ccp4 study week-end 2012 - univie.ac.at€¦ · ccp4 study week-end 2012 programme (titles may not...

CCP4 study week-end 2012

Highlights from the

CCP4 Study Week-End 2012University of Warwick

Tim GrüneUniversity of Göttingen

Dept. of Structural Chemistry

http://[email protected]

Tim Grüne 1/26

http://shelx.uni-ac.gwdg.de


Programme

(titles may not be original but short content descriptions)

Thursday 5 JanuaryR. Keegan What’s New in CCP4J. Turkenburg/ K. McAuley Opening the MeetingZ. Dauer Effective data collection and processingJ. Holton Signal vs. Noise . . .A. Leslie Autoindexing: theory & practiceJ. Pflugrath d*TrekP. Evans How good are my data, and what’s the resolution?K. Diederichs Data quality measures that are or should be usedJ.-P. Abrahams Processing electron diffraction data . . .T. White Crystallographic data processing for FELsA. McCarthy The mini-kappa goniometer headG. Evans µ-sized X-ray beams and their application

Tim Grüne 2/26


Programme

(titles may not be original but short content descriptions)

Friday 6 JanuaryA. Popov Features and developments of BESTG. Winter automated processing with xia2N. Sauter new methods in phenix for the FELT. Skarzynski Evolution of home sourcesE. Gordon How the ESRF helps industry and how they help usA. Wagner Advances in automated sample mountingM. Mueller Optimal data collection for PilatusT. Krojer (SGC) squeezing the most from every crystalD. Stuart In-situ data collection at synchrotronsW. Hendrickson Multi-crystal anomalous data for low resolution phasing

Tim Grüne 3/26


Main Focus of this Talk

• New Programs: Ample, Zanuda, Nautilus

• Z. Dauter / J. Holton “Take-home messages”

• Armin Mueller (Diamond-I23): optical tweezers for automated crystal mounting.

• Rob Nicholls’ ProSmart: external restraints for refmac5

• Phil Evans/ Kay Diederichs: How to set the resolution limit

• M. Mueller: fine-slicing for Pilatus

Tim Grüne 4/26


New Programs

Ample (Jaclyn Bibby): Ab initio method for macromolecules

1. monte-carlo based modelling of starting fragments (rosetta)

2. 1,000s – 10,000s of models⇒ clustering of solutions

3. MR (phaser), solution check with shelxe

• Resolution range: about 2 Å

• Good performance with α-helical proteins

• 40–80h runtime as pipeline (rosetta→ clustering→ phaser→ shelxe)

• Supposed to also work for nucleic acids

Tim Grüne 5/26


New Programs

Zanuda (Andrey Lebedev): Refinement based checking of space group

• Intended in cases of putative twinning or pseudo-translation (wrong origin)

• e.g. Rfree stuck at 40 %

• example: 1YUP deposited with P1; Zanuda→ P21

• Not intended for checking for screw-axes (probably better use phaser)

• accessible through server at YSBL

Tim Grüne 6/26


New Programs

Nautilus (Kevin Cowtan): Autobuidling of nucleic acids

• similar to buccaneer: features of density, points of low and of high density (like knuspr)

• backbone building based on phosphater and sugars

• new: takes sequence into account (initial release: poly-U only)

• resolution as low as 3.2–3.5 Å

The current documentation (http://www.ysbl.york.ac.uk/˜cowtan/nautilus/nautilus.html) still refers to the initialrelease (poly-U), and users might have to wait until the release of ccp4 6.3.0.

Tim Grüne 7/26

http://www.ysbl.york.ac.uk/~cowtan/nautilus/nautilus.html


“Take-Home Messages” . . .

Z. Dauter, in discussion with E. Garman

• Data collection is the last experiment

• goals for

native data high resolution (, multiplicity)

Molecular replacement medium resolution, no overloads

heavy atom derivative medium resolution, no overloads

anomalous signal accurate, complete low resolution (avoid radiation damage)

• Generally: data completeness comes before resolution — rather drop a few 0.1 Å than destroy the crystalwith radiation before data collection is complete

Tim Grüne 8/26


“Take-Home Messages” . . .

James Holton

Main Reasons for Failure of Structure Solution

• Overlaps

• Radiation damage

• Signal to noise

Complete presentation available at http://bl831.als.lbl.gov/˜jamesh/powerpoint/CCP4SW SvN 2012.ppt

Tim Grüne 9/26

http://bl831.als.lbl.gov/~jamesh/powerpoint/CCP4SW_SvN_2012.ppt


James Holton: Avoiding Overlaps

Slide courtesy James Holton

Or use a mini-κ, now available at many synchrotrons.

Spot separation is smallest when the long unit cell axis points parallel to the beam.Tim Grüne 10/26


James Holton: Detecting Radiation Damage


Tim Grüne 11/26


James Holton: Chemicals Amplify the Dose


Tim Grüne 12/26


James Holton: Signal to Noise / Exposure Time

Slides courtesy James Holton

Tim Grüne 13/26


James Holton: Exposure Time and Refinement


Tim Grüne 14/26


Armin Wagner: Optical Tweezers for small crystals

Armin Wagner: Beamline I23 at Diamond Light Source

Optical Trapping

• A Ashkin (1969)– First successful acceleration and

trapping of particles by radiation pressure

A Ashkin PNAS (1997) 94, 4853-4860

slide courtesy A. Wagner

Tim Grüne 15/26


Armin Wagner: Optical Tweezers for small crystals

Gradient Trapsa

a

b

blaser beam

high NA objective

FaFb

– Scattering and gradient forces are balanced in a tightly focussed laser beam using a high numerical aperture objective lens to form a stable 3-dimensional trap

Fscat

bea

m d

irect

ion

Fgrad

Andy Ward (CLF)

slide courtesy A. Wagner, movie available at http://youtu.be/Th7F27xqULc

Tim Grüne 16/26

http://youtu.be/Th7F27xqULc


Rob Nicholls: ProSmart

http://www.ysbl.york.ac.uk/mxstat/Rob/index.html

“ProSMART (Procrustes Structural Matching Alignment and Restraint Tool) is a software tool designedfor the conformation-independent structural comparison of protein chains.”

• Pairwise alignment of protein sequences based on local conformation — distances within 9 residuesfragments are generated and compared

• Preparation of input file for refmac5 with external restraints

• Useful at low resolution if high resolution structure exists

• visualisation of conservation available in latest version of ccp4mg

• Recommendation: Use latest version refmac5.7 (http://www.ysbl.york.ac.uk/refmac/latest refmac.html)

Tim Grüne 17/26

http://www.ysbl.york.ac.uk/mxstat/Rob/index.html

http://www.ysbl.york.ac.uk/refmac/latest_refmac.html


Rob Nicholls: ProSmart

Refmac input script:

EXTERNAL WEIGHT SCALE 100

@../input_files/restraints_chainA.text

exte dist first chain A resi 36 ins . atom N second chain A resi 36 ins . atom O value 3.50046 sigma 0.1

Refmac log-file example:

---------------------------------------

Standard External All

Bonds: 44085 13716 57801

Angles: 78639 0 78639

Chirals: 4585 0 4585

Planes: 8544 0 8544

Torsions: 18235 0 18235

---------------------------------------

Tim Grüne 18/26


ProSmart Caveat: Model Bias

My experience with ProSmart in case of low resolution and low similarity:

Strong Model Bias during refinement

Work-around:

1. Run blast search (http://blast.ncbi.nlm.nih.gov)against PDB and download multiple sequencealignment

2. Use “ConSurf” (http://consurf.tau.ac.il) to assignsimilarity scores to B-factor column in model PDBfile

3. Cut out conserved atoms4. run ProSmart with modified PDB-file

Tim Grüne 19/26

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://consurf.tau.ac.il


Discussion: How to set Data Set Resolution

Phil Evans

How to define “Resolution”? Cut data where adding the next resolution shell does not add information to thedata anymore.

How to determine “Information”?

Rmerge/Rmeas: Useful figures, but not for resolution cut-off!

I/σI > 2: Good, but dependent on proper determination of σI

CC1/2: (Called CC Imean in Scala log file)

• Randomly divide data in to two equal sets

• Calculate CC of equivalent reflections between both sets

• Cut resolution e.g. where CC1/2 < 50%

Independent of σI-values;

Tim Grüne 20/26


Phil Evans: Tests w.r.t CC1/2

Anisotropy

Many (perhaps most) datasets are anisotropicThe principal directions of anisotropy are defined by symmetry (axes or planes), except in the monoclinic and triclinic systems, in which we can calculate the orthogonal principle directions

We can then analyse half-dataset CCs or I/σ(I) in cones around the principle axes, or as projections on to the axes

Cones

Projections

I/σ(I) in cones1.91Å

2.15Å

2.00Å

Anisotropic cutoffs are probably a Bad Thing, since it leads to strange series termination errors and problem with intensity statistics

So where should we cut the data?Maybe at some compromise point

Monday, 9 January 2012

slide courtesy Phil Evans

Tim Grüne 21/26



Case 1:656 amino acids, omit sugars, shake structure, refine at different resolutionsSpace group R32 (aka H32)

2.1Å

Half-dataset CCs:overallalong c*perpendicular to c*

1.8Å 2.0Å 2.2Å 2.4Å

Conclusion: not a huge difference

2.4Å

Some very preliminary tests of the effect of different resolution cutoffs

2mFo-DFc

mFo-DFc

figures made with ccp4mg



Tim Grüne 22/26



3.0Å 2.4Å 2.0Å 1.8Å

Rfree 0.294 Rfree 0.282 Rfree 0.285 Rfree 0.290

2.2Å

Rfree 0.284

figures made with ccp4mg

Case 2: 3 molecules/asu, omit 22/276 residues from each molecule, model build with Arp/warp at different resolutions

0

23

47

70

1.8 1.9 2 2.1 2.2 2.3 2.4

Number of residues built and sequenced

Cones

1.91Å

2.15Å

2.00Å2.00Å

R & Rfree after initial refinement

Conclusion: there is not a huge difference



Tim Grüne 23/26


Marcus Mueller: effect of fine-slicing on data quality with the Pilatus detector

(published: Mueller et al, Acta Cryst D68, 2012, p. 42–56)

• Collected 94 data sets from Insulin, Lysozyme, Thaumatin, Thermolysin at SLS, Pilatus 6M

• variation of ∆φ (0.01◦ – 1.28◦)

• data processed with XDS

• data publicly available (we have a copy)

Tim Grüne 24/26


Marcus Mueller: effect of fine-slicing on data quality with the Pilatus detector

• Statistics in highest resolution shell• left x-axis: ∆φ/mosaicity; right y-axis:

normalised to best value in each series• minimum reached at ∆φ ≈ 0.5σφ (σφ:

from Gaussian profile fitting in XDS)• Overall statistics: less pronounced

Tim Grüne 25/26


Acknowledgments

The following people made their talk material available to me to prepare this seminar:

James Holton http://bl831.als.lbl.gov/˜jamesh/powerpoint/CCP4SW SvN 2012.ppt

Armin Wagner http://youtu.be/Th7F27xqULc

Phil Evans

Kay Diederichs

Tim Grüne 26/26

http://bl831.als.lbl.gov/~jamesh/powerpoint/CCP4SW_SvN_2012.ppt

http://youtu.be/Th7F27xqULc

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