Medical Research Council

W?RC Labountory of Molecular Bio!ogy
University Postgraduate Medical School
Hills !3on.d, Cambridge, C52 2QH
England

telephone Cambridge ( 0223 ) 48011
telex i 81532

28 September 1976

Dr. F. H. C. Crick, F.R.S.,
The Salk Institute,
Post Office Box 1809,
San Diego,
California 92112, U.S.A.

Dear Francis,

   Thank you for your letter of 17 September, with the enclosed internal
memo.  The latter is very clear and it is good to have your ideas written
down.

   I believe I may now have an explanation of the packing in the crystals
which relates to other information and ideas on chromatin structure.
I would be very glad to have your opinion soon on this as I am considering
mentioning these ideas in the paper on crystals (so far not beyond the
draft stage!).

    The unit cell dimensions are a = 110 8, b = 193 8, c = 340 2, and
all odd reflections are missing on the principal    reciprocal lattice
lines,  suggesting a halving in projection onto.these lines.  My inter-
pretation is as follows.  Columns of nucleosomes parallel to the c axis
are hexagonally packed to give the pseudo-hexagonal cell of the ab plane.
Neighbouring columns are staggered by half a nucleosome to account for
the halving of the OOR reflections.  There are three nucleosome cores in
the repeat distance of 340 8, successive particles being rotated through
120o.

    How does this relate to other ideas?  Along a natural nucleofilament,
the successive nucleosomes must be related by translation only so that
they can form a solenoid in which,  as you have pointed out, all nucleo-
somes must be equivalent,   relative to the helical axis.    Consider an
ideal solenoid which has the minimum amount of DNA, i.e. just sufficient
to make two turns of helix, without the additional loop which you
postulate as an excrescence looping around the Hl on the outside of the
nucleofilament.  The number of base pairs to two turns is say 170 - 180
(we need to know the minimum repeat size in different species - physarum
is 171).  Now what happens in a Zachau reconstitution which gives a
repeat of about 140 base pairs?  Presumably the nucfeosomes pack more
tightly into the "screwed down" position and there will now be
140/170 x 2 turns per bead.   X/e know that the DNA does not have to be
continuous for nucleosome association,  so one assumes that the packing
in the columns in the crystals is the same as in Zachau's reconstituted
material.  If there were 1 2/3 turns of DNA per 140 base pair nucleosome
core,  and the packing was such as to make the DlGA geometrically
continuous, then the 120"scrow along the 340 x axis would come out

nclillroll~7


Dr . F. H. C. Crick, F.R.S.

-2-

28 September 1976

   If this picture is right, then the inter-nucleosome spacing in
the screwed down position is 113 2 but,  in the natural position in a
nucleofilament,
i.e. to 130 2.  this spacing would be increased by a factor of 6/5,
           If one takes the Sperling and Tardieu determination of
mass per unit length,  indeed one finds this is the spacing of
nucleosomes.  Moreover, Chambon has always quoted the diameter of a
nucleosome (by which I think he means inter-nucleosome spacing) in the
SV40 complex as 125 - 135 8.  If this is correct, then the pitch of
the DNA helix would be 130/2 = 65 8,  a value not inconsistent with
the apparent helical character of the a axis photograph of the crystals
(at face value here the pitch would be more like 340/6 = 58 2, but

??????*?? o  Some of the curves Linda Sperling
peaks more in the 60 2 region than the 55,

the latter will of course be the second order of the spacing between
turns of the solenoid or columns of the crystal.

   This picture is of course over-simplified.  From looking at the
X-ray photographs,  it seems likely that the ideal structure described
above is perturbed.   Moreover it would be neater if the nucleosome
particles contained 150 base pairs, i.e.  1 6/9 turns of a helix kinked
B la Sobell.,
OntO the aby" This would also give a three fold character in projection
          The minimum nucleofilament structure containing two
turns would then have 180 base pairs.   Is this all too neat?

         Everybody-is now back after holidays and conferences, but we miss
       Daniela, who will be at home with her baby for three to six months,
       and Barbara has been ill,  so we have not made much progress in finding
         \  out just what the nature of the histone degradation is that makes the
ii !:I ~,,&'$%?-i'. big crystals.
& \ct-$a            I am pretty convinced that the packing in the micro-

            c rystals (i.e. the hexagonal layers of which we had e.m.s and powder
      photographs) which are made by intact core particles, is different from
       that of the large crystals.  The simplest explanation is that neigh-

yf& b c     ouring columns pack in a register to give a sheet structure, but the
   \  packing isn't as neat so that the crystals"don't grow as long.
w ,+-.J i

    More news.  Ulf Laemmli came and, by his new technique, produced
some very long 300 2 fibres which John Finch has looked at in e.m.
and by X-rays.  The X-rays don't show anything new because, I think,
the solenoids are twisted and need straightening.  One assumes that in
the nuclei, solenoids are not perfect, but take UP  various modifications
as the gene is packed.  However,  the material is certainly a great
advance, and we hope to be able to get a good estimate of the mass per
unit length (we may find how to straighten it out in solution) and also
use it for making oriented specimens for X-ray work.  The material is
fairly stable because one doesn't need to add magnesium: it seems to
contain a fair number of non-histone proteins and probably represents
the most intact extracted material anybody has so far obtained.   We are
not advertising our work on this new material because Uli Laemmli wants
a breathing space to study the physical chemistry, and, likewise, we
need to get the feel of the material which will take a few months, but
I think we do have a way in now to higher order structures in a more
na,tural state.


Dr. F. B. C. Crick, F.R.S.        -3-

28 September 1976

   However the number of things that needs to be done is still enormous.
We need to know the mass per unit length of the Zachau reconstituted
material,  the distance between nucleosomes in this and in natural nucleo-
filaments and so on, so that even dealing with the ordered structures
alone is going to keep our hands full.

   Len is going to write up his stuff, but he is trying to obtain
some good gel patterns, all taken on the same specimen, for publication.

   By the way, what do you think of this idea!  It is quite striking
that the DNAase I digestion pattern of the degraded nucleosome cores
is so similar to that of the particles with intact histones.   On the
assumption that the amino acids come from the N-terminus, this would
mean that the so called tails of the histones are not vital to packing
the DNA onto the core  - this will be done by other interactions.
What then are the tails for?  Presumably to enable nucleosomes to
pack side by side in the contacts necessary to form solenoids.   This
would explain why we get a different packing in the crystals when the
tails are removed.  Clearly to prove the idea will be difficult, but
I am thinking of ways of testing this point.

   Enjoy sunny California.  Here it has been raining almost every day
and the grass is green and high.

Yours ever,