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PRESENT AND FUTURE DEVELOPMENTS OF X-RAY STRUCTURE DETERMINATION OF
PROTEINS AND VIRUSES
R. Fourme
To cite this version:
R. Fourme. PRESENT AND FUTURE DEVELOPMENTS OF X-RAY STRUCTURE DETERMI-
NATION OF PROTEINS AND VIRUSES. Journal de Physique Colloques, 1987, 48 (C9), pp.C9-9-
C9-18. �10.1051/jphyscol:1987902�. �jpa-00227197�
JOURNAL DE PHYSIQUE
Colloque C9, suppldment au n012, Tome 48, ddcembre 1987
PRESENT AND FUTURE DEVELOPMENTS OF X-RAY STRUCTURE DETERMINATION OF PROTEINS AND VIRUSES
R. FOURME
LURE, Universite Paris-Sud, (CNRS, MRES, CEA), Bbt. 2 0 9 0 , F - 9 1 4 0 5 Orsay Cedex, France
Resume:
La d i f f r a c t i o n des rayons X est l a seule methode d'utilisation generale qui permette l a determination de l a s t r u c t u r e tridimensionnelle d'une macromolecule
a
r e s o l u t i o n atomique. E l l e a apporte des r e s u l t a t s essentiels en recherche fondamentale, et son importance va croissant dans l e domaine applique et notamment pour l'ingenierie des proteines. Sa puissance et sa rapidite ont progresse de maniere spectaculaire depuis quelques annees. Ces p r o g r e s portent notamment s u r l e s methodes de cristallisation; s u r l a technologie des sources de rayons X et des detecteurs bidimensionnels, favorisant l e renouveau des methodes d'enregistrement des donnbes de diffraction; s u r des avancees theorlques dans I'utilisation et I'integration de toutes les informations physiques ou experimentales pour resoudre l e problhme des phases; s u r l e graphisme moleculalre, outil puissant d'analyse, de synthese et de communication; l a progression des outils inforrnatiques a egalement joue un r o l e essentiel. Cette evolution est rapidement presentee, ainsi que l e s perspectives a c o u r t et moyen terme.Summaru:
Single c r y s t a l X-ray d i f f r a c t i o n i s t h e unique general purpose method by which t h e 3D-structure of a macromolecule can be investigated t o atomic resolution. I t s Importance, quite obvious f o r fundamental research, i s increasing i n applied research, especially protein engineering. The power and speed of the method have made spectacular p r o g r e s s within t h e l a s t few years. This concerns: crystallization; X-ray sources and area detectors, which favoured a renewal i n data collection methods; t h e o r e t i c a l advances i n t h e use and integration of a v a r i e t y of experimental and physical constraints f o r the solution of the phase problem; m o l e c u l a r graphics, a powerful t o o l f o r analysis, synthesis and communication. The availability of high performance hardware and software has also been c r u c i a l . A n overview of these advances i s presented, together wlth prospects f o r the near future.
X-ray d i f f r a c t i o n methods provide us with images of biological macromolecules t o atomic o r n e a r l y atomic resolution. Direct imaging of a single macromolecule with X-rays i s not possible f o r several reasons: the scattering c r o s s section of l i g h t atoms f o r X-rays i s so weak and the radiation sensitivity of the specimen so l a r g e that the dose required t o get an image with a good signal-to-noise r a t i o (SNR) would destroy the specimen. I n addition, devices acting as a lens a r e now available f o r soft X-rays, but not f o r the 1A range electromagnetic radiation. The solution t o these v a r i o u s problems i s w e l l known: a single c r y s t a l i s used- i.e. a r e g u l a r t h r e e dimensional a r r a y of say 10"
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1987902
macromolecules- instead of a single macromolecule.
The situation i s d i f f e r e n t with electrons: h e r e lenses a r e available and the scattering c r o s s section i s l a r g e r . The major problem i s again radiation sensitivity and, hence, the SNR problem. I n fact, much of the r e s e a r c h e f f o r t i n the l a s t 20 years has been focussed on enhancing t h e SNR values of the images. Averaging of l a r g e numbers of noisy images f r o m individual molecules was the basic idea f o r obtaining good SNR images. By recording a l a r g e number of individual m o l e c u l a r images using a low t o t a l e l e c t r o n exposure p e r molecule, one can obtain images of intact molecules and yet have a good SNR r e s u l t i n the f o r m of t o t a l average. I n X-ray crystallography, the radiation load i s shared by a l l individual molecules i n the c r y s t a l during the r e g i s t r a t i o n of the diffractogram; post-registration averaging of m o l e c u l a r images i n e l e c t r o n microscopy i s the equivalent of t h i s p r i n c i p l e of
"shared suffering" The processed e l e c t r o n microscope images a r e various projections of the molecule, f r o m which a 30 reconstruction i s attempted. This approach i s evolving i n t o a rapid and r e l i a b l e technique t o determine at l e a s t l o w resolution (15-40
A)
molecularstructures, especially on l a r g e macromolecular assemblies /I/.
3D s t r u c t u r e s o f macromolecules i n solution can also be t a c k l e d by NMR techniques;
but this approach i s l i m i t e d t o proteins of low m o l e c u l a r weight.
Accordingly, X-ray single c r y s t a l d i f f r a c t i o n i s c u r r e n t l y the unique general purpose method by which the 3D s t r u c t u r e of a macromolecule can be investigated t o atomic, o r n e a r l y atomic, resolution. The development of biological c r y s t a l l o g r a p h y durlng t h e l a s t few y e a r s has been quite impressive. The f r a c t i o n of papers devoted t o biological c r y s t a l l o g r a p h y i n international meetings i s steadily increasing : fo r instance, about 30% of t h e contributions submitted t o the 1987 International Congress of Crystallography at Perth, Australia, w e r e r e l a t e d t o the field.
There a r e several reasons f o r t h i s development. The modern language of enzymology and m o l e c u l a r biology owes much of i t s sophistication t o the success of X-ray diffraction.
This technique has revealed the detailed a r c h i t e c t u r e of many proteins, enzymes, nucleic acids, complexes between proteins and nucleic acids, v i r u s e s . Molecular assemblies, such as a photosynthetic centre, have r e c e n t l y been solved; and t h e r e a r e prospects t o solve t h e s t r u c t u r e of the complete ribosome- t h e organite which synthesizes proteins i n cells. But a new dimension of biological c r y s t a l l o g r a p h y i s emerging: i t s d i r e c t intervention i n biotechtology, with c r u c i a l i n d u s t r i a l and social stakes. Let us c i t e Blow et a l /2/: "Protein engineering i s t h e production of a novel o r a l t e r e d protein- a 'factitious' protein- by t h e creation o r manipulation of a gene which c o n t r o l s i t s synthesis, f o r a specific purpose..
.
Effective p r o t e i n engineering depends on an appreciation of the structure-function relationships of the factitious protein, and so depends l a r g e l y on c r y s t a l l o g r a p h y f o r s t r u c t u r a l data. I n the f i r s t place, this w i l l come f r o m t h e s t r u c t u r e of the wild-type protein. Without t h i s knowledge, redesign of a p r o t e i n i s a m a t t e r of guesswork, and could often be done b e t t e r by evolution under a c o n t r o l l e d selective pressure, as i n a chemostat...
Crystallography i s also important t o make a s t r u c t u r a l check of a newly engineered protein, t o see whether t h e design has worked out as planned..." .
There a r e various ways t o describe the status of biological crystallography. A s a complete p r o j e c t i s a s e r i a l sequence of s t e p s , we have chosen t o describe v e r y b r i e f l y recent p r o g r e s s and t r e n d s relevant t o each step.
C r u s t a l s
Crystallization of macromolecules i s a p r e r e q u i s i t e t o any s t r u c t u r a l study and may be a limiting f a c t o r . A l a r g e amount of observations has been gathered and semi-empirical p r o t o c o l s have emerged with the numerous macromolecules and macromolecular systems which have been amenable t o c r y s t a l l i z a t i o n . But t h e r e i s a recent i n t e r e s t of physicists and biologists t o b e t t e r understand the fundamental processes whlch govern the nucleation phase, t h e growth phase and the cessation of growth.
Lack of c o n t r o l of p u r i t y i s a common cause of unsuccessful c r y s t a l l i z a t i o n . The necessity of " c r y s t a l l o g r a p h y grade" p u r i t y i s now f i r m l y established / 3 / : t h i s means p u r i t y i n t e r m s of contaminating molecules o r ions, but also i n t e r m s of sequence i n t e g r i t y and conformational homogeneity. Recent p r o g r e s s i n chromatographic and genetic engineering technologies a r e helping t o satisfy these requirements.
Among recent achievements, we c i t e the c r y s t a l l i z a t i o n i n t h r e e dimensions of i n t e g r a l membrane proteins /4,5/ i n a suitable detergent system such as p-octyl glucoside;
t h e c r y s t a l l i z a t i o n of the complex of a s p a r t y l tRNA and a s p a r t y l tRNA synthetase; and f i n a l l y t h e c r y s t a l l i z a t i o n of the l a r g e 50s subunit of the ribosome, a p a r t i c l e on which biosynthesis occurs i n a l l organisms, which consists of the assembly of 32 proteins and 2 RNA chains with a t o t a l molecular weight (MW) of 1.6 x l o 6 Daltons.
Future prospects include standardization and robotization of c r y s t a l l i z a t i o n methods, i n o r d e r either t o get high reproducibility i n experiments o r t o p e r f o r m c o n t r o l l e d variations of a selected parameter; c r y s t a l l i z a t i o n i n systems with phase separation, i n gels o r i n z e r o gravity; and b e t t e r knowledge of solution properties of macromolecules and physical-chemical properties of precipitants.
D i f f r a c t i o n data c o l l e c t i o n
The mathemetical r e l a t i o n between a sample which s c a t t e r s X-rays and the scattered waves i s the F o u r i e r t r a n s f o r m . F o r a single crystal, which has a n e a r l y p e r f e c t l y perlodic e l e c t r o n density, the F o u r i e r t r a n s f o r m i s essentially z e r o everywhere except at r e c i p r o c a l l a t t i c e nodes. Once the set of moduli and phases of complex numbers (called s t r u c t u r e factors) at each node a r e known, the average e l e c t r o n density of the sample i s obtained by i n v e r s e F o u r i e r transformation. I n fact, detectors a r e not sensitive t o t h e phase (even i f that were not so, the phases would be scrambled by X-ray source incoherence and the mosaic s t r u c t u r e of macromolecular c r y s t a l s ) and measure only intensities of Bragg reflections, which a r e proportional t o the square of t h e s t r u c t u r e f a c t o r moduli. F o r t o t a l l y unknown structures, the phase problem i s solved i n n e a r l y a l l cases by inducing intensity changes of Bragg reflections either by l a b e l l i n g t h e molecules I n the native c r y s t a l with heavy atoms (multiple isomorphous replacement method o r MIR) o r by anomalous dispersion: what i s done i n both cases i s basically triangulation, with a s t a t i s t i c a l treatment of e r r o r s .
Data f r o m the native c r y s t a l and s e v e r a l derivatives have t o be measured; f o r each of them, thousands t o m i l l i o n s of reflections, depending on the complexity of the s t r u c t u r e and on the resolution, have t o be measured with a sufficient redundancy. Crystal degradation under the X-ray exposure i s a f u r t h e r complication. Hence, t h e measurement of diffraction data i s a considerable task. I n the past few years, the status of data collection has evolved quite remarkably; this i s due t o progress i n sources as w e l l as detectors.
li) X-rau sources
Data c o l l e c t i o n i s s t r o n g l y dependent on available X-ray sources. F o r macromolecular crystallography, the ideal source must have a high spectral b r i l l i a n c e i n the wavelength range f r o m 0.5A t o 2.9,. Rotating anode tubes were a significant progress with respect t o conventional sealed tubes; but t h e i r s p e c t r a l b r i l l i a n c e i s limited f o r thermal and mechanical reasons and only the c h a r a c t e r i s t i c K, o r Kp emission lines a r e of p r a c t i c a l use. The availability of synchrotron radiation emitted by multi-GeV electron/positron storage rings was the r e a l breakthrough. A conventional dipolar magnet source has a s p e c t r a l b r i l l i a n c e which i s 2 o r d e r s of magnitude higher than a rotating anode tube ; a multipole wiggler brings a f u r t h e r gain of 1 t o 2 o r d e r s of magnitude. To f i x ideas, a r o t a t i o n camera using phosphor plates and the radiation f r o m the 5 pole superconducting wiggler w i l l c o l l e c t data about
lo4
f a s t e r than a camera using f i l m s and a rotating anode tube, so that a complete d i f f r a c t i o n data set f o r a c r y s t a l of a l a r g e p r o t e i n (MW 100,000) w i l l r e q u i r e a t o t a l exposure time of a few minutes. It t u r n s out that radiation damage, f o r a fixed dose, i s s m a l l e r f o r higher dose rates; accordingly, not only a r e exposure times dramatically s h o r t e r with SR sources, but a l s o m o r e information can be obtained f r o m each c r y s t a l . This l a t t e r fact, together with the improved SNR obtained with SR sources, explains why d i f f r a c t i o n data can i n general be collected t o higher resolution with a SR source than with a conventional source, thus leading t o m o r e detailed e l e c t r o n density maps. Another useful p r o p e r t y of SR i s i t s continuous spectrum: the wavelength of the X-ray beam may be chosen i n o r d e r t o produce strong anomalous dispersion effects;another applicatio,n of tuneability, at least with wiggler sources, i s the use of wavelengths s h o r t e r than the usual 1.5A radiation i n o r d e r t o reduce the absorption of X-rays by the c r y s t a l , thus improving both the quality of data and c r y s t a l lifetime.
fii) d e t e c t o r s
The ideal detector should count a l l photons emitted by the c r y s t a l during exposure;
f r o m t h i s r a w data, it should e x t r a c t i n nealy r e a l time the backpround-corrected intensities of Bragg reflections.
The f o u r c l r c l e diffractometer with a single scintil?ation counter i s of l o w efficiency i n p r o t e i n crystallography, because it measures one r e f l e c t i o n at a time. It i s now mainly used f o r p r e l i m i n a r y studies at l o w resolution and search f o r heavy atom derivatives.
The simultaneous recording of many r e f l e c t i o n s became possible with the r o t a t i o n camera using a f i l m as high spatial resolution area detector. I n the rotation method, the c r y s t a l exposed t o X-rays i s slowly rotated and each cassette of a carousel i s exposed t o the photons emitted during a rotation of the c r y s t a l over a range of about 1'. Films a r e scanned with a computer-controlled densitometer, producing digital data which a r e analysed with sophisticated programs t o obtain intensity data.
New integrating area detectors have r e c e n t l y appeared (manufactured by Kodak and Fuji companies); i n t h i s detector system, an X-ray image i s t e m p o r a r i l y s t o r e d as a distribution of F-centers i n a phosphostimulable phosphor ( B ~ F B ~ : E U ~ + ) ; the latent image i s read out by measuring the intensity of fluorescence stimulated by a He-Ne l a s e r beam scanned over the s u r f a c e of the plate. Phosphor plates have a 100% efficiency f o r 0.7- 1.5A X-rays, a spatial resolution of 100pm, a dynamic range of 1:10~ and no count r a t e limitations / 6 / ; they can be used i n the same way as f i l m s but they a r e f r e e of t h e i r
drawbacks, except that t h e image remains r e l i a n t on a m a t e r i a l medium.
Diffractometers with electronic area detectors (EAD) have also been built, and several systems of programs f o r on-line analysis a r e now available, at least i n a somewhat preliminary form. EAD have distinct advantages: they provide a l i s t of s t r u c t u r e factors, ready f o r use; as each image i s just information s t o r e d i n memory chips, time information i s not l o s t and they make it possible t o r e c o r d as many electronic images as needed t o optimize the SNR i n each p a r t i c u l a r case. Most of these detectors a r e m u l t i w i r e proportional counters (MWPC) which function i n a pulse-counting mode and have a count r a t e l i m i t e d t o 30,000-400,000 cps. One commercial instrument i s an integrating TV detector, which can cope with intensities higher than MWPC but with a limited dynamic range, spatial distortion and nonuniformity of response. Integrating electronic detectors based on l a r g e CCD a r r a y s could have a higher count r a t e and a m o r e uniform response than c u r r e n t detectors. This development Is c r u c i a l and challenging, especially i n view of the high fluxes expected f o r t h e coming generation of 6 GeV storage rings.
liii) Data c o l l e c t i o n methods
I n p r o t e i n crystallography, the X-ray radiation i s usually monochromatic (in SR experiments, t y p i c a l values a r e h=1.4A and 6h/h=l0-~) and the exploration of r e c i p r o c a l space i s done by rotating the c r y s t a l . This method i s inefficient because it makes no use of most of incoming photons. Taking into account the smooth continuum of SR, it was tempting t o t r y a different strategy: use a broad bandpass and l e t the s t i l l c r y s t a l select i n the spectrum a l l wavelengths which satisfy t h e Bragg relation. This i s just the o l d Laue method, wnich has been revisited. Experiments have shown that e x t r a o r d i n a r i l y c l e a r and sharp d i f f r a c t i o n p i c t u r e s of protein c r y s t a l s could be obtained with v e r y s h o r t exposure times /7,8/; it was also demonstrated, using number theory, that (contrary t o intuition), a l a r g e f r a c t i o n of r e f l e c t i o n s obtained with white radiation a r e unique (1.e. without overlap of several o r d e r s of diffraction)/W. Simulations have also shown that the spatial overlap of reflections i s not a too severe problem and that
,
with a few different settings of a crystal, it was possible t o c o l l e c t the major p a r t of i t s diffractogram. The s o f t w a r e used f o r monochromatic radiation methods was adapted t o polychromatic radiation, so as t o obtain a l i s t of s t r u c t u r e f a c t o r s of reasonable accuracy, suitable f o r s t r u c t u r e analysis.With most powerful SR sources, focussing optics and detection by phosphor plates, d i f f r a c t i o n data can now be collected i n the millisecond time range by the Laue method. A new field, investigation of time-dependent properties of a macromolecular crystal, i s now open. I n 15 years, f r o m the diffractometer on a sealed anode tube t o a phosphor p l a t e Laue setup on a SR source, the data collection r a t e has improved by 9-10 o r d e r s of magnitude!
The ~ h a s e ~ r o b l e m
I n o r d e r t o c a l c u l s t e t h e e l e c t r o n density, we need t o know s t r u c t u r e f a c t o r s F(hkl):
that i s we need t o know both the amplitude F(hk1) and the phase a(hkl) of t h e s t r u c t u r e factor. The basic equation i s
p(xy Z) = 1 /V
XIX
F(hkl)expia(hkl)exp-2ni(hx+ky+lz) h k lAs emphasized previously, only the intensities, and hence the amplitudes of the diffracted r a y s may be measured. The phase information i s l o s t . This problem of phase determination i s t h e basic problem i n any s t r u c t u r e determination. Mathematically, the problem i s indeterminate, because the set of s t r u c t u r e f a c t o r amplitudes f r o m the native
c r y s t a l does not determine unequivocally the s t r u c t u r e . To solve it, the c r y s t a l l o g r a p h e r uses some additional information, which bears constraints on the s t r u c t u r a l model.
Diffraction data themselves can be considered as a set of experimental constraints; other informations a r e general physical c r i t e r i a l i k e positivity of the e l e c t r o n density function.
(1) E x D e r l r n e n t a l c o n s t r a f n t s ( 1 . e . based on d i f f r a c t i o n data only)- may be sufficient t o derive phases which a r e good enough t o compute an i n t e r p r e t a b l e e l e c t r o n density map.
The basic idea i s that a s m a l l change i s made t o the diffracting unit by the preparation of heavy atom derivatives (multiple isomorphous derivative method, o r MIR), making some s o r t of triangulation posssible. To take e r r o r s i n t o account, a phase probability distribution i s calculated; t h e phase calculated as t h e centroid of the distribution i s taken as the "best "
phase" and t h e amplitude t e r m i s given a weight that depends on the probable e r r o r s on the phase; t h e map calculated f r o m the weighted amplitudes and the "best" phases w i l l have the minlmum l e a s t squares e r r o r .
Although the MIR method remains the basic method of phase determination, the preparation of several derivatives i s often a tedious job, and, i n general, the derivative s t r u c t u r e s a r e not p e r f e c t l y isomorphous t o the native s t r u c t u r e . There i s another method t o mimic s e v e r a l p e r f e c t l y isomorphous derivatives with a single sample, j u s t by tuning the wavelength of the X-ray radiation. This method r e l i e s on anomalous scattering by a few label atoms bound n a t u r a l l y o r a r t i f i c i a l l y t o t h e molecule. Anomalous dispersion i s p a r t i c u l a r l y pronounced at absorption edges; that is, when the X-ray energy approaches the c h a r a c t e r i s t i c energy l e v e l s of atomic orbitals; the diffractogram i s thus recorded at several wavelengths close t o one of the absorption edge of the label atoms. The tunability of SR sources has stimulated research on anomalous dispersion methods. Although the idea i s simple, t h e p r a c t i c a l experiments a r e d i f f i c u l t because small intensity changes must be accurately measured; only a few s t r u c t u r e s have now been solved by m u l t i p l e wavelength anomalous d i f f r a c t i o n method (MAD), but the prospect Is bright. Our group solved the s t r u c t u r e of a calcium-binding protein, parvalbumin, f r o m anomalous dispersion by terbium at t h r e e wavelengths close t o the L I I I absorption edge/lO/. Another group has used selenium as anomalous scatterer, with wavelengths close t o the K absorption edge of selenium; i n o r d e r t o solve the s t r u c t u r e of streptavidin, selenomethionine was i n c o r p o r e d i n streptavidin i n the place of (su1fur)methionine by recombinant DNA t e c h n i q u e d l l/.
(ii) Phusical c o n s t r a i n t s a r e based on o u r a-priori knowledge of the c h a r a c t e r i s t i c s of t h e density function. Methods which apply these constraints a r e c a l l e d i n general density modification methods (DM)
.
It i s out of our scope t o give details about these methods.There a r e s e v e r a l possible ways t o design DM algorithms, whlch can be roughly be divided i n t w o categories
1- i t e r a t i v e alaorithms. I n these algorithms, constraints a r e imposed alternatively i n r e a l and r e c i p r o c a l spaces, so that one searches f o r a map that has an optimal agreement with them. Constraints include positivity of t h e e l e c t r o n density; high r e s o l u t i o n atomicity;
uniformity of solvent regions; continuity of t h e biopolymer chain; and known non- c r y s t a l l o g r a p h i c symmetry. They may be extremely powerful; i n particular:
- non-crystallographic symmetry obliges v a r i o u s points i n the unit cell, not r e l a t e d by space g r o u p operations, t o have the same e l e c t r o n density values. This method has been c r u c i a l t o solve the s t r u c t u r e of those macromolecular assemblies which a r e b u i l t f r o m identical building blocks, such as v i r u s e s .
-in macromolecular crystals, between 30 t o 85% of the c r y s t a l a r e occupied by the solvent, so t h a t t h e existence of f l a t solvent regions also places s t r o n g constraints on the s t r u c t u r e f a c t o r phases, especially i f the solvent content i s high. I n o r d e r t o implement it, the m o l e c u l a r boundaries a r e identified and t h e density i n the solvent regions I s replaced by i t s mean value. During the l a s t few years, many papers have appeared on techniques dealing with solvent flatness and especially about t h e definition of molecular boundary (see e.g./12/).
2- maximimum e n t r o ~ u method (MEM). A n alternative way t o t r e a t t h e data i s t o use the method of maximum entropy (MEM), which selects the most uniform e l e c t r o n density distribution f r o m among a l l those that f i t t h e observed s t r u c t u r e amplitude data t o within the experimental e r r o r s ; t h e r e should t h e r e f o r e be no features i n t h e map that a r e not supported by the data. W o r k along those lines l e d t o a revision of t h e classical s t a t i s t i c a l methods of phase determination ( known as d i r e c t methods, and used f o r the solution of the s t r u c t u r e of thousands of molecules with up t o about 150 atoms) which widens t h e i r theoretical foundations and consolidates t h e i r p r a c t i c a l implementation (Bricogne /l3/).
This method can use both native and heavy atom derivative d i f f r a c t i o n data and can make f u l l use of knowledge about p r i o r information on the solution, which a r e combined with the d i f f r a c t i o n data i n a coordinated way. According t o Bricogne (this i s also the author's opinion), t h i s unified phase determination strategy i s l i k e l y t o b r i n g macromolecular s t r u c t u r e s within the reach of d i r e c t methods. Woolfson has expressed r e c e n t l y a different appreciation about MEM /14/:
". .
.entropy maximisation i s adding nothing completely new t o the c r y s t a l l o g r a p h i c scene, and since it involves a great deal of effort, perhaps nothing useful."M o l e c u l a r a r a ~ h i c modeling
The e l e c t r o n density map produced by the i n i t i a l phasing r a r e l y provides atomic resolution
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The next step of t h e analysis i s the construction of an atomic model which f i t s reasonably t o t h e e l e c t r o n density; f r o m t h i s model, a tentative set of atomic coordinates i s obtained. I n practice, t h i s i n i t i a l model i s progressively improved through many cycles of a procedure i n which the c r y s t a l l o g r a p h e r i n t e r a c t s with the model through computer graphics then proceeds t o some kind of automatic refinement procedure by a computer.The f i r s t p r o t e i n model (myoglobin) was b u i l t i n 1959 out of metal r o d s at a scale of 5cm/A and l a t e r 2cm/A; coloured paper c l i p s w e r e fixed t o the v e r t i c a l r o d s t o mark e l e c t r o n density values.These w i r e models allowed bond rotations which could be fixed by tightening screws. The standard model-building t o o l f o r s e v e r a l y e a r s was the Richards box, which used a semi-silvered m i r r o r t o overlap the e l e c t r o n density (drawn as contours on plastic sheets) and t h e w i r e model. Then electronic devices were designed by 197q.
Since then, computer graphics has become a standard t o o l f o r crystallographers; it has reached through hardware and software developments a high degree of sophistication and i n t e r a c t i v i t y . The graphics display allows t h e model t o show t h e c r y s t a l l o g r a p h e r a p a r t of i t s e l e c t r o n density such that he can decide how a m o l e c u l a r fragment best f i t s . The aim of a m o l e c u l a r f i t t i n g program i s t o c r e a t e the necessary environment t o a l l o w the c r y s t a l l o g r a p h e r t o decide what atoms he wants i n what piece of density.
Perhaps 10 e l e c t r o n density f i t t i n g program systems a r e i n active use.0ne of the most popular system I s FROD0/15/. This system i s a general modeling system. It incorporates now various extensions which make use of the v a s t l y increased computing power of t h e 32-bit computer which c o n t r o l s the display. One of them i s r e a l space
refinement, an extension of the molecular fragment r o t a t e / t r a n s l a t e menu option; the
fragment moves as a r i g i d body t o maximize the g r i d sum convolution . ~ Q ~ where ~ ~ ~ Q ~ ~ ~ ,
Qcalc i s t h e calculated density obtained f r o m a gaussian function and pobs the observed g r i d density (i.e. calculated with c u r r e n t phases). Another extension of FRODO i s a set of tools that pickout, i n r e a l time at the graphics terminal, the best match between an input set of Ca-coordinates and a data base of s t r u c t u r e s . Tests suggest that a l l p a r t s of a new p r o t e i n already exist i n the Protein Data Bank!
Clearly, density f i t t i n g programs evolve t o combine sophisticated graphic tools, data base information, a r t i f i c i a l intelligence and on-line refinement. The r e s u l t w i l l be the semi-automatic production i n s h o r t e r times of accurate models.
Density f i t t i n g programs r e s o r t t o the activity of analysis, which i s only one aspect of molecular graphic modeling. Let us c i t e Olson /16/ j u s t t o emphasize that molecular graphics i s now a discipline in itself, at t h e f r o n t i e r of many other sciences and a r t .
"
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Computer graphic molecular modeling supports t h r e e general activities:synthesis, analysis and communication. I n each of these a tivities, it i s the high bandwith of information transmission and t h e t h r e e dimensional cont x t of the visual images that give u t i l i t y t o t h e computer graphic representation..
. J
I n t h e activity of synthesis, t h e modeling i s used t o b u i l d o r extend existing models by combining information and knowledge f r o m a v a r i e t y of sources f o r i t s construction..
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Analysis r e q u i r e s selective display of experimental and/or computional r e s u l t s i n a comprehensible framework. Display and comparison of any number of macromolecular properties such as chemical composition, connectivity, m o l e c u l a r shape, electrostatic p r o p e r t i e s o r mobility c h a r a c t e r i s t i c s a l l f a l l i n t o the domain of modeling analysis..
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Communication i s c r i t i c a l i n science i f ideas a r e t o compete and take hold.Computer graphics provide a powerful medium f o r conveying complex three-dimensional relationships i n t h e s t r u c t u r e and function of biological molecules. It i s imperative that this information i s comprehensible not only t o the s t r u c t u r a l scientist but also t o t h e l a r g e r body of scientists whose expertise can add data and knowledge t o increase o v e r a l l understanding..
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M o l e c u l a r dunamics
The diffractogram of a p r o t e i n c r y s t a l contains information, not only on the time and space-averaged structure, but also on the dynamics of molecules i n the c r y s t a l lattice.
(1) Atomic t e m ~ e r a t u r e f a c t o r s a r e parameters which a r e adjusted during the course of s t r u c t u r e refinement. Those temperature f a c t o r s give indications on the degree of motion i n various p a r t s of the s t r u c t u r e . A colour-coded representation of the atoms on a graphic display i s useful t o visualize r e s u l t s . Such studies a r e often coupled with m o l e c u l a r dynamics calculations and normal-mode analysis.
(ii) X-rau diffuse s c a t t e r i n g i s a promising field. A s diffuse scattering i s
lo2-lo4
times weaker than Bragg reflections, a SR source i s r e q u i r e d f o r data collection. This technique can y i e l d information on the atomic and molecular displacements, provided that they a r e correlated. It has been recently applied at LURE t o hen egg-white lysozyme. I n t h i s pioneering work/l7/, t h e major features of diffuse scattering were explained by a simple model of rigid-body m o l e c u l a r displacements c o r r e l a t e d along short rows of aligned molecules i n t w o perpendicular directions.Landmarks
This survey of modern biological crystallography w i l l be i l l u s t r a t e d by a few examples:
(I)- Photosunthetic reaction c e n t r k o f t h e p u r p l e bacterium Rhodopseudomonas v i r i d l s /18/.
This reaction c e n t r e consists of s e v e r a l p r o t e i n subunits and a c-type cytochrom with f o u r covalently linked haem groups. Other prosthetic groups associated with the reaction center a r e f o u r bacteriochlorophyll b, two bacteriophyophytin b, two quinones, one non-haem f e r r o u s i r o n and one carotenoid. This work i s t h e f i r s t description of the high resolution s t r u c t u r e of an i n t e g r a l membrane protein; it i s also one of the l a r g e s t s t r u c t u r e s which has e v e r been solved (10288 independent non-hydrogen atoms without non-crystallographic symmetry) and it has now been highly refined (R=0,19). The detailed a r c h i t e c t u r e of t h i s complex i s obviously a major progress f o r r e s e a r c h on photosynthesis.
Rhinovirus 14 has an external diameter of about 300A and f o r m s icosahedral shells.
I t s MW i s around 8 . 5 ~ 1 0 ~ . The shell i s b u i l t of 6 0 protomers, each composed of four proteins; t h e molecular weight of a protomer i s 94,000. The s t r u c t u r e was solved f r o m data collected with SR radiation, using MIR techniques, symmetry averaging (which I s especially powerful since t h e r e a r e 60 copies of t h e protomer) and a Cyber 205 supercomputer/l9/
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Poliovirus i s a closely r e l a t e d virus; data w e r e collected with a conventional X-ray source and a Vax 750 midicomputer was used f o r calculations/20/.
(iiil- Catalusis i n t h e crustal: s y n c h r o t r o n radiation studies with glycogen phosphorylase.
This work demonstrates t h e feasibility of time-resolved studies on a complex enzyme, glycogen phosphorylase b (MW about 100,000 Daltons) which catalyses t h e f i r s t step i n the breakdown of glycogen; it i s the key enzyme through which hormonal, nervous and metabolic signals a r e r e l a y e d t o meet the energy requirements of the muscle cell; the s t r u c t u r e had previously been solved by standard methods.
Direct observation of t h e p r o g r e s s of a catalysed reaction i n c r y s t a l s of phosphorylase has been made possible through fast data c o l l e c t i o n with SR; a complete data set data t o high resolution could be measured i n times as s h o r t as 25 minutes with monochromatic radiation. I n a series of time-resolved studies i n which the c o n t r o l properties of the enzyme were exploited i n o r d e r t o slow down t h e reaction, t h e conversion of heptenitol t o heptulose-2-phosphate, t h e phosphorylysis of maltoheptaose t o y i e l d glucose-1-phosphate and the oligosaccharide synthesis reaction involving m a l t o t r i o s e and glucose-1-phosphate have been monitored i n t h e c r y s t a l . Changes i n t h e e l e c t r o n density i n the difference F o u r i e r maps were observed as t h e r e a c t i o n proceeded, not only at the catalytic s i t e but also t h e a l l o s t e r i c and glycogen storage sites /21/. Similar experiments have been done by the same group with an improved time resolution using the Laue method with SR /22/.
References
/1/ Van Heel, M., i n Crystallography i n Molecular Biology ( M o r a s D. et al, Ed., Plenum Press, New York) (1987) 89.
/2/ Blow, D.M., Brick, P., Brown, K. A.
,
Fersht, A., R., Winter, G., i n Crystallography i n Molecular Biology ( M o r a s D. et al, Ed., Plenum Press, New York) (1987) 241./3/ Giege, R., Dock, A., C., Kern, D., Lorber, B., Thierry, J. C. and Moras, D., J. of Cryst. Growth
76
(1986) 554./4/ Michel, H. and Oesterhelt, D., Proc. Nat. Acad. Sci. USA Z ( 1 9 8 0 ) 1283.
/5/ Garavito, R. M. and Rosenbusch, J., P., J. Cell Biol.
B
(1980) 327./6/ Miyahara, J., Takahashi, K., Amemiya, Y., Kamiya, N., Satow, Y., Nucl.
I n s t r u m . Methods
A236
(1986) 572./7/ Moffat, K., Szebenyi, D. and Bilderback, D., SclenceZZ3(1984) 1423.
/8/ Helliwell, J., J. Mol. Struct. m ( 1 9 8 5 ) 63.
/9/ Cruickshank, D., W., J., Helliwell, J. and Moffat, K., Acta Cryst., A43 (1 987) 656.
/ l o / -
Kahn, R., Fourme, R., Bosshard, R., Chiadmi, M., Rlsler, J., L., Dideberg, O., and W ery, J., P., FEBS Lett. (1985) 133.11 1/ Smith, J., L., Pahler, A., Krishna Murthy, H., M. and Hendrickson W., A,, Collected A b s t r a c t s of Fourteenth International Congress of
Crystallography (1987), a b s t r a c t 01.2-3.
/12/ Podjarny, A., D., i n Crystallography i n Molecular Biology ( M o r a s D. et al, Ed., Plenum Press, New York) (1987) 63.
/13/ Bricogne, G., A c t a Cryst. m ( 1 9 8 4 ) 410.
/14/ Woolfson, M., M., Acta Cryst M ( l 9 8 7 ) 593.
/15/ Jones, T., A., J. Appl. Cryst.
U
(1978) 268./16/ Olson, A., J., i n Crystallography i n Molecular Biology ( Moras D. et al, Ed., Plenum Press, New York) (1987) 131.
/17/ Doucet, J. and Benoit, J., P., Nature 323 (1987) 643.
/18/ Deisenhofer, J., Epp, O., Miki, K., Huber, R. and Michel, H., Nature U ( 1 9 8 5 ) 618.
/19/ Rossmann, M. G.
,
Arnold, E., Erickson, J. ,W., Fankenberger, E., A., Griffith, J.P., Hecht, H., J., Johnson, J. E., Kamer, G., Luo, M., Mosser, A., G., Rueckert, R., R., Sherry, B. and G. Vriend, Nature X ( 1 9 8 5 ) 1 4 5 .
/20/ Hogle J.,M., Chow, M. andFilmanD. J., Science a ( 1 9 8 5 ) 1358.
/21/ Hajdu, J., Acharya, K., R., Stuart D., I., McLaughlin, P., J., Barford, D., Oikonomakos, N., G., Kleln, H. and Johnson, L., N. The EM60 J. 6(1987) 539.
/22/ Hadju, J., Machin, P., A., Campbell, J., W:, Greenough, T., J., Clifton, J., J., Zurek, S., Gover, S., Johnson, L., N. and Elder, M., Nature329(1987) 178.
