{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T01:57:36Z","timestamp":1791338256407,"version":"4.3.1"},"reference-count":35,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5539","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[2001,9,28]]},"abstract":"<jats:p>\n                    The seven-subunit Arp2\/3 complex choreographs the formation of branched actin networks at the leading edge of migrating cells. When activated by Wiskott-Aldrich Syndrome protein (WASp), the Arp2\/3 complex initiates actin filament branches from the sides of existing filaments. Electron cryomicroscopy and three-dimensional reconstruction of\n                    <jats:italic>Acanthamoeba castellanii<\/jats:italic>\n                    and\n                    <jats:italic>Saccharomyces cerevisiae<\/jats:italic>\n                    Arp2\/3 complexes bound to the WASp carboxy-terminal domain reveal asymmetric, oblate ellipsoids. Image analysis of actin branches indicates that the complex binds the side of the mother filament, and Arp2 and Arp3 (for actin-related protein) are the first two subunits of the daughter filament. Comparison to the actin-free, WASp-activated complexes suggests that branch initiation involves large-scale structural rearrangements within Arp2\/3.\n                  <\/jats:p>","DOI":"10.1126\/science.1063025","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T05:39:52Z","timestamp":1027748392000},"page":"2456-2459","source":"Crossref","is-referenced-by-count":227,"title":["Structure of Arp2\/3 Complex in Its Activated State and in Actin Filament Branch Junctions"],"prefix":"10.1126","volume":"293","author":[{"given":"Niels","family":"Volkmann","sequence":"first","affiliation":[{"name":"The Burnham Institute, La Jolla, CA 92037, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kurt J.","family":"Amann","sequence":"additional","affiliation":[{"name":"Structural Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Svetla","family":"Stoilova-McPhie","sequence":"additional","affiliation":[{"name":"The Burnham Institute, La Jolla, CA 92037, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Coumaran","family":"Egile","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dirk C.","family":"Winter","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Larnele","family":"Hazelwood","sequence":"additional","affiliation":[{"name":"The Burnham Institute, La Jolla, CA 92037, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John E.","family":"Heuser","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Washington University, School of Medicine, St. Louis, MO 63110, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rong","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas D.","family":"Pollard","sequence":"additional","affiliation":[{"name":"Structural Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dorit","family":"Hanein","sequence":"additional","affiliation":[{"name":"The Burnham Institute, La Jolla, CA 92037, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.145.5.1009"},{"key":"e_1_3_1_3_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.11.6181"},{"key":"e_1_3_1_4_2","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.9.4.841"},{"key":"e_1_3_1_5_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.131.2.385"},{"key":"e_1_3_1_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(99)80243-7"},{"key":"e_1_3_1_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80732-1"},{"key":"e_1_3_1_8_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.146.6.1319"},{"key":"e_1_3_1_9_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.7.3739"},{"key":"e_1_3_1_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0959-440X(99)80034-7"},{"key":"e_1_3_1_11_2","unstructured":"Acanthamoeba Arp2\/3 complex was purified by poly- l -proline (26) and gel filtration chromatography as described (22). Scar WA the 65 COOH-terminal residues of human Scar1 protein (8) was expressed in bacteria from pMW172 (27) without induction and was purified as described (22). The preparations [buffer: 150 mM KCl 10 mM Tris (pH 8.0) 100 \u03bcM CaCl 2 200 \u03bcM ATP 500 \u03bcM dithiothreitol (DTT)] were applied to copper grids coated with Quantifoil holey carbon films (Quantifoil Micro Tools GmbH Jena Germany) and plunged in liquid ethane. Images were obtained under low-dose conditions with the use of a Tecnai 12 electron microscope (FEI Electron Optics) and DH626 cryo-holder (Gatan Inc.) at a nominal magnification of 67 000 at 2.5 \u03bcm defocus. The micrographs were digitized with a SCAI scanner [Z(I Imaging Corporation Englewood CO)] with pixel size of 0.4 nm on the sample. Shadowed specimens were prepared as described (2). Handedness of the shadowed specimen was determined using the 5.1-nm left-handed actin repeat and the 37.5-nm right-handed half-twist crossover repeat both visible in the images (Fig. 2D)."},{"key":"e_1_3_1_12_2","unstructured":"Image processing of single particles of Acanthamoeba Arp2\/3 complex. Interactive selection from 46 images yielded 4414 particles (Fig. 1A). The particles were processed and analyzed using the EMAN (28) and COAN (29) software packages. A correction for the contrast transfer function was applied for all images. Absolute scaling was performed using the known helical symmetry of actin filaments present in the same images. For 3D reconstruction particles were aligned to each other using the reference-free method classified into 20 to 25 classes and averaged. Briefly particles that appear to be similar (by dot product) to one another were grouped together into a predefined number of groups and then the particles within each group were mutually aligned and averaged. This generated a class average for each group that should represent one characteristic view of the particle. Several of these averages were then selected manually and passed to a Fourier common-lines routine that determines the relative orientations (Euler angles) of all selected averages which are then combined to calculate an initial 3D model. This initial model was then iteratively refined against the data. Convergence was achieved when the Fourier shell correlation between successive iterations stabilized. Three independent starting models were generated from different subsets of the Acanthamoeba data set. Several rounds (15 to 25) of refinement were performed for each of these models using the complete data set. The three calculations converged to the same structure (Fig. 1D) as suggested by the high correlation values."},{"key":"e_1_3_1_13_2","doi-asserted-by":"publisher","DOI":"10.1038\/386088a0"},{"key":"e_1_3_1_14_2","unstructured":"Scar WA-Cys containing a single cysteine residue at its NH 2 -terminus was purified as described (22) and then reacted with equimolar monoamino Nanogold (Nanoprobes) in 100 mM NaCl and 10 mM NaPO 4 (pH 7.0) for 16 hours at 4\u00b0C. Nanogold-labeled WA was further purified by S-100 gel filtration chromatography in 100 mM NaCl 10 mM Tris-HCl (pH 8.0) and 2 mM DTT. Images of frozen-hydrated Arp2\/3 complex labeled with nanogold-labeled WA were recorded in focal pairs at 0.5 and 2.5 \u03bcm defocus using low-dose conditions. The position of the gold label within the reconstruction was calculated by backprojection from 10 class averages of gold-decorated particles (generated by superposition of the focal-pair images) after alignment to projections calculated from the undecorated reconstruction."},{"key":"e_1_3_1_15_2","unstructured":"The budding yeast Saccharomyces cerevisiae Arp2\/3 complex was purified as described (30). Bee1-WA the 125 COOH-terminal residues of yeast Beep1 was purified as described (31 32). Phalloidin-stabilized actin filaments were incubated with 25 nM Arp2\/3 complex and 50 nM Bee1-WA domain in UBA buffer [50 mM Hepes (pH 7.5) 100 mM KCl 3 mM MgCl 2 1 mM EGTA 0.2 mM ATP] for 5 min at 22\u00b0C. The pellet containing actin filaments and attached complexes was resuspended in UBA buffer and applied to copper grids coated with triafol holey carbon films (pelleting: Beckman-Coulter TLA-100 rotor at 70 000 rpm for 10 min at 4\u00b0C). The samples were stained with aqueous uranyl acetate air-dried and imaged at 2 \u03bcm defocus. The yeast data set consisted of 1157 particles selected from 13 images and was processed following the procedure described for Acanthamoeba (11) which yielded a reconstruction very similar to that of Acanthamoeba (Fig. 1D)."},{"key":"e_1_3_1_16_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.136.2.331"},{"key":"e_1_3_1_17_2","unstructured":"Actin was isolated from rabbit skeletal muscle acetone powder (33) and further purified by gel filtration on Sephacryl S-300 in Ca-G buffer (22). Branches were prepared by polymerizing 4 \u03bcM Mg-ATP actin in the presence of 50 nM Arp2\/3 and 200 nM Scar WA in Mg-G buffer [2 mM imidazole (pH 7.0) 200 \u03bcM ATP 100 \u03bcM MgCl 2 500 \u03bcM DTT] containing 1\u00d7 KMEI [50 mM KCl 100 \u03bcM MgCl 2 1 mM EGTA 10 mM imidazole (pH 7.0)] for 2 min at 22\u00b0C followed by addition of 4 \u03bcM phalloidin 1 min before application to the Quantifoil grids. For the reconstruction of the branch junction 167 clearly visible branches were interactively selected from 127 images. A reference-free alignment of these images gave class averages with branch angles of \u223c70\u00b0. The class with the clearest definition of actin monomers in the filaments was selected for reference-based alignment. To overcome the blurring of details due to small differences in branching angles we generated three references emphasizing different portions of the structure. One reference focused on the mother filament one on the daughter filament and one on the central density of the branch junction. The complete data set was aligned to each of the references. Independent 2D averages were calculated and then merged (Fig. 2B). Atomic models of actin filaments (19) were fitted to the projection density of the mother and daughter filaments using COAN (29)."},{"key":"e_1_3_1_18_2","unstructured":"For myosin decoration actin filaments were polymerized in the presence of Arp2\/3 complex (25 nM) and Bee1-WA domain (50 nM) in UBA buffer (32). Samples were applied to the triafol grids and incubated with expressed smooth muscle myosin catalytic domain (no light chains) (34) prior to staining."},{"key":"e_1_3_1_19_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.139.2.397"},{"key":"e_1_3_1_20_2","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.1993.1628"},{"key":"e_1_3_1_21_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.274.46.32531"},{"key":"e_1_3_1_22_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.13.7288"},{"key":"e_1_3_1_23_2","doi-asserted-by":"publisher","DOI":"10.1038\/35060104"},{"key":"e_1_3_1_24_2","doi-asserted-by":"publisher","DOI":"10.1038\/35017011"},{"key":"e_1_3_1_25_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(98)00015-3"},{"key":"e_1_3_1_26_2","doi-asserted-by":"publisher","DOI":"10.1038\/35010008"},{"key":"e_1_3_1_27_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.127.1.107"},{"key":"e_1_3_1_28_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.109.2.593"},{"key":"e_1_3_1_29_2","doi-asserted-by":"publisher","DOI":"10.1006\/jsbi.1999.4174"},{"key":"e_1_3_1_30_2","doi-asserted-by":"publisher","DOI":"10.1006\/jsbi.1998.4074"},{"key":"e_1_3_1_31_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(06)00223-5"},{"key":"e_1_3_1_32_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.136.3.649"},{"key":"e_1_3_1_33_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(99)80218-8"},{"key":"e_1_3_1_34_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(18)62016-2"},{"key":"e_1_3_1_35_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(17)31894-X"},{"key":"e_1_3_1_36_2","unstructured":"We are grateful to G. Ouyang for technical assistance to K. M. Trybus and S. Lowey for the myosin sample to S. Almo for critical reading of the manuscript and to R. Liddington for helpful discussions and valuable comments on the manuscript. Supported by NIH research grants (T.D.P. R.L.) an NIH postdoctoral fellowship (K.J.A.) and the Human Frontier Science Program (C.E.). D.H. is a Pew Scholar in the Biomedical Sciences."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.1063025","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,9]],"date-time":"2024-01-09T17:22:29Z","timestamp":1704820949000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.1063025"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2001,9,28]]},"references-count":35,"journal-issue":{"issue":"5539","published-print":{"date-parts":[[2001,9,28]]}},"alternative-id":["10.1126\/science.1063025"],"URL":"https:\/\/doi.org\/10.1126\/science.1063025","relation":{"has-review":[{"id-type":"doi","id":"10.3410\/f.1001819.8060","asserted-by":"object"},{"id-type":"doi","id":"10.3410\/f.1001819.12957","asserted-by":"object"},{"id-type":"doi","id":"10.3410\/f.1001819.12409","asserted-by":"object"},{"id-type":"doi","id":"10.3410\/f.1001819.16864","asserted-by":"object"}]},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2001,9,28]]}}}