Ultramicroscopy 4 (1979) 4 8 1 - 4 8 2 © North-Holland Publishing Company

REPORT

DIRECT IMAGING OF ATOMS IN CRYSTALS AND MOLECULES

Michael BEER Department of Biophysics. Johns HopkbTs University, Baltimore, Mao,land 21218, USA

and

Peter OTTENSMEYER Physics Dil, ision, Ontario Cancer Institute, Toronto, Ontario, Canada

Received 11 September 1979

A conference on "Direct Imaging of Atoms in Crystals and Molecules" sponsored by the Nobel Committee was held August 6 -10 in the IBM Nordic Education Center on the island of kiding6, outside Stockholm. Thirty-three participants and several observers, while enjoying the finest of hospitality in beautiful surroundings, discussed the recent successes in atomic imaging and their inapor- tance in studies of the structure of biological mate- rials, inorganic solids and metals. We shall here review aspects of the meeting which appear most relevant to biology.

Visibility of single heavy atoms is now firmly estab- lished; A.V. Crewe, M. Beer, F.P. Ottensmeyer, H. Hashimoto, B. Jouffrey and J. Wall all showed clear micrographs of individual atoms using dark-field scan- ning or fixed-beam electron microscopy. Time-lapse cinematography on the one hand permitted the study of the diffusion of atoms on the substrate surface; on the other hand it demonstrated a remarkable lack of motion of many individual atoms under often extremely high electron bombardment (104 e/A 2 or higher). Study of the visibility of clusters of atoms on an amorphous carbon foil by Wall suggested that atomic resolution may still be required to reveal their presence, since carbon film structural noise at spa- tial frequencies corresponding to clusters is, in fact, stronger than at frequencies corresponding to the size of individual atoms. Jouffrey indicated that

boron films were somewhat superior to amorphous carbon.

At the molecular level radiation damage to the structure becomes a problem. A. Klug presented the work of Unwin and Henderson on the two- dimensional crystal of the purple membrane pro- tein as an example of an excellent solution to this problem. Here low-dose high-noise information is combined into high-resolution information making use of the extensive redundancy of the two-dimen- sional crystal resulting in a model structure with 7 A resolution. At least 40 substances are being investigated in many laboratories with similar tech- niques, and about a dozen are already giving good two- dimensional rafts. Undoubtedly further protein structures will be elucidated from these in the near future.

For organic crystals it has been established that radiation exposures in excess of about 1 e/A 2 result in loss of crystallinity, while energy loss spectro- scopy indicated a loss of aromatic absorption in orga- nic molecules between 1 and 50 e/.~ 2. Nevertheless Ottensmeyer indicated that repeatedly similar 5 A detail in individual protamine molecules and in smaller polypeptides could be observed even at exposures.of 1000 e /h 2. The proportion of favorably oriented protamine molecules in which such high resolution detail could be recognized in what must be the crumbling remains of the three-dimensional

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482 hi. Beer, P. Ottensmeyer / Report

structure decreased from close to 100% at 100 e/A 2 using the STEM to about 15% at 1000 e/A 2 using the TEM dark field. Combining the EM images, the primary sequence and physico-chemical data on the molecule, Ottensmeyer derived a self-consistent space-filling atomic model of this protein, corro- borated partially by heavy atom labelling of its amino terminus. Questions were raised about the possible bias in the selection of images. The consensus was that this approach must be further investigated, since it appears to be successful even though radiation sensitivity studies in other systems suggest damage to structure at the 5 A resolution at such levels of irra- diation.

Beer described several selective heavy atom rea- gents suitable for high resolution structural investi- gations. Reactions for nucleic acids-are now avail- able for binding one osmium atom to every pyrimidine, or one to every thymine (or uracil) or one to every thymine and adenine. These labelling reactions might be used to recognize particular sections of nucleic acid molecules of known sequence and help establish the exact binding sites of proteins on a nucleic acid.

The complex of platinum and the peptide glycyl-L-methionine when reacted with collagen

bound specifically to the methionine residues. In their reactions with glycoproteins and various saccharides a chelate-osmate complex stained the sugar residues but not the proteins. This specificity promises important application in the study of glycoproteins.

Electron microscopic studies of labelled poly- nucleotides indicated that the movement of heavy atoms limits the accuracy of the inferred positions of marked groups to perhaps 5 to 10

Possibly the most exciting development was the result of J. Dubochet indicating that at 4.2 K in the superconducting microscope of I. Dietrich organic crystals were a factor of 20 to 300 times more resistant to radiation than at room temperature.

This result suggests that the damage dose of individual molecules in crystals may be of the order of 100 e/A 2 at 4.2 K, sufficiently high to enable the fomlation of a 5 A resolution image in STEM dark field while the molecule is still relatively well preserved. The reasons for the discrepancy between Dubochet's results at 4.2 K and those of earlier workers at similar temperatures must still be examined before unbridled joy can sweep through the EM community.