Structural Biology: A Collaborative NecessityOr:

Collaborative Computing – does it have a future?Or:

What should MX Software deliver now?

• West Coast Crystallographic Meeting

• Monterey: March 11th 2007

How has MX changed in 2007?

• The Internet – questions can be asked and answered; information found

• Much more work done much faster, so desperate need for organisation of information

• Better languages, Faster Computers, Better graphics

• But still need good appropriate algorithms..

Has CCP4 helped develop New Algorithms?

• Yes… maybe, but developers are free spirits – very little “contracted” software

• CCP4MG• Coot• Acorn• New density modification from KDC

• CCP4 provides distribution and support; author keeps copyright, References flagged

What helps algorithm development?

• Common Data structures ( formats?)

•Library routines for data handling, crystallographic operations ( Symmetry, FFTs, etc) :

Libraries must be accessible to developers and there needs to be a way to update them to add routines, and to debug. They must be well documented and curated.

Cooperation – is it possible? desirable?

• Advantages•Can speed up developments if library routines are well documented and accessible :

•Shared efforts for maintenance and distribution extends the code lifetime

• Common style helps users

•Organising crystallographic data is not easy and requirements change– maybe we can agree on and provide a better standard?

•Mtz model carries vital information with it.

Cooperation – is it possible? desirable?

• Disadvantages•Time consuming – consultation essential :

•Needs commitment by developers of algorithms and libraries– often faster to make a quick cludgey fix than read library; new routines may need to be added to libraries

•Harder to get credit, raise funds

•Licensing issues!

Friendly Discussion Amongst Developers??

The Future - Automation ?

• Chemists now use crystallography as a tool and the software is robust.

• MX will often be used in the same way in future– a handy technique the user cannot be expected to understand or criticise.

•Obviously, automation modules must be designed by good crystallographers

( How will the good crystallographers be trained?)

What level of knowledge to assume- Discuss?

• Assessing the experiment• Some understanding of crystal lattices, symmetry, point

groups & spacegroups• Something about intensity statistics (at least that they exist!) • I think it is important to know the structure factor equation

• Much basic information in

http://www.ccp4.ac.uk/docs.php

• (But there are still pathological cases.. See CCP4BB!)

• Acquiring some crystallographic know-how. How much time will people devote to this?

Extracts from York tutorials given by Johan Turkenburg (most slides taken from the web)

16 slides I thought essential follow!

Crystal: unit cell + lattice + symmetry

The unit cell in three dimensions.The unit cell is defined by three vectors a, b, and c, and three angles , , .

a

b

c

is angle between b and c; between a and c; between a and b

Unit cells are usually defined in terms of the lengths of the three vectors and the three angles. For example, a=94.2Å, b=72.6Å, c=30.1Å, =90°, =102.1°, =90°.

Crystal System

Minimum Symmetry

Bravais Lattices

Unit Cell Geometry

1. Triclinic None 1. Primitive (P) a b c;

2. Monoclinic One 2fold axis

2. Primitive (P)3. Base-Centered (C)

a b c; = = 90

3. Orthorhombic Three orthogonal 2fold axes

4. Primitive (P)5. Base-Centered (C)6. Body-Centered (I)7. Face-Centered (F)

a b c; = = = 90

4. Tetragonal One 4fold axis

8. Primitive (P)9. Body-Centered (I)

a = b c; = = = 90

5. Trigonal One 3fold axis

10. Primitive (P) a = b c; = = 90, = 120

11. Rhombohedral (R) a = b = c; = = 90

6. Hexagonal One 6fold axis

10. Primitive (P) a = b c; = = 90, = 120

7. Cubic Four 3fold axes

12. Primitive (P)13. Body-Centered (I)14. Face-Centered (F)

a = b = c; = = = 90

The Seven Crystal SystemsThe 230 space groups can be grouped into seven crystal systems

Owing to symmetry requirementssome unit cells may not be primitive:

Therefore we can have:

• P - primitive• I – body centred • A, B, C – face centred• F – all-face centred unit cells

ab

cP I

F

C B A

In total only 14 different combinations of a, b, c and can exist = 14 Bravais’ lattices

Symmetry Operators and ElementsApart from the identity and translational symmetry, protein crystals can only contain the following symmetry elements:

Proper rotation: Rotate by 360°/n. n = 2 3 4 or 6

Screw rotation: Rotate by 360°/n & translate by d(m/n); d= unit cell edge.

Proper Rotations

Two-fold

Three-fold

Four-fold

Six-fold

Symbol (n)

2

3

4

6

Screw Rotations

Symbol (nm)

21

31, 32

41, 42, 43

61, 62, 63, 64, 65

Space group diagram P212121

Know where Int Tab A is!!

Indexing Conventions: http://www.ccp4.ac.uk/dist/html/reindexing.html

Example: • Reindexing (CCP4: General) - information about changing indexing regime • etc• All P3i and H3:

(h,k,l) not equivalent to (-h,-k,l) or (k,h,-l) or (-k,-h,-l) so we need to check all 4 possibilities:

• real axes:(a,b,c) and (-a,-b,c) and (b,a,-c) and (-b,-a,c)• reciprocal axes: (a*,b*,c*) and (-a*,-b*,c*) and (b*,a*,-c*) and (-b*,-a*,c*)• i.e. reindex (h,k,l) to (-h,-k,l) or (h,k,l) to (k,h,-l) or (h,k,l) to (-k,-h,-l).

• N.B. For trigonal space groups, symmetry equivalent reflections can be conveniently described as (h,k,l), (k,i,l) and (i,h,l) where i=-(h+k).

• Replacing the 4 basic sets with a symmetry equivalent gives a bewildering range of possibilities!.

Many choices of Asymmetric unit and unit cell See http://www.ccp4.ac.uk/dist/html/alternate_origins.html

Unit cell = The smallest volume from which the entire crystal can be constructed by translation only.

Diffraction Geometry

Diffraction lattice and symmetry does not mirror crystal symmetry exactly:

Use Reciprocal Space definitions to describe it..

• First we need to define the relation between real space and reciprocal space. (Ie crystal lattice and diffraction space)

• This requires us to look at Bragg planes and Miller indices.

Definitions used for reciprocal space• To go from real to reciprocal space we define a set of

axes a*, b* and c* such that:• a* is perpendicular to b and c (b.a* = c.a* = 0)• b* is perpendicular to a and c (a.b* = c.b* = 0)• c* is perpendicular to a and b (a.c* = b.c* = 0)• a.a* = b.b* = c.c* = 1

• For orthogonal system, the length of a* is 1/(length a)• The length of a reciprocal vector d* is related to the

interplanar spacing in real space as 1/d

Structure Factor EquationVery useful IF you know atom positions

Very useful for understanding crystallography

Adding one (or more) atoms in known positions changes the structure factor in a known way

FP

Native

Derivative

FPH

Alternate representation: Structure factor can be represented by 2-d vectors.

Symmetry in reciprocal space

•No translations

•So point groups!

•But: Centrosymmetry: Friedel’s law Ihkl = I-h-k-l

•=> 11 Laue groups

Systematic absences• Translational symmetry such as screw axes and lattice

centring, leads to some reflections being ‘absent’. This can be shown using Structure Factor Formula

• If a space group has a 21 screw axis along b, then this will affect the reflections 0k0: only k=2n observed

• If a space group has a 62 or 64 screw axis along c, then this will affect the reflections 00l: only l=(6/2)n observed

• Beware – a non-crystallographic translation of(0.2,0.3,1/3) will ALSO give these absences

Centric and Acentric reflections Centrosymmetric zones

• If Ihkl = I-h-k-l for a subset of reflections under the space group symmetry without invoking Friedel’s law, then these reflections are centric.

• In P21: Ihkl = I-hk-l so for all k=0 reflections, Ih0l = I-h0-l • • Most reflections are acentric• This is relevant because:

1. Centric and Acentric intensities have different statistical properties

2. Centric phases must be or +180.

Does CCP4 help guide Users through this?

• We hope so, but the best critics are the users themselves

• In general some knowledge is assumed

• As far as possible programmers try to illustrate important information by presenting it graphically.

• Links to documentation where possible

CCP4 Main Page

• CCP4 Documentation• Individual Program Documentation • Tutorials • Maths for Protein Crystallographers • Crystallographic guidance • Roadmaps through the Suite • Talks

Example from CCP4 Main Page

• CCP4 Documentation• Individual program documentation

• CCP4 Tutorial

• Maths for Protein Crystallographers• Eleanor Dodson prepared a document containing all the

maths a protein crystallographer might need. It helps to have this all together, and available on the web, so Maria Turkenburg developed it further. It is distributed with the suite as a set of documents in which certain symbols are represented by small .gif-pictures. They are available here:

• Basic Maths for Protein Crystallographers

An aside- Project Book-keeping

• There is an urgent need for data management.

Each specific application program needs to define its requirements and its product along with a book-keeping header

e.g. Protein production needs sequence, so does automatic model building – how to pass this info on via intervening steps

– from laboratory to beamline to structure solution ?

Brief Introduction to the Graphical User Interface• Designed - to keep a record of what has been done within a project

directory/folder It is far from perfect but at least it exists!

- to provide easy access to the tasks required for each crystallographic module

- to provide diagnostic information, mostly via graphs, summaries, and as a last resort, log files

-CANNOT “manage” your work pattern! You must do

that..

GUI Structure Solution Modules• Data Processing• Experimental phasing• Molecular Replacement• Density Improvement• Model building• Refinement• Structure Analysis• Validation and Deposition• Reflection, Coordinate, Map and Graphical utilities• Clipper applications• Program list• Needs logical up-dating! Currently underway –

CCP4BB request for feedback soon..

Lots of Graphical analysis from CCP4 software

Scala Analysis(Scaling and Merging)

Use hklview to see diffraction zones

Intensity DistributionsThe structure factor equation means we can predict

some properties of all INTENSITY distributions

• These should be inspected as soon as data are processed

• Intensity distribution v resolution • Wilson Plot• Moments

• SFCHECK good too

Intensity Analysis

Intensity Analysis

Lead to: Refinement problems

What level of knowledge to assume- Validation

• Assessing how well the model describes the experiment and fits with expectation - COOT lists many tools

• Protein geometry – Ramachandran plots Need a tool not part of refinement

• Sensible contacts (Molprobity, PISA etc) • Density Fit• Unmodelled map features• Critical facilities

Solving the structure – Automation as it is now

• From data through experimental phases to model

• ShelxD/ShelxE: Decisions made within program, based on good methodology.

• Solve & Resolve. Decisions made by programmer, based on expert knowledge.

• AutoSharp: links several programs using scripting. Decisions made at scripting level?, based on expert knowledge.

More Automation Procedures• From homologous model to final model.• Molrep: Input experimental data and model –

output model of asymmetric unit. (Mr Bump – Balbes)

• Arp-Warp/Refmac5. Model building using refinement & map interpretation. This uses a GUI to set a protocol, interpreted into a C-shell script.

• Some CCP4 GUI tasks

Automation Thoughts• Should procedures aim to be “black boxes”?• Yees – but I think there are too many difficult

cases for this..

• Can MX be automated? Will Automation lead to rigidity?

• There is a danger of this – not so serious if the approach is modular, linked by scripting..

• Will automation destroy our critical facilities