Near-atomic resolution for one state of F-actin.

Actin functions as a helical polymer, F-actin, but attempts to build an atomic model for this filament have been hampered by the fact that the filament cannot be crystallized and by structural heterogeneity. We have used a direct electron detector, cryo-electron microscopy, and the forces imposed on actin filaments in thin films to reconstruct one state of the filament at 4.7 Å resolution, which allows for building a reliable pseudo-atomic model of F-actin. We also report a different state of the filament where actin protomers adopt a conformation observed in the crystal structure of the G-actin-profilin complex with an open ATP-binding cleft. Comparison of the two structural states provides insights into ATP-hydrolysis and filament dynamics. The atomic model provides a framework for understanding why every buried residue in actin has been under intense selective pressure.

[1]  S. Scheres,et al.  Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles , 2013, eLife.

[2]  C. Schutt,et al.  The structure of an open state of beta-actin at 2.65 A resolution. , 1996, Journal of molecular biology.

[3]  R. Levine,et al.  Phosphorylation of actin Tyr-53 inhibits filament nucleation and elongation and destabilizes filaments , 2006, Proceedings of the National Academy of Sciences.

[4]  L. Otterbein,et al.  The Crystal Structure of Uncomplexed Actin in the ADP State , 2001, Science.

[5]  Michael S. Spilman,et al.  ResLog plots as an empirical metric of the quality of cryo-EM reconstructions. , 2014, Journal of structural biology.

[6]  Ruei-Jiun Hung,et al.  Direct Redox Regulation of F-Actin Assembly and Disassembly by Mical , 2011, Science.

[7]  Israel S. Fernández,et al.  Structure of the Mammalian Ribosome-Sec61 Complex to 3.4 Å Resolution , 2014, Cell.

[8]  S. Scheres,et al.  Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine , 2014, eLife.

[9]  I. Boldogh,et al.  A Mammalian Actin Substitution in Yeast Actin (H372R) Causes a Suppressible Mitochondria/Vacuole Phenotype* , 2005, Journal of Biological Chemistry.

[10]  D. Julius,et al.  Structure of the TRPV1 ion channel determined by electron cryo-microscopy , 2013, Nature.

[11]  T. Pollard,et al.  Polymerization and structure of nucleotide-free actin filaments. , 2000, Journal of molecular biology.

[12]  C. Altenbach,et al.  Hydrophobic loop dynamics and actin filament stability. , 2006, Biochemistry.

[13]  G. Bruns,et al.  Structure, chromosome location, and expression of the human smooth muscle (enteric type) gamma-actin gene: evolution of six human actin genes , 1991, Molecular and cellular biology.

[14]  E. Egelman Ambiguities in helical reconstruction , 2014, eLife.

[15]  Edward H. Egelman,et al.  Actin allostery again? , 2001, Nature Structural Biology.

[16]  E. Egelman,et al.  Actin Filaments as Tension Sensors , 2012, Current Biology.

[17]  Alan Brown,et al.  Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine, large subunit , 2014 .

[18]  A Leith,et al.  SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. , 1996, Journal of structural biology.

[19]  Steven B Marston,et al.  Genotype–phenotype correlations in ACTA1 mutations that cause congenital myopathies , 2009, Neuromuscular Disorders.

[20]  W. Chiu,et al.  Structure of the acrosomal bundle , 2004, Nature.

[21]  W. Chiu,et al.  Crystallographic conformers of actin in a biologically active bundle of filaments. , 2008, Journal of molecular biology.

[22]  Keiichi Namba,et al.  Direct visualization of secondary structures of F-actin by electron cryomicroscopy , 2010, Nature.

[23]  N. Laing,et al.  Myopathy mutations in α-skeletal-muscle actin cause a range of molecular defects , 2004, Journal of Cell Science.

[24]  Wah Chiu,et al.  Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function , 1997, The Journal of cell biology.

[25]  N. Grigorieff,et al.  Accurate determination of local defocus and specimen tilt in electron microscopy. , 2003, Journal of structural biology.

[26]  J. Moraczewska,et al.  The actin/actin interactions involving the N-terminus of the DNase-I-binding loop are crucial for stabilization of the actin filament. , 1993, European journal of biochemistry.

[27]  J. Israelachvili,et al.  Force amplification response of actin filaments under confined compression , 2009, Proceedings of the National Academy of Sciences.

[28]  K. Trybus,et al.  Expression of a nonpolymerizable actin mutant in Sf9 cells. , 2004, Biochemistry.

[29]  P. Rubenstein,et al.  Insights into the effects of disease‐causing mutations in human actins , 2014, Cytoskeleton.

[30]  F. Oosawa,et al.  POLYMERIZATION OF ACTIN FREE FROM NUCLEOTIDES AND DIVALENT CATIONS. , 1965, Biochimica et biophysica acta.

[31]  E. Egelman,et al.  Actin hydrophobic loop 262-274 and filament nucleation and elongation. , 2008, Journal of molecular biology.

[32]  Ueli Aebi,et al.  A Correlative Analysis of Actin Filament Assembly, Structure, and Dynamics , 1997, The Journal of cell biology.

[33]  D. DeRosier,et al.  The Fourier transform of actin and other helical systems with cumulative random angular disorder , 1982 .

[34]  Katherine A. Fitzgerald,et al.  Unified Polymerization Mechanism for the Assembly of ASC-Dependent Inflammasomes , 2014, Cell.

[35]  G. Schröder,et al.  Remodeling of actin filaments by ADF/cofilin proteins , 2011, Proceedings of the National Academy of Sciences.

[36]  Locking the hydrophobic loop 262-274 to G-actin surface by a disulfide bridge prevents filament formation. , 2002, Biochemistry.

[37]  Steven C Almo,et al.  Polylysine Induces an Antiparallel Actin Dimer That Nucleates Filament Assembly , 2002, The Journal of Biological Chemistry.

[38]  U. Aebi,et al.  Probing actin polymerization by intermolecular cross-linking , 1988, The Journal of cell biology.

[39]  Edward H Egelman,et al.  Actin's prokaryotic homologs. , 2003, Current opinion in structural biology.

[40]  Alan Brown,et al.  Structure of the Yeast Mitochondrial Large Ribosomal Subunit , 2014, Science.

[41]  E. Egelman A robust algorithm for the reconstruction of helical filaments using single-particle methods. , 2000, Ultramicroscopy.

[42]  E. Egelman,et al.  Actin-destabilizing factors disrupt filaments by means of a time reversal of polymerization. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[43]  R. Dominguez,et al.  Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding , 2008, Proceedings of the National Academy of Sciences.

[44]  P. Penczek,et al.  Molecular imprinting as a signal-activation mechanism of the viral RNA sensor RIG-I. , 2014, Molecular cell.

[45]  E. Egelman,et al.  A change in actin conformation associated with filament instability after Pi release. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[46]  W Chiu,et al.  EMAN: semiautomated software for high-resolution single-particle reconstructions. , 1999, Journal of structural biology.

[47]  K. Henrick,et al.  Inference of macromolecular assemblies from crystalline state. , 2007, Journal of molecular biology.

[48]  V. Ramakrishnan,et al.  Initiation of Translation by Cricket Paralysis Virus IRES Requires Its Translocation in the Ribosome , 2014, Cell.

[49]  Yuichiro Maéda,et al.  The nature of the globular- to fibrous-actin transition , 2009, Nature.

[50]  Yanyu Zhao,et al.  Three-dimensional structure of human γ-secretase , 2014, Nature.

[51]  D. Agard,et al.  Electron counting and beam-induced motion correction enable near atomic resolution single particle cryoEM , 2013, Nature Methods.

[52]  Ikuko Fujiwara,et al.  Microscopic analysis of polymerization dynamics with individual actin filaments , 2002, Nature Cell Biology.

[53]  K. Trybus,et al.  Crystal Structures of Expressed Non-polymerizable Monomeric Actin in the ADP and ATP States* , 2006, Journal of Biological Chemistry.

[54]  M. Maloney,et al.  ADF/Cofilin-actin rods in neurodegenerative diseases. , 2010, Current Alzheimer research.

[55]  W. Chiu,et al.  Direct electron detection yields cryo-EM reconstructions at resolutions beyond 3/4 Nyquist frequency. , 2012, Journal of structural biology.

[56]  E. Reisler,et al.  Structural connectivity in actin: effect of C-terminal modifications on the properties of actin. , 1994, Biophysical journal.

[57]  Gürol M. Süel,et al.  Evolutionarily conserved networks of residues mediate allosteric communication in proteins , 2003, Nature Structural Biology.

[58]  K. Hideg,et al.  Conformational dynamics of loop 262-274 in G- and F-actin. , 2006, Biochemistry.

[59]  J. Pogliano,et al.  Phylogenetic analysis identifies many uncharacterized actin‐like proteins (Alps) in bacteria: regulated polymerization, dynamic instability and treadmilling in Alp7A , 2009, Molecular microbiology.

[60]  D. DeRosier,et al.  F-actin is a helix with a random variable twist , 1982, Nature.

[61]  W. Kabsch,et al.  Atomic model of the actin filament , 1990, Nature.

[62]  Willy Wriggers,et al.  Actin Depolymerizing Factor Stabilizes an Existing State of F-Actin and Can Change the Tilt of F-Actin Subunits , 2001, The Journal of cell biology.

[63]  Jan Löwe,et al.  F‐actin‐like filaments formed by plasmid segregation protein ParM , 2002, The EMBO journal.

[64]  Edward H. Egelman,et al.  A New Internal Mode in F-Actin Helps Explain the Remarkable Evolutionary Conservation of Actin's Sequence and Structure , 2002, Current Biology.

[65]  Edward H. Egelman,et al.  Structural Polymorphism in F-actin , 2010, Nature Structural &Molecular Biology.

[66]  D. Drummond,et al.  Molecular genetics of actin function. , 1993, The Biochemical journal.

[67]  H. Mannherz,et al.  An antiparallel actin dimer is associated with the endocytic pathway in mammalian cells. , 2012, Journal of structural biology.

[68]  Jan Löwe,et al.  Prokaryotic origin of the actin cytoskeleton , 2001, Nature.