Structure of Keyhole Limpet Hemocyanin Type 1 (KLH1) at 15 A Ê Resolution by Electron Cryomicroscopy and Angular Reconstitution{ Elena V. Orlova 1,2 , Prakash Dube 1,3 , J. Robin Harris 4 , Erich Beckman 2 Friedrich Zemlin 2 , Ju È rgen Markl 4 and Marin van Heel 1 * 1 Imperial College of Science Medicine and Technology Department of Biochemistry London SW7 2AY, UK 2 Fritz Haber Institute of the Max Planck Society Faradayweg 4-6, D14195 Berlin Germany 3 Max Planck Institute for Molecular Genetics, Ihnestraûe 73, D14195 Berlin, Germany 4 Institute of Zoology University of Mainz D-55099 Mainz, Germany A three-dimensional reconstruction of keyhole limpet hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid helium temperatures and single particle image processing. The use of a high-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) am- monium molybdate (pH 7.0), enables high-resolution electron micro- graphs to be recorded close to focus, i.e. with excellent transfer of high- resolution information, while maintaining enough image contrast to loca- lise the individual macromolecules in the images. When low-pass ®ltered to 45 A Ê resolution, the new 15 A Ê resolution reconstruction is very simi- lar to the earlier reconstructions of gastropodan hemocyanins of speci- mens embedded in vitreous ice. The map shows much detail and reveals many new symmetry elements in this very large cylindrical molluscan hemocyanin. The full KLH1 didecamer has D5 pointgroup symmetry, yet within the KLH1 decameric half-molecules local 2-fold axes have emerged that make the wall of the KLH1 decamer, in spite of its having an exact C5 symmetry only, resemble the D5-symmetric wall of the deca- meric cephalopod hemocyanins. In fact, the outside of each tier of this six-tiered gastropodan hemocyanin was found to have an approximate D5 symmetry. Local 2-fold axes also relate the ``functional units'' within the dimeric ``morphological units'' of the wall and the collar areas of the 8 MDa KLH1 molecule. Certain local-symmetry-related surface motifs may be present up to 60 times on the outside wall of this highly sym- metric cylindrical hemocyanin. Keyhole limpet hemocyanin is used clini- cally as an immunostimulant. The very strong immune reaction elicited by this hemocyanin may be associated with its intricate hierarchy of local-symmetry components. # 1997 Academic Press Limited Keywords: keyhole limpet hemocyanin; electron cryomicroscopy; angular reconstitution; single particle analysis; high contrast embedding *Corresponding author Introduction General background of KLH The hemocyanins are large molecular mass, cop- per-containing, oxygen-transporting proteins found in the hemolymph of many molluscs and arthro- pods (for a review, see van Holde & Miller, 1995). Whereas the family of arthropod hemocyanins con- sists of assemblies containing between one and eight hexameric building blocks (Markl & Decker, 1992; van Heel & Dube, 1994), the molluscan hemocyanins are organised as hollow cylindrical oligomers with external diameters of 370 A Ê . The {This article is dedicated to the memory of Anneke van Heel. Abbreviations used: FU, functional unit; KLH, keyhole limpet hemoyamin; 2D, two-dimensional; 3D, three-dimensional; FEG, ®eld-emission gun; PhCTF, phase contrast transfer function; SNR, signal-to-noise ratio; FT, Fourier transform; FSC, Fourier shell correlation; FRC, Fourier ring correlation; FSPR, Fourier shell phage residual; DPR, differential phase residual; MSA, multivariate statistical analysis. J. Mol. Biol. (1997) 271, 417±437 0022±2836/97/330417±21 $25.00/0/mb971182 # 1997 Academic Press Limited