A New Approach to EM of Helical A New Approach to EM of Helical Polymers Yields New Insights Polymers Yields New Insights � Helical polymers are ubiquitous in biology Helical polymers are ubiquitous in biology – – � � This is because a helical structure results from This is because a helical structure results from � simplest bonding rule between any two objects! simplest bonding rule between any two objects! � Other interactions (symmetrical Other interactions (symmetrical dimer dimer, , trimer trimer, , � tetramer, etc.) are more restrictive tetramer, etc.) are more restrictive � Helical polymers have played an important Helical polymers have played an important � role in the development of structural biology role in the development of structural biology � DeRosier & DeRosier & Klug Klug, 1968 , 1968 � � Yonekura Yonekura et al. et al. , 2003 , 2003 � � Miyazawa Miyazawa et al. et al. , 2003 , 2003 �
Most Helical Polymers Have Been Refractory to Most Helical Polymers Have Been Refractory to High Resolution EM Studies (and are High Resolution EM Studies (and are incompatible with crystallization)! incompatible with crystallization)! � Disorder or variability Disorder or variability � � Heterogeneity Heterogeneity � � Weak Scattering Weak Scattering �
New method: Iterative Helical Real Space Reconstruction New method: Iterative Helical Real Space Reconstruction and ~ 35 other papers published or in press and ~ 35 other papers published or in press
What are the problems with the traditional approach? What are the problems with the traditional approach? For most real filaments, never straight over long distances For most real filaments, never straight over long distances � � can correct for small curvature with “ “straightening straightening” ” algorithm ( algorithm ( i.e i.e ., Egelman, 1986), ., Egelman, 1986), can correct for small curvature with � � but this can introduce artifacts but this can introduce artifacts Indexing can be problematic – Indexing can be problematic – both difficult and ambiguous both difficult and ambiguous � � Absence of space group means that disorder can accumulate, with Absence of space group means that disorder can accumulate, with liquid liquid- -like order like order � � Many polymers can have great variability, so that Fourier- -Bessel methods are useless Bessel methods are useless Many polymers can have great variability, so that Fourier � � No reason for there to be simple symmetry expressed as ratio of small integers small integers No reason for there to be simple symmetry expressed as ratio of � � Very small changes in symmetry can lead to very large changes in the the “ “selection rule selection rule” ” Very small changes in symmetry can lead to very large changes in � � Example of actin: “ Example of actin: “u/t u/t = 13/6 = 13/6” ”, c=355 , c=355 Å Å, , Δϕ Δϕ =166.1538 =166.1538º º � � but Δ ( Δϕ )=0.128º º, , Δϕ =166.2818º º, , u/t u/t=1299/600, c=35,463 =1299/600, c=35,463 Å Å! ! but Δ ( Δϕ )=0.128 Δϕ =166.2818 � � Bessel overlap can occur for many real structures at low resolution ion Bessel overlap can occur for many real structures at low resolut � �
Iterative Helical Real Space Reconstruction Method Egelman (2000), Ultramicroscopy 85, 225-234 Helically symmetric 3D volume rotate azimuthally by 4° increment, impose helical symmetry project onto 2D image Δφ, Δ z 90 reference projections (helical parameters) 0° 4° 8° 12° 16° 20° 24° 28° 32°... multi-reference set of raw images determine helical symmetry by alignment (thousands of segments from least squares fit helical filaments) In-plane rotation, x-shift, y-shift Asymmetric 3D volume parameters back projection Aligned, rotated images; azimuthal angular assignments
generator creates IHRSR script creates IHRSR script generator
imposed imposed asymmetric asymmetric Most important part symmetry symmetry reconstruction reconstruction of cycle… symmetry search symmetry search
Iterative Helical Real Space Reconstruction Method Advantages of method over Fourier- Advantages of method over Fourier -Bessel approach: Bessel approach: • Overcomes problems of straightening • Overcomes problems of straightening • Can work with very weakly scattering specimens • Can work with very weakly scattering specimens • bacterial • bacterial pili pili, filamentous phage , filamentous phage • Can deal with disordered or heterogeneous • Can deal with disordered or heterogeneous filaments filaments • RecA/RAD51/Dmc1, • RecA/RAD51/Dmc1, actin actin, , ParM ParM • Is transparent to the almost intractable problem • Is transparent to the almost intractable problem of Bessel overlap of Bessel overlap • myosin thick filament • myosin thick filament • Is easier, both conceptually and in practice • Is easier, both conceptually and in practice Disadvantages: Disadvantages: • None • None
Archaeal flagellum flagellum Archaeal shows homology with shows homology with bacterial Type IV bacterial Type IV pilus pilus
Unambiguous fit of Unambiguous fit of monomeric crystal crystal monomeric subunit from Neisseria Neisseria subunit from gonorrhoeae gonorrhoeae ~ 3.6 subunits per turn of a 37 Å pitch helix Craig et al. , Mol. Cell 23, 651-662 (2006)
Packing of crystal Packing of crystal subunit from Neisseria Neisseria subunit from gonorrhoeae gonorrhoeae Craig et al. , Mol. Cell 23, 651-662 (2006)
EspA of of Enteropathogenic Enteropathogenic E. coli E. coli EspA
EspA of of Enteropathogenic Enteropathogenic E. coli E. coli EspA ~60,000 segments (each ~ 240 Å Å in length) analyzed in length) analyzed ~60,000 segments (each ~ 240 Most variation determined to be due to variable axial rise Most variation determined to be due to variable axial rise Wang Wang et al et al ., Structure ., Structure 14 14 , 1189 , 1189- -96 (2006) 96 (2006)
EspA: heterogeneity in axial rise : heterogeneity in axial rise EspA Right-handed 6-start Left-handed 5-start Grey: 5.3 Å Yellow: 4.2 Å Wang Wang et al et al ., Structure ., Structure 14 14 , 1189 , 1189- -96 (2006) 96 (2006)
What role does such plasticity play? What role does such plasticity play? � EspA EspA needle extensions are quite long needle extensions are quite long � (~ 0.6 μ μ ) and must remain intact in a high ) and must remain intact in a high (~ 0.6 shear environment shear environment � Which is more resistant to breakage Which is more resistant to breakage – – a a � rigid glass tube or a flexible rubber rigid glass tube or a flexible rubber tube? tube? � What happens when a rubber tube is What happens when a rubber tube is � stretched? stretched? � Homology suggests that flexibility of Homology suggests that flexibility of � flagellar hook may arise by similar means hook may arise by similar means flagellar
Variable “ “twist twist” ” in F in F- -actin actin Variable
Variable tilt and twist seen in actin actin- -scruin scruin bundle bundle Variable tilt and twist seen in Nature 431 Nature 431 , 2 Sept. 2004 , 2 Sept. 2004
“Movie Movie” ” of tilt of tilt “ shows: shows: 1) rearrangement of 1) rearrangement of contacts contacts 2) change in twist 2) change in twist (from 167º º to 154 to 154º º) ) (from 167 3) propeller rotation 3) propeller rotation of actin domains of actin domains
Unprecedented Unprecedented resolution in resolution in looking at looking at isolated actin actin isolated filaments and filaments and complexes complexes F-actin decorated with ABD2 of fimbrin, ~ 12 Å shows unambiguously that there is not a conserved mode of interaction of Calponin Homology (CH) domains with actin
Unprecedented Unprecedented resolution in resolution in looking at looking at isolated actin actin isolated filaments and filaments and complexes complexes pure F-actin, ~ 9 Å
Different G-actin structures reinforce concept of subdomain 2 being a switch Kabsch et al. et al. Kabsch (1990), actin actin- - (1990), DNase I complex I complex DNase Otterbein et al. et al. Otterbein (2001), modified (2001), modified G- -actin actin G
Unprecedented resolution in looking at isolated actin filaments and complexes pure F-actin, ~ 9 Å
Conserved family of proteins Conserved family of proteins
Dramatic support for conformational changes in actin actin Dramatic support for conformational changes in subunit comes from ParM ParM structures! structures! subunit comes from van den Ent et al. , “F-actin-like filaments formed by plasmid segregation protein ParM”, EMBO J. (2002)
Bacterial ParM ParM filaments filaments Bacterial negative stain unstained frozen-hydrated
The ParM ParM Paradox Paradox The
Variability in twist in ParM greater than F-actin
Unstained frozen- hydrated ParM filaments by cryo-EM
Unstained frozen- hydrated ParM filaments by cryo-EM
Helical hand confirmed by quick Helical hand confirmed by quick freeze/deep etch EM freeze/deep etch EM ParM F-actin ParM F-actin
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