β fold stabilized by three disulfide bonds (Figure 1B). The EC domain (C515-F664) houses two canonical helix-loop-helix EF-hands (EF1 and EF2) that each contain a Ca2+-binding site with seven-oxygen coordination; the EF-hands are connected by a short helical turn (D517FEV520) that packs adjacent to an elongated bundle of helices (αA, αBC, αC) (Figures 1B and 1C). A disulfide bond between C634 and C650 stabilizes EF2, with a second disulfide bond between C515 and C626 connecting EF1 to the linker (K510-T514) between the FS and EC domains. A large cleft separates the FS and EC domains. The interface between the two domains is small (∼200 Å2) and contains only a few residues from strand β5 of the FS domain and from helix αF and the loop connecting the two EF-hands in the EC domain (Figure 1B). Two hevin molecules (mol A and mol B) embrace each other tightly in the asymmetric unit of the crystal structure so that helices αA and αC from the EC domain of mol B interact with the EC domain and Kazal subdomain of the neighboring mol A, with Y539 from helix αA fitting like a key in the cleft; likewise, helices αA, and αBC from the EC domain of mol A insert into mol B in a similar manner (Figures 1D and S2A). Because mol A and mol B are stabilized by an additional Ca2+ ion binding between them, and the monomers adopt similarly “open” configurations between the FS and EC domains (Figures S2A and S2B), we tested whether the hevin FS-EC tandem behaves as a dimer in solution. However, in analytical ultracentrifugation, size-exclusion chromatography (SEC), and cross-linking studies, hevin FS-EC (hevin_C2) is monomeric in solution, even in an acidic buffer similar to that used for crystallization and/or in presence of increasing amounts of Ca2+ (Figures S2C–S2E). To gauge whether the FS and EC domains, which are kept apart in the crystal structure, are able to associate in solution, we used SEC and demonstrated that the isolated FS (hevin_C5) and EC (hevin_C6) domains do not associate in solution, at least not without the linker between the two domains (Figure 1E). Taken together, the hevin FS-EC tandem forms an elongated structure that exists as a monomer in solution, and the two domains appear independent of each other, suggesting that they can function autonomously.Table 1Data collection and refinement statisticsCrystalNative 1Native 2Holmium derivativeData collectionWavelength (Å)1.000011.000011.53532Space groupI212121I212121I212121Cell dimensions a, b, c (Å)57.66, 132.29, 149.2357.63, 131.78, 148.6257.75, 130.97, 148.00 α, β, γ (º)90, 90, 9090, 90, 9090, 90, 90Resolution (Å)33.00–2.27 (2.31–2.27)30.39–2.34 (2.38–2.34)43.00–3.41 (3.47–3.41)Mean I/σ(I)36.7 (1.9)40.0 (1.9)20.9 (1.0)Rmerge0.063 (1.266)0.062 (1.339)0.070 (0.889)Rpim0.019 (0.449)0.018 (0.490)0.029 (0.491)CC½0.998 (0.696)0.979 (0.672)0.972 (0.637)Unique reflections26,686 (1,309)23,942 (1,191)7,865 (322)Completeness (%)99.8 (99.1)99.9 (100.0)98.5 (85.2)Multiplicity12.7 (7.9)12.1 (8.3)6.7 (3.3)PhasingResolution (Å)–43.00–3.41No. of sites–5Bayes CC–49.7 ± 18.3Figure of merit–0.300RefinementResolution (Å)33.00–2.27 (2.36–2.27)Rwork/Rfree18.85/24.07 (22.75/31.33)Reflections used Rwork/Rfree25,958/1,298 (2,098/110)Non-hydrogen atoms3,636Protein3,403Water155Other78B factors (Å2), overall42.2 Protein42.3 Water37.9 Other50.4RMSD Bond lengths (Å)0.009 Bond angles (º)1.09Ramachandran plot (%) Favored97.3 Allowed2.7 Disallowed0.0Rotamer outliers, n (%)1 (0.27)CC, correlation coefficient; RMSD, root-mean-square deviation. Open table in a new tab CC, correlation coefficient; RMSD, root-mean-square deviation. The hevin FS-EC and SPARC FS-EC (Hohenester et al., 1997Hohenester E. Maurer P. Timpl R. Crystal structure of a pair of follistatin-like and EF-hand calcium-binding domains in BM-40.EMBO J. 1997; 16: 3778-3786Crossref PubMed Scopus (136) Google Scholar) structures feature striking differences despite their high level of sequence identity (56% for the FS domain and 63.3% for the EC domain, respectively) (Figures 2A and 2B ). Although the hevin and SPARC FS domain have similar folds (root-mean-square deviation [RMSD] = 1.2 Å for 79 Cα superimposable residues) (Figure 2C, left), the hevin FS domain is rotated away from the EC domain by ∼40º into an “open” form compared with SPARC (PDB: 1BMO) (Figure 2C, right). Consequently, the hevin FS-EC interface is smaller (∼200 Å2) compared with the SPARC FS-EC interface (∼370 Å2) even though most residues at the interface are conserved (Figure 2A). Unexpectedly, the hevin and SPARC EC domains show dramatic structural differences; only 94 Cα residues out of 128 (hevin) and 150 (SPARC) residues observed in the respective crystal structures superimpose with an RMSD of 1.5 Å (Figure 2C, right). The two EF-hands form a structurally conserved core, differing only in their connecting segment (Figure 2D). However, the region between the FS domain and EF-hand tandem, I512-N584 (I135-N223 in SPARC) is completely reorganized so that helices αA′, αA, αBC, and αC (here referred to as “variable helices”) now point away from the EF-hands (Figure 2D). Despite the high sequence identity (45 residues out of the 73-residue stretch I512-N584), the region containing these variable helices has completely rearranged: the single, contiguous helix αA in SPARC is broken up in hevin into two kinked helices (αA′-αA); helix αB in hevin is disordered; and helices αBC and αC adopt a different fold (Figure 2D). Interestingly, in SPARC, helices αA, αB, and αC play an important role in maintaining high-affinity Ca2+ binding of the EF-hand pair, in particular helix αA, a long amphiphilic helix that inserts into the hydrophobic core of the EF-hand pair (Hohenester et al., 1996Hohenester E. Maurer P. Hohenadl C. Timpl R. Jansonius J.N. Engel J. Structure of a novel extracellular Ca(2+)-binding module in BM-40.Nat. Struct. Biol. 1996; 3: 67-73Crossref PubMed Scopus (132) Google Scholar). Thus, despite significant sequence conservation, the hevin EC domain

De novo protein structure determination by heavy-atom soaking in lipidic cubic phase and SIRAS phasing using serial synchrotron crystallography.

During the past few years, serial crystallography methods have undergone continuous development and serial data collection has become well established at high-intensity synchrotron-radiation beamlines and XFEL radiation sources. However, the application of experimental phasing to serial crystallography data has remained a challenging task owing to the inherent inaccuracy of the diffraction data. Here, a particularly gentle method for incorporating heavy atoms into micrometre-sized crystals utilizing lipidic cubic phase (LCP) as a carrier medium is reported. Soaking in LCP prior to data collection offers a new, efficient and gentle approach for preparing heavy-atom-derivative crystals directly before diffraction data collection using serial crystallography methods. This approach supports effective phasing by utilizing a reasonably low number of diffraction patterns. Using synchrotron radiation and exploiting the anomalous scattering signal of mercury for single isomorphous replacement with anomalous scattering (SIRAS) phasing resulted in high-quality electron-density maps that were sufficient for building a complete structural model of proteinase K at 1.9 Å resolution using automatic model-building tools.

Introducing site-specific cysteines into nanobodies for mercury labelling allows de novo phasing of their crystal structures.

The generation of high-quality protein crystals and the loss of phase information during an X-ray crystallography diffraction experiment represent the major bottlenecks in the determination of novel protein structures. A generic method for introducing Hg atoms into any crystal independent of the presence of free cysteines in the target protein could considerably facilitate the process of obtaining unbiased experimental phases. Nanobodies (single-domain antibodies) have recently been shown to promote the crystallization and structure determination of flexible proteins and complexes. To extend the usability of nanobodies for crystallographic work, variants of the Nb36 nanobody with a single free cysteine at one of four framework-residue positions were developed. These cysteines could be labelled with fluorophores or Hg. For one cysteine variant (Nb36-C85) two nanobody structures were experimentally phased using single-wavelength anomalous dispersion (SAD) and single isomorphous replacement with anomalous signal (SIRAS), taking advantage of radiation-induced changes in Cys-Hg bonding. Importantly, Hg labelling influenced neither the interaction of Nb36 with its antigen complement C5 nor its structure. The results suggest that Cys-Hg-labelled nanobodies may become efficient tools for obtaining de novo phase information during the structure determination of nanobody-protein complexes.

Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

Serial crystallographic analysis of protein isomorphous replacement data from a mixture of native and derivative microcrystals.

A post-experimental identification/purification procedure similar to that described in Zhang et al. [(2015), IUCrJ, 2, 322-326] has been proposed for use in the treatment of multiphase protein serial crystallography (SX) diffraction snapshots. As a proof of concept, the procedure was tested using theoretical serial femtosecond crystallography (SFX) data from a mixture containing native and derivatized crystals of a protein. Two known proteins were taken as examples. Multiphase diffraction snapshots were subjected to two rounds of indexing using the program CrystFEL [White et al. (2012). J. Appl. Cryst. 45, 335-341]. In the first round, an ab initio indexing was performed to derive a set of approximate primitive unit-cell parameters, which are roughly the average of those from the native protein and the derivative. These parameters were then used in a second round of indexing as input to CrystFEL. The results were then used to separate the diffraction snapshots into two subsets corresponding to the native and the derivative. For each test sample, integration of the two subsets of snapshots separately led to two sets of three-dimensional diffraction intensities, one belonging to the native and the other to the derivative. Based on these two sets of intensities, a conventional single isomorphous replacement (SIR) procedure solved the structure easily.

An isomorphous replacement method for efficient de novo phasing for serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) holds great potential for structure determination of challenging proteins that are not amenable to producing large well diffracting crystals. Efficient de novo phasing methods are highly demanding and as such most SFX structures have been determined by molecular replacement methods. Here we employed single isomorphous replacement with anomalous scattering (SIRAS) for phasing and demonstrate successful application to SFX de novo phasing. Only about 20,000 patterns in total were needed for SIRAS phasing while single wavelength anomalous dispersion (SAD) phasing was unsuccessful with more than 80,000 patterns of derivative crystals. We employed high energy X-rays from SACLA (12.6 keV) to take advantage of the large anomalous enhancement near the LIII absorption edge of Hg, which is one of the most widely used heavy atoms for phasing in conventional protein crystallography. Hard XFEL is of benefit for de novo phasing in the use of routinely used heavy atoms and high resolution data collection.

Solution of the phase problem at non-atomic resolution by the phantom derivative method.

For a given unknown crystal structure (the target), n random structures, arbitrarily designed without any care for their chemical consistency and usually uncorrelated with the target, are sheltered in the same unit cell as the target structure and submitted to the same space-group symmetry. (These are called ancil structures.) The composite structures, whose electron densities are the sum of the target and of the ancil electron densities, are denoted derivatives. No observed diffraction amplitudes are available for them: in order to emphasize their unreal nature, the term phantom is added. The paper describes the theoretical basis by which the phantom derivative method may be used to phase the target structure. It may be guessed that 100-300 ancil structures may be sufficient for phasing a target structure, so that the phasing technique may be denoted as the multiple phantom derivative method. Ancil phases and amplitudes may be initially combined with observed target magnitudes to estimate amplitudes and phases of the corresponding phantom derivative. From them suitable algorithms allow one to obtain poor target phase estimates, which are often improved by combining the indications arising from each derivative. Probabilistic criteria are described to recognize the most reliable target phase estimates. The method is cyclic: the target phase estimates just obtained are used to improve amplitudes and phases of each derivative, which, in their turn, are employed to provide better target phase estimates. The method is a fully ab initio method, because it needs only the experimental data of the target structure. The term derivative is maintained with reference to SIR-MIR (single isomorphous replacement-multiple isomorphous replacement) techniques, even if its meaning is different: therefore the reader should think of the phantom derivative method more as a new method than as a variant of SIR-MIR techniques. The differences are much greater than the analogies. The paper also describes how phantom derivatives may be used for improving structure models obtained via other ab initio or non-ab initio techniques. The method is expected to be insensitive to the structural complexity of the target and to the target experimental data resolution, provided it is better than 4-6 Å.

Crystal structure of the fibre head domain of bovine adenovirus 4, a ruminant atadenovirus.

BackgroundIn adenoviruses, primary host cell recognition is generally performed by the head domains of their homo-trimeric fibre proteins. This first interaction is reversible. A secondary, irreversible interaction subsequently takes place via other adenovirus capsid proteins and leads to a productive infection. Although many fibre head structures are known for human mastadenoviruses, not many animal adenovirus fibre head structures have been determined, especially not from those belonging to adenovirus genera other than Mastadenovirus.MethodsWe constructed an expression vector for the fibre head domain from a ruminant atadenovirus, bovine adenovirus 4 (BAdV-4), consisting of amino acids 414–535, expressed the protein in Escherichia coli, purified it by metal affinity and cation exchange chromatography and crystallized it. The structure was solved using single isomorphous replacement plus anomalous dispersion of a mercury derivative and refined against native data that extended to 1.2 Å resolution.ResultsLike in other adenoviruses, the BAdV-4 fibre head monomer contains a beta-sandwich consisting of ABCJ and GHID sheets. The topology is identical to the fibre head of the other studied atadenovirus, snake adenovirus 1 (SnAdV-1), including the alpha-helix in the DG-loop, despite of them having a sequence identity of only 15 %. There are also differences which may have implications for ligand binding. Beta-strands G and H are longer and differences in several surface-loops and surface charge are observed.ConclusionsChimeric adenovirus fibres have been used to retarget adenovirus-based anti-cancer and gene therapy vectors. Ovine adenovirus 7 (OAdV-7), another ruminant atadenovirus, is intensively tested as a basis for such a vector. Here, we present the high-resolution atomic structure of the BAdV-4 fibre head domain, the second atadenovirus fibre head structure known and the first of an atadenovirus that infects a mammalian host. Future research should focus on the receptor-binding properties of these fibre head domains.

Minireview: A Comeback of Hg‐Derivatives in Protein Crystallography with Cys‐Modification

AbstractWhile a majority of protein crystal structures are solved by using molecular replacement (MR), it is limited by the available homologous models with sufficient sequence identity. Another popular method is multi‐wavelength anomalous diffraction (MAD) that uses Se‐Met and synchrotron radiations. However, proteins containing Se‐Met are sometimes difficult to obtain and/or crystallize. Consequently, other heavy‐atom derivatives are still used in crystal structure determination, while single isomorphous replacement (SIR) and single wavelength anomalous diffraction (SAD) are more popular than conventional multiple isomorphous replacement (MIR). Here, some recent cases of structure determination in which Hg serves as the only heavy‐atom type are discussed. For Hg‐derivatives, data collection does not need absorption spectrum scanning and it can be carried out using an in‐house X‐ray source. Derivatization with Hg can start directly by soaking the native crystals in Hg‐containing solutions, or use a specific mutant in the active site of an enzyme if it lacks intrinsic free cysteine residues. The rapid advance of bioinformatics and protein engineering has made this strategy of cysteine‐dependent Hg‐phasing by MIR, SIR and SAD an effective method of choice besides MR and MAD for crystal structure determination.

Determination of the structure of human α-l-iduronidase and structural basis of mucopolysaccharidosis type Ι

In this study, we tried to determine the crystal structure of human α-l-iduronidase (hIDUA) and elucidate the structural basis of mucopolysaccharidosis type I (MPS I). We crystallized and solved the structure of hIDUA by the single isomorphous replacement with anomalous scattering method. A deglycosylation study was performed to examine the involvement of N-glycan in the substrate interaction. Then, we built structural models of mutant hIDUA due to 57 amino acid substitutions that had been identified in 27 severe, 1 severeintermediate, 13 intermediate, 1 attenuated-intermediate and 15 attenuated type MPS I patients by means of homology modeling using the crystal hIDUA structure as a template. The structural changes were examined by calculating the root-mean-square distances (RMSDs) and the number of atoms influenced by the amino acid replacements. We solved the crystal structures of apoand iduronic acid -bound hIDUA consisting of three domains: a triosephosphate isomerase barrel fold, a β-sandwich domain and an immunoglobulin-like domain. The mannose residue of the N-glycan attached to N372 constituted a part of the substrate-binding pocket and interacted directly with a substrate. The result of deglycosylation study showed that the hIDUA enzyme activity was highly correlated with the N-glycan attached to N372. Structural analysis revealed that W306 interacts with the N-glycan at N372 and the W306L mutation may cause MPS I through the effect on the conformation of theN-glycan atN372. Further structural study revealed that the RMSD values and the numbers of atoms influenced by amino acid substitutions found in severeMPS I patients were larger than those in intermediate and attenuated MPS I ones, suggesting that the structural changes of hIDUA tended to be correlated with the severity of the disease. Coloring of atoms influenced by an amino acid substitution was performed in each case. On the basis of these results, we constructed a database and disclosed the structural information involving MPS I. Structural investigation is useful for elucidating the basis of MPS I.

Desensitization to galsulfase for the treatment of recurrent infusion association reactions in a child with MPS VI

In this study, we tried to determine the crystal structure of human α-l-iduronidase (hIDUA) and elucidate the structural basis of mucopolysaccharidosis type I (MPS I). We crystallized and solved the structure of hIDUA by the single isomorphous replacement with anomalous scattering method. A deglycosylation study was performed to examine the involvement of N-glycan in the substrate interaction. Then, we built structural models of mutant hIDUA due to 57 amino acid substitutions that had been identified in 27 severe, 1 severeintermediate, 13 intermediate, 1 attenuated-intermediate and 15 attenuated type MPS I patients by means of homology modeling using the crystal hIDUA structure as a template. The structural changes were examined by calculating the root-mean-square distances (RMSDs) and the number of atoms influenced by the amino acid replacements. We solved the crystal structures of apoand iduronic acid -bound hIDUA consisting of three domains: a triosephosphate isomerase barrel fold, a β-sandwich domain and an immunoglobulin-like domain. The mannose residue of the N-glycan attached to N372 constituted a part of the substrate-binding pocket and interacted directly with a substrate. The result of deglycosylation study showed that the hIDUA enzyme activity was highly correlated with the N-glycan attached to N372. Structural analysis revealed that W306 interacts with the N-glycan at N372 and the W306L mutation may cause MPS I through the effect on the conformation of theN-glycan atN372. Further structural study revealed that the RMSD values and the numbers of atoms influenced by amino acid substitutions found in severeMPS I patients were larger than those in intermediate and attenuated MPS I ones, suggesting that the structural changes of hIDUA tended to be correlated with the severity of the disease. Coloring of atoms influenced by an amino acid substitution was performed in each case. On the basis of these results, we constructed a database and disclosed the structural information involving MPS I. Structural investigation is useful for elucidating the basis of MPS I.

Levomepromazine as a treatment for non-epileptic movement disorder in advanced neurodegenerative lysosomal disorders

In this study, we tried to determine the crystal structure of human α-l-iduronidase (hIDUA) and elucidate the structural basis of mucopolysaccharidosis type I (MPS I). We crystallized and solved the structure of hIDUA by the single isomorphous replacement with anomalous scattering method. A deglycosylation study was performed to examine the involvement of N-glycan in the substrate interaction. Then, we built structural models of mutant hIDUA due to 57 amino acid substitutions that had been identified in 27 severe, 1 severeintermediate, 13 intermediate, 1 attenuated-intermediate and 15 attenuated type MPS I patients by means of homology modeling using the crystal hIDUA structure as a template. The structural changes were examined by calculating the root-mean-square distances (RMSDs) and the number of atoms influenced by the amino acid replacements. We solved the crystal structures of apoand iduronic acid -bound hIDUA consisting of three domains: a triosephosphate isomerase barrel fold, a β-sandwich domain and an immunoglobulin-like domain. The mannose residue of the N-glycan attached to N372 constituted a part of the substrate-binding pocket and interacted directly with a substrate. The result of deglycosylation study showed that the hIDUA enzyme activity was highly correlated with the N-glycan attached to N372. Structural analysis revealed that W306 interacts with the N-glycan at N372 and the W306L mutation may cause MPS I through the effect on the conformation of theN-glycan atN372. Further structural study revealed that the RMSD values and the numbers of atoms influenced by amino acid substitutions found in severeMPS I patients were larger than those in intermediate and attenuated MPS I ones, suggesting that the structural changes of hIDUA tended to be correlated with the severity of the disease. Coloring of atoms influenced by an amino acid substitution was performed in each case. On the basis of these results, we constructed a database and disclosed the structural information involving MPS I. Structural investigation is useful for elucidating the basis of MPS I.

A case study on the treatment of protein SIRAS data.

A case study has been made on the treatment of the SIRAS (single isomorphous replacement with anomalous scattering) data of the originally unknown protein LegC3N. An alternative treatment has been proposed which led to improved results in this particular test case. The treatment involves iterative direct-method SAD (single-wavelength anomalous diffraction) phasing and direct-method-aided model completion, both of which are implanted in the IPCAS (Iterative Protein Crystal-structure Automatic Solution) pipeline. Apart from the experimental data, a simulated SIRAS data set for LegC3N with the derivative data truncated to 5.0 Å resolution has also been tested. SAD phasing and phase/model extension in PHENIX without direct methods failed to solve the structure using these simulated SIRAS data. However, the procedure proposed here involving direct methods in both SAD phasing and phase/model extension led to a nearly complete structure model. This shows the potential ability of treating SIRAS data with a derivative diffracting to lower resolution.

Alternative phasing method in macromolecular crystallography

Selenium is the most widely used heavy atom for experimental phasing, either by single anomalous scattering (SAD) or multiple-wavelength anomalous diffraction (MAD) procedures. The use of the single isomorphous replacement (SIR) or single isomorphous replacement with anomalous scattering (SIRAS) phasing procedure with selenomethionine (Mse) containing proteins is not so commonly used, as it requires isomorphous native data. Several non-redundant X-ray diffraction data sets from various Mse derivatised protein crystals were collected at energies far below the absorption edge before and after exposing the crystal to ultraviolet (UV) radiation with 266 nm lasers. A detailed analysis revealed that significant changes in diffracted intensities were induced by ultraviolet irradiation whilst retaining crystal isomorphism. These intensity changes allowed the crystal structures to be solved by the radiation damage-induced phasing (RIP) technique [1]. These can be coupled with the anomalous signal from the dataset collected at the selenium absorption edge to obtain SIRAS phases in a UV-RIPAS phasing experiment [2]. Inspection of the crystal structures and electron-density maps demonstrated that covalent bonds between selenium and carbon at all sites located in the core of the proteins or in a hydrophobic environment were much more susceptible to UV radiation-induced cleavage than other bonds typically present in Mse proteins. The rapid UV radiation-induced bond cleavage opens a reliable new paradigm for phasing at synchrotron [1,2] and at in-house X-ray source [3].

The first protein Structure Determinations

Max Perutz had been studying the structure of haemoglobin for 21 years using highly original and creative techniques, when I arrived in Cambridge in the summer of 1958. Most recently he had explored the use of isomorphous replacement by substituting specific amino acids with heavy metal compounds. Together with Ann Cullis, he had collected three dimensional diffraction data on native haemoglobin crystals and on three heavy atom derivatives. But now Max needed help in analyzing this data. I had had experience in determining three small aromatic hydrocarbon structures as a graduate student with J.M. Robertson at the University of Glasgow using only mechanical hand calculators, a slide rule and Beevers-Lipson strips. At the University of Minnesota, as a post doc with Bill Lipscomb, I had learned how to program one of the earliest commercial computers and solved a plant terpenoid structure in three dimensions. Thus, when I joined Max's lab, I was soon busy programming the bran new "home build" EDSAC2 computer to determine and refine the heavy atom parameters for phase determination by isomorphous replacement. When we calculated the 5.5Å resolution horse oxy haemoglobin electron density map in the summer of 1959 we were amazed to see that the two polypeptide chains of haemoglobin each had the same fold as that of myoglobin, another oxygen carrying protein determined by John Kendrew at 6Å resolution two years earlier at the Cavendish. John and his group were extending the structure of myoglobin to 2Å resolution. The high resolution map of myglobin was calculated using my Fourier program the week after the low resolution haemoglobin map. We then saw the atomic structure of α-helices as had been predicted by Linus Pauling. Seeing evolution for the first time at a molecular level has inspired my thinking and choice of research topics ever since. In the following few years, stimulated by these discoveries , David Blow (who had returned from the US later that summer after two years of post-doc studies) and I developed the single isomorphous replacement method, the use of anomalous dispersion for phase determination, phase combination procedures and, most importantly, molecular replacement.

Structure of the 3.3MDa, in vitro assembled, hubless bacteriophage T4 baseplate.

The bacteriophage T4 baseplate is the control center of the virus, where the recognition of an Escherichiacoli host by the long tail fibers is translated into a signal to initiate infection. The short tail fibers unfold from the baseplate for firm attachment to the host, followed by shrinkage of the tail sheath that causes the tail tube to enter and cross the periplasmic space ending with injection of the genome into the host. During this process, the 6.5MDa baseplate changes its structure from a "dome" shape to a "star" shape. An in vitro assembled hubless baseplate has been crystallized. It consists of six copies of the recombinantly expressed trimeric gene product (gp) 10, monomeric gp7, dimeric gp8, dimeric gp6 and monomeric gp53. The diffraction pattern extends, at most, to 4.0Å resolution. The known partial structures of gp10, gp8, and gp6 and their relative position in the baseplate derived from earlier electron microscopy studies were used for molecular replacement. An electron density map has been calculated based on molecular replacement, single isomorphous replacement with anomalous dispersion data and 2-fold non-crystallographic symmetry averaging between two baseplate wedges in the crystallographic asymmetric unit. The current electron density map indicates that there are structural changes in the gp6, gp8, and gp10 oligomers compared to their structures when separately crystallized. Additional density is also visible corresponding to gp7, gp53 and the unknown parts of gp10 and gp6.

Exploiting subtle structural differences in heavy-atom derivatives for experimental phasing.

Structure determination using the single isomorphous replacement (SIR) or single-wavelength anomalous diffraction (SAD) methods with weak derivatives remains very challenging. In a recent structure determination of glycoprotein E2 from bovine viral diarrhea virus, three isomorphous uranium-derivative data sets were merged to obtain partially interpretable initial experimental maps. Small differences between them were then exploited by treating them as three independent SAD data sets plus three circular pairwise SIR data sets to improve the experimental maps. Here, how such subtle structural differences were exploited for experimental phasing is described in detail. The basis for why this approach works is also provided: the effective resolution of isomorphous signals between highly isomorphous derivatives is often much higher than the effective resolution of the anomalous signals of individual derivative data sets. Hence, the new phasing approaches outlined here will be generally applicable to structure determinations involving weak derivatives.