In vitro evolution of an influenza broadly neutralizing antibody is modulated by hemagglutinin receptor specificity
Identifieur interne : 000272 ( Pmc/Checkpoint ); précédent : 000271; suivant : 000273In vitro evolution of an influenza broadly neutralizing antibody is modulated by hemagglutinin receptor specificity
Auteurs : Nicholas C. Wu [États-Unis] ; Geramie Grande [États-Unis] ; Hannah L. Turner [États-Unis] ; Andrew B. Ward [États-Unis] ; Jia Xie [États-Unis] ; Richard A. Lerner [États-Unis] ; Ian A. Wilson [États-Unis]Source :
- Nature Communications [ 2041-1723 ] ; 2017.
Abstract
The relatively recent discovery and characterization of human broadly neutralizing antibodies (bnAbs) against influenza virus provide valuable insights into antiviral and vaccine development. However, the factors that influence the evolution of high-affinity bnAbs remain elusive. We therefore explore the functional sequence space of bnAb C05, which targets the receptor-binding site (RBS) of influenza haemagglutinin (HA) via a long CDR H3. We combine saturation mutagenesis with yeast display to enrich for C05 variants of CDR H3 that bind to H1 and H3 HAs. The C05 variants evolve up to 20-fold higher affinity but increase specificity to each HA subtype used in the selection. Structural analysis reveals that the fine specificity is strongly influenced by a highly conserved substitution that regulates receptor binding in different subtypes. Overall, this study suggests that subtle natural variations in the HA RBS between subtypes and species may differentially influence the evolution of high-affinity bnAbs.
Url:
DOI: 10.1038/ncomms15371
PubMed: 28504265
PubMed Central: 5440694
Affiliations:
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Wu</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Integrative Structural and Computational Biology, The Scripps Research Institute</institution>, La Jolla, California 92037,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Grande, Geramie" sort="Grande, Geramie" uniqKey="Grande G" first="Geramie" last="Grande">Geramie Grande</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Chemistry, The Scripps Research Institute</institution>, La Jolla, California 92037,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Turner, Hannah L" sort="Turner, Hannah L" uniqKey="Turner H" first="Hannah L." last="Turner">Hannah L. 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Ward</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Integrative Structural and Computational Biology, The Scripps Research Institute</institution>, La Jolla, California 92037,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Xie, Jia" sort="Xie, Jia" uniqKey="Xie J" first="Jia" last="Xie">Jia Xie</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Chemistry, The Scripps Research Institute</institution>, La Jolla, California 92037,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Lerner, Richard A" sort="Lerner, Richard A" uniqKey="Lerner R" first="Richard A." last="Lerner">Richard A. Lerner</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Chemistry, The Scripps Research Institute</institution>, La Jolla, California 92037,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Wilson, Ian A" sort="Wilson, Ian A" uniqKey="Wilson I" first="Ian A." last="Wilson">Ian A. Wilson</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Integrative Structural and Computational Biology, The Scripps Research Institute</institution>, La Jolla, California 92037,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a3"><institution>The Skaggs Institute for Chemical Biology, The Scripps Research Institute</institution>, La Jolla, California 92037,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Nature Communications</title><idno type="eISSN">2041-1723</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The relatively recent discovery and characterization of human broadly neutralizing antibodies (bnAbs) against influenza virus provide valuable insights into antiviral and vaccine development. However, the factors that influence the evolution of high-affinity bnAbs remain elusive. We therefore explore the functional sequence space of bnAb C05, which targets the receptor-binding site (RBS) of influenza haemagglutinin (HA) via a long CDR H3. We combine saturation mutagenesis with yeast display to enrich for C05 variants of CDR H3 that bind to H1 and H3 HAs. The C05 variants evolve up to 20-fold higher affinity but increase specificity to each HA subtype used in the selection. Structural analysis reveals that the fine specificity is strongly influenced by a highly conserved substitution that regulates receptor binding in different subtypes. Overall, this study suggests that subtle natural variations in the HA RBS between subtypes and species may differentially influence the evolution of high-affinity bnAbs.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Neher, R A" uniqKey="Neher R">R. A. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Nat Commun</journal-id><journal-id journal-id-type="iso-abbrev">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type="epub">2041-1723</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">28504265</article-id><article-id pub-id-type="pmc">5440694</article-id><article-id pub-id-type="pii">ncomms15371</article-id><article-id pub-id-type="doi">10.1038/ncomms15371</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title><italic>In vitro</italic> evolution of an influenza broadly neutralizing antibody is modulated by hemagglutinin receptor specificity</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Wu</surname><given-names>Nicholas C.</given-names></name><xref ref-type="aff" rid="a1">1</xref><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0002-9078-6697</contrib-id></contrib><contrib contrib-type="author"><name><surname>Grande</surname><given-names>Geramie</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Turner</surname><given-names>Hannah L.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ward</surname><given-names>Andrew B.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Xie</surname><given-names>Jia</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Lerner</surname><given-names>Richard A.</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Wilson</surname><given-names>Ian A.</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a3">3</xref><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0002-6469-2419</contrib-id></contrib><aff id="a1"><label>1</label><institution>Department of Integrative Structural and Computational Biology, The Scripps Research Institute</institution>, La Jolla, California 92037,<country>USA</country></aff><aff id="a2"><label>2</label><institution>Department of Chemistry, The Scripps Research Institute</institution>, La Jolla, California 92037,<country>USA</country></aff><aff id="a3"><label>3</label><institution>The Skaggs Institute for Chemical Biology, The Scripps Research Institute</institution>, La Jolla, California 92037,<country>USA</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>jiaxie@scripps.edu</email></corresp><corresp id="c2"><label>b</label><email>wilson@scripps.edu</email></corresp></author-notes><pub-date pub-type="epub"><day>15</day><month>05</month><year>2017</year></pub-date><pub-date pub-type="collection"><year>2017</year></pub-date><volume>8</volume><elocation-id>15371</elocation-id><history><date date-type="received"><day>20</day><month>10</month><year>2016</year></date><date date-type="accepted"><day>24</day><month>03</month><year>2017</year></date></history><permissions><copyright-statement>Copyright © 2017, The Author(s)</copyright-statement><copyright-year>2017</copyright-year><copyright-holder>The Author(s)</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract><p>The relatively recent discovery and characterization of human broadly neutralizing antibodies (bnAbs) against influenza virus provide valuable insights into antiviral and vaccine development. However, the factors that influence the evolution of high-affinity bnAbs remain elusive. We therefore explore the functional sequence space of bnAb C05, which targets the receptor-binding site (RBS) of influenza haemagglutinin (HA) via a long CDR H3. We combine saturation mutagenesis with yeast display to enrich for C05 variants of CDR H3 that bind to H1 and H3 HAs. The C05 variants evolve up to 20-fold higher affinity but increase specificity to each HA subtype used in the selection. Structural analysis reveals that the fine specificity is strongly influenced by a highly conserved substitution that regulates receptor binding in different subtypes. Overall, this study suggests that subtle natural variations in the HA RBS between subtypes and species may differentially influence the evolution of high-affinity bnAbs.</p></abstract><abstract abstract-type="web-summary"><p>Broadly neutralizing antibodies (bnAbs) against influenza hemagglutinin (HA) have yielded insights for antiviral development. Here, the authors employ saturated mutagenesis of the paratope region of a bnAb combined with yeast display screening using H1 and H3 HAs, and find that a tradeoff exists between Ab affinity and breadth that influenced by disparate modes of receptor binding.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>Schematic representation of the yeast display screening.</title><p>(<bold>a</bold>) Schematic representation of the dual promoter yeast expression plasmid encoding the C05 Fab is shown. The arrows indicate the locations of the SfiI sites employed for cloning. (<bold>b</bold>) Saturation mutagenesis was applied to the six amino acids of C05 CDR H3 that interact closely or are buried in the receptor-binding site of influenza hemagglutinin (HA). A C05 Fab plasmid mutant library, which consists of at least 10<sup>8</sup> clones, was created. The plasmid mutant library was transformed into yeast strain EBY100 to generate a yeast surface display library. Different variants of C05 Fab were displayed on yeast cells. This yeast surface display library was then subjected to selection for HA-binding affinity. We used fluorescence-activated cell sorting (FACS) to enrich yeast cells that were able to interact with PE-conjugated influenza HA. The post-selection pool was then expanded and subjected to another round of selection. For each of H1, H3, and H5 HAs, three rounds of selection was performed. Variants that were able to bind to the HA would enrich in occurrence frequency throughout the screening process. Variants with higher affinity would enrich to a higher frequency. The plasmid mutant library and each of the post-selection mutant libraries were next-generation sequenced to monitor the frequency change of each variant.</p></caption><graphic xlink:href="ncomms15371-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>Monitoring the selection process by next-generation sequencing.</title><p>(<bold>a</bold>) The occurrence frequency of each amino acid at each of the 6 residues of interest is shown as a heatmap. (<bold>b</bold>) For each round of selection, a sequence logo was generated using the amino-acid sequences of the top 10 variants with the highest occurrence frequency.</p></caption><graphic xlink:href="ncomms15371-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>Validation for C05 variants binding against H1 HA and H3 HA.</title><p>(<bold>a</bold>) Supernatant of 293T cells transfected with plasmids encoding the indicated C05 Fab variants was used to estimate the affinity of C05 Fab variants against immobilized SI06 HA (H1) and A/Perth/16/2009 (Perth09) HA (H3). The relative <italic>K</italic><sub>d</sub> of WT C05 Fab was set as 1, which is indicated by the green line. For visualizing purpose, the relative <italic>K</italic><sub>d</sub> is capped at 100. Variants that had an H3-specific binding profile and cited in the main text are boxed. (<bold>b</bold>) The effect of changing position 100d from Ala to Ser on binding kinetics was investigated. Four pairs of C05 variants, in which variants within each pair were related by an Ala to Ser substitution at position 100d, were included in our validation experiment (<bold>a</bold>). We computed the fold change in <italic>k</italic>on and <italic>k</italic>off by comparing the binding kinetics in the variant that carried a Ser at position 4 against the variant that carried an Ala at position 4. For visualization, for a given pair of variants (<italic>i</italic>→<italic>j</italic>), the fold change in <italic>k</italic>on was calculated as on rate of <italic>j</italic> divided by on rate of <italic>i</italic>, whereas fold change in <italic>k</italic><sub>off</sub> was calculated as off rate of <italic>i</italic> divided by off rate of <italic>j</italic>, such that a positive value at the log scale indicates a positive effect in binding. (<bold>c</bold>) The occurrence frequencies of all observed variants in the sequencing data (a total of 848,630) in round 3 post-selection library and round 1 post-selection library are compared and shown as a scatterplot. Variants with >0.4% occurrence frequency in H1 round 3 post-selection library and <0.003% in H3 round 3 post-selection library were classified as ‘H1 specialists' (shaded in red in the scatterplot). Variants with >0.3% occurrence frequency in H3 round 3 post-selection library and <0.001% in H1 round 3 post-selection library were classified as ‘H3 specialists' (shaded in lilac in the scatterplot).</p></caption><graphic xlink:href="ncomms15371-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>Interaction between VPGSGW and HK68/H3 HA1.</title><p>(<bold>a</bold>) The crystal structure of VPGSGW in complex with HA1 exhibits a similar conformation to that of C05 WT in complex with HA1 (PDB: 4FP8)<xref ref-type="bibr" rid="b21">21</xref>. (<bold>b</bold>) There are two VPGSGW-HA1 complexes in the asymmetric unit of the crystal structure. The orientations of the serine at 100d differ between those two complexes. Intramolecular hydrogen bonds are shown for the two different conformations of CDR H3 of VPGSGW. (<bold>c</bold>) Interaction of the HA RBS (grey) with CDR H3 (pink) is shown. Hydrogen bonds are represented by green dashed lines. Water molecules are represented by red spheres. A putative sodium ion is represented by the purple sphere. For clarity, only G100c, S100d, G100e, and W100f on the CDR H3 of VPGSGW are displayed.</p></caption><graphic xlink:href="ncomms15371-f4"></graphic></fig><fig id="f5"><label>Figure 5</label><caption><title>E190D in the HA RBS favors binding against VPGSGW.</title><p>The hydrogen bond network involving S100d of the CDR H3 of VPGSGW is shown for (<bold>a</bold>) binding against HA RBS of HK68/H3, and (<bold>b</bold>) binding against HA RBS of HK68/H3 that carried mutation E190D, which is modelled based on the crystal structure of VPGSGW-HK68/H3 HA1. A putative sodium ion is represented by the purple sphere. For visual clarity, only G100c, S100d, G100e, and W100f on the Fab are displayed. (<bold>c</bold>) The affinities of WT C05 Fab (VVSAGW) and a C05 Fab variant (VPGSGW) against the HA from A/Hong Kong/1/1968 (wild type: E190; E190D mutant: D190) are shown as a bar chart. (<bold>d</bold>,<bold>e</bold>) The neutralizing activity of WT C05 and VPGSGW in IgG format against (<bold>d</bold>) SI06-HA/WSN virus, and (<bold>e</bold>) A/Aichi/2/68 virus were measured by cell viability assay. SI06-HA/WSN virus was generated based on WSN, in which the HA ectodomain was replaced by that from SI06 (see Methods). Colour code is the same as that of <bold>c</bold>. Mean value across three replicates is shown and the error bar represents the s.d. The large error bar in VPGSGW at 100 μg ml<sup>−1</sup> against A/Aichi/2/68 virus is due to complete protection in one but not in the other two replicates.</p></caption><graphic xlink:href="ncomms15371-f5"></graphic></fig><table-wrap position="float" id="t1"><label>Table 1</label><caption><title>Binding affinity of C05 variants against hemagglutinins from different influenza strains.</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"><italic><bold>K</bold></italic><sub><bold>d</bold></sub><bold>in nM</bold></th><th align="center" valign="top" charoff="50"><bold>Residue 190</bold></th><th align="center" valign="top" charoff="50"><bold>VVSAGW (WT)</bold></th><th align="center" valign="top" charoff="50"><bold>VPGSGW</bold></th><th align="center" valign="top" charoff="50"><bold>VVSSGW</bold></th><th align="center" valign="top" charoff="50"><bold>VTGSGW</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">A/Solomon Islands/3/2006 (H1N1)</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">91.4±0.9</td><td align="center" valign="top" charoff="50">15.0±0.2</td><td align="center" valign="top" charoff="50">4.6±0.6</td><td align="center" valign="top" charoff="50">115.8±1.9</td></tr><tr><td align="left" valign="top" charoff="50">A/New Caledonia/20/1999 (H1N1)</td><td align="center" valign="top" charoff="50">N</td><td align="center" valign="top" charoff="50">6.3±0.1</td><td align="center" valign="top" charoff="50">0.6±0.0</td><td align="center" valign="top" charoff="50">2.9±0.1</td><td align="center" valign="top" charoff="50">1.1±0.2</td></tr><tr><td align="left" valign="top" charoff="50">A/Beijing/262/1995 (H1N1)</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">23.1±0.6</td><td align="center" valign="top" charoff="50">12.6±0.7</td><td align="center" valign="top" charoff="50">259.3±2.8</td><td align="center" valign="top" charoff="50">182.3±0.5</td></tr><tr><td align="left" valign="top" charoff="50">A/WSN/1933 (H1N1)</td><td align="center" valign="top" charoff="50">E</td><td align="center" valign="top" charoff="50">169.4±1.0</td><td align="center" valign="top" charoff="50">719.6±2.9</td><td align="center" valign="top" charoff="50">727.7±20.5</td><td align="center" valign="top" charoff="50">1,539±42</td></tr><tr><td align="left" valign="top" charoff="50">A/Japan/305/1957 (H2N2)</td><td align="center" valign="top" charoff="50">E</td><td align="center" valign="top" charoff="50">1,701±46</td><td align="center" valign="top" charoff="50">>5,000</td><td align="center" valign="top" charoff="50">>5,000</td><td align="center" valign="top" charoff="50">>5,000</td></tr><tr><td align="left" valign="top" charoff="50">A/Hong Kong/1/1968 (H3N2)</td><td align="center" valign="top" charoff="50">E</td><td align="center" valign="top" charoff="50">327.8±12.0</td><td align="center" valign="top" charoff="50">958.8±45.6</td><td align="center" valign="top" charoff="50">1,100±94</td><td align="center" valign="top" charoff="50">1,860±83</td></tr><tr><td align="left" valign="top" charoff="50">A/Panama/2007/1999 (H3N2)</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">926.3±12.1</td><td align="center" valign="top" charoff="50">1,306±12</td><td align="center" valign="top" charoff="50">1,290±71</td><td align="center" valign="top" charoff="50">4,335±207</td></tr><tr><td align="left" valign="top" charoff="50">A/Perth/16/2009 (H3N2)<xref ref-type="fn" rid="t1-fn1">*</xref></td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">23.4±0.1</td><td align="center" valign="top" charoff="50">43.6±1.0</td><td align="center" valign="top" charoff="50">43.7±0.5</td><td align="center" valign="top" charoff="50">125.1±1.2</td></tr></tbody></table><table-wrap-foot><fn id="t1-fn1"><p><sup>*</sup>C05 is able to make additional interaction with A/Perth/16/2009 (H3N2) via CDR H1 (ref. <xref ref-type="bibr" rid="b21">21</xref>).</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t2"><label>Table 2</label><caption><title>X-ray data collection and refinement statistics.</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center"></col></colgroup><tbody valign="top"><tr><td align="left" valign="top" charoff="50"><italic>Data collection</italic></td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"> Beamline</td><td align="center" valign="top" charoff="50">SSRL 12-2</td></tr><tr><td align="left" valign="top" charoff="50"> Wavelength (Å)</td><td align="center" valign="top" charoff="50">0.9795</td></tr><tr><td align="left" valign="top" charoff="50"> Space group</td><td align="center" valign="top" charoff="50">P4<sub>3</sub></td></tr><tr><td align="left" valign="top" charoff="50"> Unit cell parameters (Å)</td><td align="center" valign="top" charoff="50">a=b=88.3, c=255.0</td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="center" valign="top" charoff="50">50-1.97 (2.04-1.97)<xref ref-type="fn" rid="t2-fn2">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> Unique reflections</td><td align="center" valign="top" charoff="50">136,341 (13,627)<xref ref-type="fn" rid="t2-fn2">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> Redundancy</td><td align="center" valign="top" charoff="50">6.3 (6.2)<xref ref-type="fn" rid="t2-fn2">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> Completeness (%)</td><td align="center" valign="top" charoff="50">99.5 (99.5)<xref ref-type="fn" rid="t2-fn2">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> <<italic>I</italic>/<italic>σ</italic><sub>I</sub>></td><td align="center" valign="top" charoff="50">15.4 (2.0)<xref ref-type="fn" rid="t2-fn2">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>sym</sub><xref ref-type="fn" rid="t2-fn3">†</xref></td><td align="center" valign="top" charoff="50">0.16 (0.82)<xref ref-type="fn" rid="t2-fn2">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>pim</sub><xref ref-type="fn" rid="t2-fn3">†</xref></td><td align="center" valign="top" charoff="50">0.07 (0.35)<xref ref-type="fn" rid="t2-fn2">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> CC<sub>1/2</sub><xref ref-type="fn" rid="t2-fn4">‡</xref></td><td align="center" valign="top" charoff="50">0.99 (0.72)<xref ref-type="fn" rid="t2-fn2">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> <italic>Z</italic><sub>a</sub><xref ref-type="fn" rid="t2-fn5">§</xref></td><td align="center" valign="top" charoff="50">2</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"><italic>Refinement statistics</italic></td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="center" valign="top" charoff="50">50–1.97</td></tr><tr><td align="left" valign="top" charoff="50"> Reflections (work)</td><td align="center" valign="top" charoff="50">130,202</td></tr><tr><td align="left" valign="top" charoff="50"> Reflections (test)</td><td align="center" valign="top" charoff="50">6,751</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>cryst</sub>(%)<xref ref-type="fn" rid="t2-fn6">‖</xref>/<italic>R</italic><sub>free</sub>(%)<xref ref-type="fn" rid="t2-fn7">¶</xref></td><td align="center" valign="top" charoff="50">18.1/20.8</td></tr><tr><td align="left" valign="top" charoff="50"> No. of atoms</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"> Protein</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"> HA1</td><td align="center" valign="top" charoff="50">3,987</td></tr><tr><td align="left" valign="top" charoff="50"> Fab</td><td align="center" valign="top" charoff="50">6,846</td></tr><tr><td align="left" valign="top" charoff="50"> Water</td><td align="center" valign="top" charoff="50">1,113</td></tr><tr><td align="left" valign="top" charoff="50"> Ion</td><td align="center" valign="top" charoff="50">1</td></tr><tr><td align="left" valign="top" charoff="50"> Glycan</td><td align="center" valign="top" charoff="50">56</td></tr><tr><td align="left" valign="top" charoff="50"> Other<xref ref-type="fn" rid="t2-fn8">#</xref></td><td align="center" valign="top" charoff="50">90</td></tr><tr><td align="left" valign="top" charoff="50"> Average <italic>B</italic>-value (Å<sup>2</sup>)</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"> Protein</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"> HA1</td><td align="center" valign="top" charoff="50">37.2</td></tr><tr><td align="left" valign="top" charoff="50"> Fab</td><td align="center" valign="top" charoff="50">30.5</td></tr><tr><td align="left" valign="top" charoff="50"> Water</td><td align="center" valign="top" charoff="50">40.0</td></tr><tr><td align="left" valign="top" charoff="50"> Ion</td><td align="center" valign="top" charoff="50">46.3</td></tr><tr><td align="left" valign="top" charoff="50"> Glycan</td><td align="center" valign="top" charoff="50">74.4</td></tr><tr><td align="left" valign="top" charoff="50"> Other<xref ref-type="fn" rid="t2-fn8">#</xref></td><td align="center" valign="top" charoff="50">77.2</td></tr><tr><td align="left" valign="top" charoff="50"> Wilson <italic>B</italic>-value (Å<sup>2</sup>)</td><td align="center" valign="top" charoff="50">22.7</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"><italic>R.m.s.d. from ideal geometry</italic></td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"> Bond length (Å)</td><td align="center" valign="top" charoff="50">0.013</td></tr><tr><td align="left" valign="top" charoff="50"> Bond angle (°)</td><td align="center" valign="top" charoff="50">1.60</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"><italic>Ramachandran statistics (%)</italic><xref ref-type="fn" rid="t2-fn9">**</xref></td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"> Favored</td><td align="center" valign="top" charoff="50">96.7</td></tr><tr><td align="left" valign="top" charoff="50"> Outliers</td><td align="center" valign="top" charoff="50">0.2</td></tr></tbody></table><table-wrap-foot><fn id="t2-fn1"><p>r.m.s.d., root mean squared deviation.</p></fn><fn id="t2-fn2"><p><sup>*</sup>Numbers in parentheses refer to the highest resolution shell.</p></fn><fn id="t2-fn3"><p><sup>†</sup><italic>R</italic><sub>sym</sub>=Σ<sub>hkl</sub>Σ<sub><italic>i</italic></sub>|<italic>I</italic><sub>hkl<italic>,i</italic></sub>−<<italic>I</italic><sub>hkl</sub>>|/Σ<sub>hkl</sub>Σ<sub><italic>i</italic></sub><italic>I</italic><sub>hkl<italic>,i</italic></sub> and <italic>R</italic><sub>pim</sub>=Σ<sub>hkl</sub>(1/(<italic>n</italic>−1))<sup>1/2</sup> Σ<sub><italic>i</italic></sub>|<italic>I</italic><sub>hkl<italic>,i</italic></sub>−<<italic>I</italic><sub>hkl</sub>>|/Σ<sub>hkl</sub>Σ<sub><italic>i</italic></sub><italic>I</italic><sub>hkl<italic>,i</italic></sub>, where <italic>I</italic><sub>hkl<italic>,i</italic></sub> is the scaled intensity of the <italic>i</italic><sup>th</sup> measurement of reflection h, k, l, <<italic>I</italic><sub>hkl</sub>> is the average intensity for that reflection, and <italic>n</italic> is the redundancy.</p></fn><fn id="t2-fn4"><p><sup>‡</sup>CC<sub>1/2</sub>=Pearson correlation coefficient between two random half datasets.</p></fn><fn id="t2-fn5"><p><sup>§</sup>Z<sub>a</sub> is the number of HA1-Fab complexes per crystallographic asymmetric unit.</p></fn><fn id="t2-fn6"><p><sup>‖</sup><italic>R</italic><sub>cryst</sub>=Σ<sub>hkl</sub>|<italic>F</italic><sub>o</sub>−<italic>F</italic><sub>c</sub>|/Σ<sub>hkl</sub>|<italic>F</italic><sub>o</sub>| × 100, where <italic>F</italic><sub>o</sub> and <italic>F</italic><sub>c</sub> are the observed and calculated structure factors, respectively.</p></fn><fn id="t2-fn7"><p><sup>¶</sup><italic>R</italic><sub>free</sub> was calculated as for <italic>R</italic><sub>cryst</sub>, but on a test set comprising 5% of the data excluded from refinement.</p></fn><fn id="t2-fn8"><p><sup>#</sup>Other includes non-water solvent and cryoprotectant.</p></fn><fn id="t2-fn9"><p><sup>**</sup>Calculated with MolProbity<xref ref-type="bibr" rid="b63">63</xref>.</p></fn></table-wrap-foot></table-wrap></floats-group></pmc><affiliations><list><country><li>États-Unis</li></country></list><tree><country name="États-Unis"><noRegion><name sortKey="Wu, Nicholas C" sort="Wu, Nicholas C" uniqKey="Wu N" first="Nicholas C." last="Wu">Nicholas C. Wu</name></noRegion><name sortKey="Grande, Geramie" sort="Grande, Geramie" uniqKey="Grande G" first="Geramie" last="Grande">Geramie Grande</name><name sortKey="Lerner, Richard A" sort="Lerner, Richard A" uniqKey="Lerner R" first="Richard A." last="Lerner">Richard A. Lerner</name><name sortKey="Turner, Hannah L" sort="Turner, Hannah L" uniqKey="Turner H" first="Hannah L." last="Turner">Hannah L. Turner</name><name sortKey="Ward, Andrew B" sort="Ward, Andrew B" uniqKey="Ward A" first="Andrew B." last="Ward">Andrew B. Ward</name><name sortKey="Wilson, Ian A" sort="Wilson, Ian A" uniqKey="Wilson I" first="Ian A." last="Wilson">Ian A. Wilson</name><name sortKey="Wilson, Ian A" sort="Wilson, Ian A" uniqKey="Wilson I" first="Ian A." last="Wilson">Ian A. Wilson</name><name sortKey="Xie, Jia" sort="Xie, Jia" uniqKey="Xie J" first="Jia" last="Xie">Jia Xie</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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