1950’s “Precession” cameras were used to take undistorted pictures of the “reciprocal lattice”. Each point in the reciprocal lattice corresponds to a plane within the crystal.

1950’s Images of the reciprocal lattice obtained from Precession photographs were used to determine the symmetry and cell dimensions for the crystal.

1950’s Weissenburg cameras were used to measure intensities of each of the reciprocal lattice points. Typical exposure of several hours to several days; a few dozen to perhaps over 100 films per crystal.

1950’s A Distorted view of the reciprocal lattice is obtained in each Weissenburg image, and each of the spots seen corresponds to a particular plane within the crystal.

1950’s A Distorted view of the reciprocal lattice is obtained in each Weissenburg image, and each of the spots seen corresponds to a particular plane within the crystal. Intensities were estimated visually using an intensity scale obtained by taking different exposure times for a given reflection.

1970’s Single-crystal Diffractometers greatly increased the speed and accuracy of data collection. The crystal and a detector were positioned so that the intensity of diffraction from each plane in the crystal could be measured automatically.

1970’s The single crystal diffractometer increased the accuracy of measurements by probably an order magnitude, and allowed much larger (and more complex) structures to be studied. Although it was computer controlled, it was limited by the serial nature (i.e. it could only measure one data point at a time). Typical crystals could require anywhere from a few days to months to be completely characterized.

1990’s Bruker-AXS SMART 6000 CCD system located in the Indiana University Molecular Structure Center at Indiana University.

Data Individual users have discretion in how raw data is archived. Typical techniques are university archival storage (HPSS), CCD’s and DVD’s.

1990’s The CCD detector consists of a fluorescent screen and a fiber optic bundle to increase the area being surveyed. In this view of a Bruker SMART6000 CCD the 4K x 4K CCD chip is located behind the circular beryllium window.

1990’s The operator’s console will allow the researcher to orient the crystal and view the resulting CCD images.

Monitoring the Experiment Polycom access.

Monitoring the Experiment Polycom access.

Monitoring the Experiment Axis web-based camera systems

A Bigger Diffraction Apparatus Synchrotron Light Source

Background Single crystals act as diffraction gratings when placed in a beam of X-rays of suitable wavelength. Basic information about the internal symmetry and basic repeat pattern can be determined by studying the diffraction properties of a crystal. The intensity of the scattered X-rays can be used to determine the coordinates of atoms within the crystal. Instrumentation consists of an X-ray source, device to hold and manipulate the crystal, and a detector suitable for measuring the positions and intensities of the diffracted X-ray beam.

Background Single crystals act as diffraction gratings when placed in a beam of X-rays of suitable wavelength. Basic information about the internal symmetry and basic repeat pattern can be determined by studying the diffraction properties of a crystal. The intensity of the scattered X-rays can be used to determine the coordinates of atoms within the crystal. Instrumentation consists of an X-ray source, device to hold and manipulate the crystal, and a detector suitable for measuring the positions and intensities of the diffracted X-ray beam.

Background Single crystals act as diffraction gratings when placed in a beam of X-rays of suitable wavelength. Basic information about the internal symmetry and basic repeat pattern can be determined by studying the diffraction properties of a crystal. The intensity of the scattered X-rays can be used to determine the coordinates of atoms within the crystal. Instrumentation consists of an X-ray source, device to hold and manipulate the crystal, and a detector suitable for measuring the positions and intensities of the diffracted X-ray beam.

Background Single crystals act as diffraction gratings when placed in a beam of X-rays of suitable wavelength. Basic information about the internal symmetry and basic repeat pattern can be determined by studying the diffraction properties of a crystal. The intensity of the scattered X-rays can be used to determine the coordinates of atoms within the crystal. Instrumentation consists of an X-ray source, device to hold and manipulate the crystal, and a detector suitable for measuring the positions and intensities of the diffracted X-ray beam.

The reciprocal lattice and the geometry of diffraction X-ray detector X-ray source

Direct beam position Direct beam shot Powder rings Ice rings Symmetry 23

Beam center off (00l off by one) dtdisplay overlay Detail of 454 Angstrom axis Systematic absences and 2-fold Beam center off (00l off by one) Beam center correct 24

What you do Pick up crystal in loop, plunge into LN2 Put crystal on magnet on goniometer head and optical align Take a diffraction image or two Look at image(s) and decide whether to proceed Collect images, index, integrate, scale 25

Si valuta la presenza di un cristallo singolo Analisi dello spettro (immagine) di diffrazione Si valuta la presenza di un cristallo singolo se il cristallo diffrange bene la massima risoluzione, la lunghezza d’onda ottimale, la distanza cristallo-rivelatore ottimale (minima sovrapposizione delle spot di diffrazione),

Indicizzazione dell’immagine di diffrazione, Si assegnano i corretti indici hkl a ciascuna spot dell’immagine. Da un sottogruppo di spot aventi intensità superiore ad un valore soglia predefinito, vengono stimati i parametri reticolari del cristallo (dimensioni) e l’appartenenza ad uno dei quattordici possibili reticoli di Bravais.

Affinamento dei parametri del cristallo e del rivelatore. Vengono affinati numerosi parametri sperimentali tra i quali l’orientamento del cristallo , del rivelatore rispetto ad un sistema di riferimento predefinito, le coordinate del punto di intersezione tra i raggi X ed il rivelatore (centro del rivelatore) e la distanza cristallo-rivelatore. La procedura di affinamento viene ripetuta per tutte le immagini della raccolta in modo da tener conto delle possibili variazioni sperimentali, quali ad esempio lo slippage del cristallo, la sua mosaicità e la sua forma .

Integrazione dei massimi di diffrazione. Per calcolare le intensità di diffrazione di ciascun riflesso, le intensità associate a ciascuno dei pixel che definiscono la spot di diffrazione assegnabile ad un determinato riflesso, sono state integrate utilizzando il metodo dell’average profile fitting applicato, successivamente, a tutte le immagini raccolte

Scalatura e merging dei dati integrati. Poiché un riflesso con indici hkl a causa, ad esempio, dell’oscillazione scelta per effettuare la raccolta dei dati o della mosaicità del cristallo, potrebbe non essere stato completamente registrato su una stessa immagine, ma solo parzialmente, si rende necessaria una ricostruzione dell’intensità totale ad esso associata.

Le intensità di quei riflessi i cui indici obbediscono alla simmetria del reticolo scelta, includendo anche le coppie di Friedel I(h,k,l) e I(-h, -k, -l) (l’operazione di diffrazione è centrosimmetrica e si trascura il contributo derivante dalla diffusione anomala), sono stati mediate fino ad ottenere una lista di riflessi indipendenti, con i valori delle intensità I e de i s(I) ad essi associati. La lista di questi valori costituisce il dato sperimentale di partenza per la determinazione della struttura mediante metodi di calcolo tipici della biocristallografia

33

How do Crystallographers Rank Crystals?? Do I have another crystal?? Is the crystal twinned? How far does the crystal diffract? Are there ice rings? Do peaks have a decent spot shapes? Can I assign a unit cell for the sample? What are the unit cell dimensions and space group? Of course, we should be concerned with finding the best crystal possible. Sometimes, real life does not allow for this and we may have to take the best we can. And ultimately, the biggest concern is the structure quality. Small deviations in crystal quality are not likely to have an extreme effect upon the final structural model, however this method allows for unattended analysis of diffraction images. I/sig(I) analysis is not sufficient Single image is probably not sufficient 34

Images - What to collect? Depends on crystal, spacegroup Spatial overlaps, mosaicity Expected statistics Overall start and end Rotation increment Exposure time 35

A fully-recorded spot is entirely recorded on one image Partials are recorded on two or more images “Fine-sliced” data has spots sampled in 3-dimensions Perhaps best processed with a 3D program (eg d*TREK, XDS) Elspeth Garman, Oxford 36

Images: fully recorded and partially recorded reflections We want to determine the intensity of a reflection, integrated over its extent in reciprocal space by rotating the crystal so that the extended reciprocal lattice point passes through the sphere Because all current detectors take a significant time to read out, we have to close the shutter & stop the rotation (simultaneously!), so our sampling of the 3-dimensional reciprocal space is in consecutive slices, typically of between about 0.1° and 1° Depending on the slice width and the reflection width a reflection may occur on one image (full or fully recorded) or on several (partial or partially recorded) Elspeth Garman Oxford via Phil Evans, MRC-LMB 37

Images - Thick vs Thin Thick Thin Fewer total images Higher X-ray background Spatial overlaps More saturation High throughput Most full reflns 2D integration More total images Less X-ray background Reduced overlaps Less saturation More readouts All partials 3D integration 38

Images - Thick vs Thin? Depends on General rule: Detector, Goniometer, Crystal, Source General rule: If mosaicity < 1, use 0.5 Else use mosaicity/2 Less if spatial overlaps Two 0.5 images of T sec each are often better than a single 1 image of 2T sec! 39

Diffraction zones are visible as lunes in a projection of reciprocal space

Data Typical experiment will consist of from 1200 – 3600 CCD images. Images can be “binned” to conserve storage space. In “normal” mode each data set is about 0.5 to 1.5GB. Typical data set can be collected in 4 hours – 36 hours.

Data Typical experiment will consist of from 1200 – 3600 CCD images. Images can be “binned” to conserve storage space. In “normal” mode each data set is about 0.5 to 1.5GB. Typical data set can be collected in 4 hours – 36 hours.

Data Typical experiment will consist of from 1200 – 3600 CCD images. Images can be “binned” to conserve storage space. In “normal” mode each data set is about 0.5 to 1.5GB. Typical data set can be collected in 4 hours – 36 hours.

Data First few frames can determine the “quality” of the diffraction and identify special problems (twinning, split crystals, etc.) Initial frames must be available in order to characterize the sample and determine data collection strategy. Preliminary structure can usually be determined after only a few hundred frames have been collected. Complete set of raw data is usually only required during the data reduction and structure solution phase of the experiment.

Data First few frames can determine the “quality” of the diffraction and identify special problems (twinning, split crystals, etc.) Initial frames must be available in order to characterize the sample and determine data collection strategy. Preliminary structure can usually be determined after only a few hundred frames have been collected. Complete set of raw data is usually only required during the data reduction and structure solution phase of the experiment.

Data First few frames can determine the “quality” of the diffraction and identify special problems (twinning, split crystals, etc.) Initial frames must be available in order to characterize the sample and determine data collection strategy. Preliminary structure can usually be determined after only a few hundred frames have been collected. Complete set of raw data is usually only required during the data reduction and structure solution phase of the experiment.

Data First few frames can determine the “quality” of the diffraction and identify special problems (twinning, split crystals, etc.) Initial frames must be available in order to characterize the sample and determine data collection strategy. Preliminary structure can usually be determined after only a few hundred frames have been collected. Complete set of raw data is usually only required during the data reduction and structure solution phase of the experiment.

Spacing between diffraction spots (after projecting back on the Ewald sphere) defines unit cell

RIDUZIONE E SCALAGGIO DEI DATI DI DIFFRAZIONE

Reflections (from images) Find X,Y,  Index Unit cell Orientation Refine Crystal Detector Source Predict / Strategy Rot start, end Completeness Integrate hkl, Intensity, I Profile fitting Scale Rmerge |2| 59

INDICIZZAZIONE La prima parte della riduzione consiste nell’indicizzazione delle diverse immagini di diffrazione, con affinamento dei parametri relativi alla cella elementare, al rivelatore, all’orientazione del cristallo, alla sua mosaicità, e integrazione e indicizzazione dei massimi di diffrazione.

Refine (crystal mosaicity) 61

Strategy Completeness and redundancy Simply predict all spots 0-360 degrees Trim away from start Trim away from end Inverse beam for anomalous? Avoid spatial overlaps Simply collect 360 degrees since it may be very fast 62

Riduzione dei dati di diffrazione Segue lo scalaggio dei dati indicizzati attraverso la determinazione della costante di scala tra immagini successive, l’affinamento della cella elementare, dell’orientazione e della mosaicità, mediante l’utilizzo dell’insieme completo dei dati.

In definitiva i dati registrati vengono elaborati così da associare intensità e indici di Miller (hkl) a ciascun riflesso. In seguito vengono mediati i valori dell’intensità dei riflessi raccolti più volte o dei riflessi correlati per simmetria. La consistenza interna dei riflessi raccolti più volte è misurata dal seguente indice: Rmerge= dove I(hkl) è l’intensità del riflesso, i definisce il riflesso, j le diverse misure dello stesso riflesso e <I(hkl)> è l’intensità media di tutte le misure per quel riflesso.

Il valore di Rmerge sarà tanto più basso quanto più simili sono le differenti misure dell’intensità di uno stesso riflesso, e, quindi, quanto migliore è la qualità dei dati. I riflessi raccolti a bassa risoluzione hanno una maggiore intensità e quindi sono misurati con maggiore precisione, man mano che si raggiunge il limite di risoluzione del cristallo i riflessi saranno sempre meno intensi e le misure avranno un errore associato maggiore: quindi l’ cresce progressivamente all’aumentare del limite di risoluzione. Avere questo andamento progressivo dell’ è indice di una raccolta dati di buona qualità. La qualità dei dati può essere valutata anche analizzando il rapporto I/σ(I), dove I è l’intensità dei riflessi e σ(I) la deviazione standard associata alla misura dei riflessi stessi. Anche in questo caso è maggiore a bassa risoluzione perché i riflessi sono più intensi e andrà progressivamente a diminuire all’aumentare del limite di risoluzione. Questo parametro risulta essere migliore dell’Rmerge in quanto, a differenza di quest’ultimo, non dipende dalla molteplicità dei dati e dalle proprietà di simmetria del cristallo. In generale, sono considerati dati di buona qualità quando l’ assume valori inferiori al 10%.

Output statistics 66 Rmerge vs Resolution -------------------------------------------------------------------------------- Resolution Average Num Num I/sig I/sig Rducd Model Rmerge Rmerge range counts rejs mults unavg avg ChiSq Eadd* shell cumul 29.29 - 3.87 6631 3864 6017 16.0 48.8 1.71 0.04 0.058 0.058 3.87 - 3.07 3842 702 5953 9.7 30.5 1.26 0.06 0.085 0.069 3.07 - 2.69 1385 110 5925 7.3 23.1 0.94 0.04 0.092 0.071 2.69 - 2.44 690 25 5896 5.5 17.1 0.81 0.02 0.111 0.074 2.44 - 2.27 379 27 5891 3.8 11.6 0.77 0.01 0.168 0.077 2.27 - 2.13 372 30 5901 2.7 7.5 1.00 0.04 0.316 0.084 2.13 - 2.03 215 21 5877 2.8 8.0 0.79 0.00 0.227 0.087 2.03 - 1.94 118 15 5878 1.9 5.4 0.80 0.00 0.350 0.089 1.94 - 1.86 111 15 5884 1.2 3.1 1.10 0.03 0.514 0.094 1.86 - 1.80 57 1 5790 1.3 3.6 0.80 0.00 0.543 0.097 29.29 - 1.80 1377 4810 59012 5.2 15.9 0.99 0.04 0.097 0.097 I/sig unavg is the mean I/sig for the unaveraged reflections in the input file. I/sig avg is the mean I/sig for the unique reflections in the output file. * When EMul == 3.57 66

Output statistics 67 Summary of data collection statistics ------------------------------------------------------------- Spacegroup P6 Unit cell dimensions 130.41 130.41 65.57 90.00 90.00 120.00 Resolution range 29.29 - 1.80 (1.86 - 1.80) Total number of reflections 636461 Number of unique reflections 59057 Average redundancy 10.78 (10.37) % completeness 99.8 (98.9) Rmerge 0.097 (0.543) Reduced ChiSquared 0.99 (0.80) Output <I/sigI> 15.9 (3.6) Note: Values in () are for the last resolution shell. 641271 reflections in data set 0 reflections rejected (|ChiSq| > 50.00) 4810 reflections total rejected ( 0.75% |Deviation|/sigma > 13.69) 123625 reflections excluded from scaling/absorption (I/sig <= 5.00) 67

Resolution 1.2 Å 2 Å 3 Å

The diffraction pattern of a unit cell a continuous Fourier transform Continuous diffraction pattern Fourier transform Diffraction experiment Electron density

Chain Tracing Electron Chain Final Density Trace Model

The Final Result http://www-structure.llnl.gov/Xray/101index.html ORIGX2 0.000000 1.000000 0.000000 0.00000 2TRX 147 ORIGX3 0.000000 0.000000 1.000000 0.00000 2TRX 148 SCALE1 0.011173 0.000000 0.004858 0.00000 2TRX 149 SCALE2 0.000000 0.019585 0.000000 0.00000 2TRX 150 SCALE3 0.000000 0.000000 0.018039 0.00000 2TRX 151 ATOM 1 N SER A 1 21.389 25.406 -4.628 1.00 23.22 2TRX 152 ATOM 2 CA SER A 1 21.628 26.691 -3.983 1.00 24.42 2TRX 153 ATOM 3 C SER A 1 20.937 26.944 -2.679 1.00 24.21 2TRX 154 ATOM 4 O SER A 1 21.072 28.079 -2.093 1.00 24.97 2TRX 155 ATOM 5 CB SER A 1 21.117 27.770 -5.002 1.00 28.27 2TRX 156 ATOM 6 OG SER A 1 22.276 27.925 -5.861 1.00 32.61 2TRX 157 ATOM 7 N ASP A 2 20.173 26.028 -2.163 1.00 21.39 2TRX 158 ATOM 8 CA ASP A 2 19.395 26.125 -0.949 1.00 21.57 2TRX 159 ATOM 9 C ASP A 2 20.264 26.214 0.297 1.00 20.89 2TRX 160 ATOM 10 O ASP A 2 19.760 26.575 1.371 1.00 21.49 2TRX 161 ATOM 11 CB ASP A 2 18.439 24.914 -0.856 1.00 22.14 2TRX 162 http://www-structure.llnl.gov/Xray/101index.html

MAD & X-ray Crystallography MAD (Multiwavelength Anomalous Dispersion Requires synchrotron beam lines Requires protein with multiple scattering centres (selenomethionine labeled) Allows rapid phasing Proteins can now be “solved” in just 1-2 days

Data and Time Flow Determine suitability for diffraction Mount Crystal on Instrument Determine suitability for diffraction First Frame Characterize crystal- Determine if split, twinned, etc. Determine cell parameters/symmetry and compare to database 40 – 90 Characterization Frames 10 s Determine preliminary structure 200 – 500 Frames 5 -10 min Integrate and refine structure Complete Set of Frame Data 1 -2 hrs Complete Structural Results 3 -36 hrs 1-30 days

Data and Time Flow Determine suitability for diffraction Mount Crystal on Instrument Determine suitability for diffraction First Frame Characterize crystal- Determine if split, twinned, etc. Determine cell parameters/symmetry and compare to database 40 – 90 Characterization Frames 10 s Determine preliminary structure 200 – 500 Frames 5 -10 min Integrate and refine structure Complete Set of Frame Data 1 -2 hrs Complete Structural Results 3 -36 hrs 1-30 days

Data and Time Flow Determine suitability for diffraction Mount Crystal on Instrument Determine suitability for diffraction First Frame Characterize crystal- Determine if split, twinned, etc. Determine cell parameters/symmetry and compare to database 40 – 90 Characterization Frames 10 s Determine preliminary structure 200 – 500 Frames 5 -10 min Integrate and refine structure Complete Set of Frame Data 1 -2 hrs Complete Structural Results 3 -36 hrs 1-30 days

Data and Time Flow Determine suitability for diffraction Mount Crystal on Instrument Determine suitability for diffraction First Frame Characterize crystal- Determine if split, twinned, etc. Determine cell parameters/symmetry and compare to database 40 – 90 Characterization Frames 10 s Determine preliminary structure 200 – 500 Frames 5 -10 min Integrate and refine structure Complete Set of Frame Data 1 -2 hrs Complete Structural Results 3 -36 hrs 1-30 days

Data and Time Flow Determine suitability for diffraction Mount Crystal on Instrument Determine suitability for diffraction First Frame Characterize crystal- Determine if split, twinned, etc. Determine cell parameters/symmetry and compare to database 40 – 90 Characterization Frames 10 s Determine preliminary structure 200 – 500 Frames 5 -10 min Integrate and refine structure Complete Set of Frame Data 1 -2 hrs Complete Structural Results 3 -36 hrs 1-30 days

Data and Time Flow Determine suitability for diffraction Mount Crystal on Instrument Determine suitability for diffraction First Frame Characterize crystal- Determine if split, twinned, etc. Determine cell parameters/symmetry and compare to database 40 – 90 Characterization Frames 10 s Determine preliminary structure 200 – 500 Frames 5 -10 min Integrate and refine structure Complete Set of Frame Data 1 -2 hrs Complete Structural Results 3 -36 hrs 1-30 days

Monitoring the Experiment Since data collection can take over one day, it is often unattended. This is especially true of “problem” crystals that are exceedingly small and may take several days. The ability to remotely monitor the experiment (and access the data) would significantly improve the data collection efficiency.

Monitoring the Experiment Since data collection can take over one day, it is often unattended. This is especially true of “problem” crystals that are exceedingly small and may take several days. The ability to remotely monitor the experiment (and access the data) would significantly improve the data collection efficiency.