Structure of the signal recognitionparticle interacting with theelongation-arrested ribosomeMario Halic1, Thomas Becker1, Martin R. Pool2, Christian M. T. Spahn3, Robert A. Grassucci4, Joachim Frank4& Roland Beckmann11Institute of Biochemistry, Charite´, University Medical School, Humboldt University of Berlin, Monbijoustrasse 2, 10117 Berlin, Germany2University of Manchester, School of Biological Sciences, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK3Institute of Medical Physics and Biophysics, Charite´, University Medical School, Humboldt University of Berlin, Ziegelstrasse 9, 10117 Berlin, Germany4Howard Hughes Medical Institute, Health Research Incorporated, Wadsworth Center, Empire State Plaza, Albany, New York 12201, and Department of BiomedicalSciences, State University of New York at Albany, Albany, New York 12222, USA...........................................................................................................................................................................................................................Cotranslational translocation of proteins across or into membranes is a vital process in all kingdoms of life. It requires that thetranslating ribosome be targeted to the membrane by the signal recognition particle (SRP), an evolutionarily conservedribonucleoprotein particle. SRP recognizes signal sequences of nascent protein chains emerging from the ribosome. Subsequentbinding of SRP leads to a pause in peptide elongation and to the ribosome docking to the membrane-bound SRP receptor. Here wepresent the structure of a targeting complex consisting of mammalian SRP bound to an active 80S ribosome carrying a signalsequence. This structure, solved to 12 A˚by cryo-electron microscopy, enables us to generate a molecular model of SRP in itsfunctional conformation. The model shows how the S domain of SRP contacts the large ribosomal subunit at the nascent chain exitsite to bind the signal sequence, and that the Alu domain reaches into the elongation-factor-binding site of the ribosome,explaining its elongation arrest activity.The existence of a signal sequence ‘binding factor’ for proteintargeting to the endoplasmic reticulum was first proposed1in1971. This binding factor was subsequently identified in a mamma-lian system as an 11S ribonucleoprotein (RNP) particle named theSRP2. This particle shows three main activities in the process ofcotranslational targeting: first, it binds to signal sequences emergingfrom the translating ribosome; second, it pauses peptide elongation;and third, it promotes protein translocation by docking to themembrane-bound SRP receptor and transferring the ribosomenascent chain complex (RNC) to the protein-conducting channel3.These activities can be assigned to the two main domains of SRPthat are separable by micrococcal nuclease treatment4. The firstdomain, the S domain, binds to signal sequences and promotestranslocation5. It includes about half of the 7S RNA of SRP (roughlynucleotides 100–250), as well as the essential proteins SRP19, SRP54and the SRP68–SRP72 (SRP68/72) heterodimer. Although SRP19 isrequired for SRP assembly6, SRP54 is the functionally most signifi-cant protein subunit of the S domain: it recognizes the signalsequence5and interacts with the SRP receptor in a GTP-dependentmanner7. SRP54 comprises an amino-terminal domain (N), acentral GTPase domain (G) and a methionine-rich carboxy-terminal domain (M)8, which anchors SRP54 to SRP RNA9.Inaddition, together with part of the RNA backbone10, the M domaincarries out the principal function of signal sequence recognition11near the peptide exit site of the large ribosomal subunit12.The second domain of SRP, the Alu domain, mediates theelongation arrest activity13. It is supposed to allow efficient targetingby providing a time window in which the nascent chain can betargeted to the translocation site14–16. The Alu domain contains the50and 30parts of 7S RNA (including the Alu-like sequences), as wellas the SRP9–SRP14 (SRP9/14) heterodimer, which is essential for itsactivity17.Little is known about the structural arrangement of the completeSRP18,19, especially when bound to the active ribosome. How canSRP recognize a signal sequence and stop elongation at the sametime? We have determined the structure of mammalian SRP boundto an elongation-arrested 80S ribosome bearing a nascent poly-peptide chain containing a signal sequence at 12 A˚resolution byusing cryo-electron microscopy (cryo-EM) and single-particlereconstruction.RNC purification and RNC–SRP reconstitutionBecause the formation of a stable complex was a prerequisite for ourstudy, we used wheat germ RNCs and canine SRP to reconstitute thetargeting complex. This well-characterized combination, which ledto the discovery of SRP, shows strong elongation arrest activity14.Assuming that this activity is a result of equally stable binding of theS and Alu domains to the ribosome, we considered that the wheatgerm–canine heterologous complex was the most suitable candidatefor structure determination.We first isolated programmed ribosomes carrying a functionalsignal sequence (RNCs) from an in vitro translation reaction20. Thenascent chain represented the first 90 amino acids of the type IImembrane protein dipeptidylpeptidase B (DPAP-B) from yeast,which contains a signal anchor sequence, and also a haemagglutinin(HA) and histidine tag. Stalled RNCs were affinity purified and usedfor reconstitution with excess amounts of purified canine SRP (seeSupplementary Information). To ensure specificity—that is, signal-sequence-dependent complex formation—we carried out sucrosedensity gradient centrifugation under high-salt conditions(500 mM potassium acetate)21, which confirmed high-affinity bind-ing of SRP to RNCs with an estimated occupancy of between 50 and90% (see Supplementary Information).Cryo-EM map and model of mammalian SRPCryo-EM and three-dimensional (3D) reconstruction of the target-ing complex shows the typical appearance of an 80S ribosome at12 A˚resolution (7.7 A˚according to the 3j criterion; see Supplemen-tary Information) with two additional densities (Fig. 1). First, atransfer RNA is visible in the P site in the ribosomal intersubunitspace. Second, a large elongate mass representing SRP stretchesfrom the peptide exit site of the 60S ribosomal subunit (S domain)articlesNATURE | VOL 427 | 26 FEBRUARY 2004 | www.nature.com/nature808© 2004Nature PublishingGroupinto the