From crystal to structure Filip Melicher S1004 Methods for structural characterization of biomolecules The aim of X-ray crystallography Workflow Step 1 Step 2 Step 3 Diffraction data Electron density calculation Electron density interpretation In-house Anode: Cu, Mo, Cr, W Target (rotating anode) Window (Be) 0.1 nm = 1 A Transition of electrons from L, M levels to K level Synchrotrons Monochromatic coherent waves Multiple wavelengths High intensity http://www.proconsystems.net/sincrotron-alba-el-acelerador-de-electrones-espanol-n-21-es Synchrotrons Monochromatic coherent waves Multiple wavelengths (tuneable) High intensity http://www.proconsysteins.net/sincrotron-alba-el-acelerador-de-electrones-espanol-n-21-es The aim of X-ray crystallography Reminder Bragg's law sin0 = HL/d Bragg's law sinö = H L/d HL=LM=w Bragg's law sin0 = HL/d HL=LM=w 2w= nA=>w= nX/2 Bragg's law sinö = H L/d HL=LM=w 2w= nA=>w= n A/2 sind = w/d Bragg's law sin 0 = HL/d HL=LM=w 2w= nX->\N- n sin 6 = w/d sin 6 = nA/2ô 2d sin 9 = nX Bragg's law Brases Dianes Í0.0.11: ľO.0.21: ľl.0.01: Í3.0.1]... (234) planes Now reciprocal space... https://phys.libretexts.org/Courses/University_of_California_Davis/UCD%3A_Biophysics_200A_ _Current_Techniques_in_Biophysics/X-ray_Protein_Crystallography Now reciprocal space... https://www.xtal.iqfr.csic.es/Cristalografia/parte_04-en.html Now reciprocal space... https://www.xtal.iqfr.csic.es/Cristalografia/parte_04-en.html https://www.xtal.iqfr.csic.es/Cristalografia/parte_04-en.html EwalcTs sphere Geometrical description of the Bragg's law in the reciprocal space. Ewald's sphere Andrew McCarthy Ewald's sphere 2d(/lfcOSin0 = nX Andrew McCarthy Ewald's sphere Ewald sphere Andrew McCarthy Limiting sphere represents the limit of resolution of your crystal given: • wavelength angle lattice distance from detector orientation of lattice relative to detector predicts: • which diffracted waves satisfy Bragg's law Data collection strategy • Trial 2 pictures of the crystal for quality check in 90 • Wavelength • Tilt angle • Rotation range • Starting angle of data collection • Intensity of the beam • Resolution? XDS r 1 XYCORR INIT □ Spatial correction for each detector pixel Detector noise, pixel variation and background estimation COLSPOT IDXREF Locate strong diffraction spots Index strong reflections Map background for next steps (trusted/untrusted regions) L XPLAN Strategy calculation INTEGRATE Determine intensity for each reflection Applies corrections, determines space group and reports on quality_: Data processing • Spot finding and autoindexing • Parameter refinement • Integration (the actual measurement of spot intensities) •Scaling and merging Scale and Merge Scale: • Illuminated volume of the crystal • Change in incident radiation intensity • Non-uniformity of detector response • Radiation damage ■ ■ ■ Merge: • Merging same or symetry equivalent reflections Scale an •Scale: • lllumin • Change • Non-ur Merge: • Mergir ID experiment ID experiment Argand diagram imaginary axis A sin a ^ Ay< 1 S\ a i real axis A cos a A = \A\ (cos a + i sin a) A = \A\ exp ia F(hki) = \FViki)\ (cosa(h/c0 +raa(M!)) F(hki)= \F(hki)\ expia^ Friedel's law - Friedel's pairs ID experiment ID experiment ID experiment ID experiment ID experiment ID experiment ID experiment ID experiment to 2D experiment 90° -ft* 1 2 3 4 5 Frekvence h ID experiment to 2D experiment 90° -ft* 1 2 3 4 5 Frekvence h [0,0] [1,0] Mathematic description 111 " r r F(hki) = Vc P(xyz) exV [2ni(hx + ky + lz)]dxdydz J J J x=0y=0z=0 It FT I I FT Pixyz) = Yj Yj \F(hk0\eXp [-2nt(hx + ky + lz) + £a(Mfc0] Mathematic description 111 = Vrj j j P(Xyz) exV [2ni(hx + fey + lz)]dxdydz x=0y=0z=0 FT I I FT ■l P(xyz) = Yj Yj \F(hk0\eXp [-2ni(hx + ky + Zz) + ia(Mfc0] Mathematic description: ID N 71=1 71=1 Sum of all structural factors \ 1 N Electron density <— P(x) =y^l^l [-2nihx+ ia(hkl-)] 71=1 Sum of all structural factors \ N Electron density 4— PI» = T^J^! [_27" hx + ia^o] I Amplitude 71=1 Sum of all structural factors V Frequency N t Electron density p(x) = y ) Kal [-2ni hx + icc^^] \ Amplitude 71=1 Sum of all structural factors V Frequency N t Electron density p(x) = T )\Fh\ [-2ni hx + icc^^] n=l ■ \ Amplitude Phase Sum of all structural factors V Frequency N t Electron density p(x) = T )\Fh\ [-2ni hx + icc^^] Length of unit cell Amplitude Pnase Methods for phasing: •Molecular replacement (70 %) •Isomorphous replacement •Anomalous scattering Molecular replacement Molecular replacement - basics O Linear intensity scale of amplitude size http://www.vsbl.vork.ac.uk/~cowtan/fourier/magic.html Molecular replacement - basics O Linear intensity scale of amplitude size El Circular rainbow scale of phases Molecular replacement - basics Molecular replacement - basics Orientation problem Finding orientation Patterson map exp [—2ni(hu + kv + Iw) ] h k i All phases ahk, are set to 0 Peaks coresponds to VECTORS BETWEEN ATOMS Patterson map exp [—2ni(hu + kv + Iw) ] molecule Patterson map Molecular replacement MR problems Isomorphous replacement Uniform binding of heavy atoms Native crystal Heavy atom derivatives 0 4p 4§l - III ^^^^^^^ w m HI 0 0 Native crystal Heavy atom derivatives erivatives 4 4 4 Number of electrons Biogenic atoms: C N O H S 6 7 8 1 16 ■ • a • • • • • • • • • • • • • • • • • I 99 9 / A change in unit-cell dimensions of 0.5% or a 0.5° rotation of the molecule produces an average 15% change in intensity! Addition of 1 Hg atom to a protein of 1000 atoms will produce an average fractional change of intensity of-25% so differences should be easy to measure and data doesn't have to be that accurate Crick, R IL C. & MagdolT, B. S. (1956). Acta Crvst 9,901-908, Native crystal Heavy atom derivatives $6. V"' fci*-' Si*-' jJKm -Jr--" sgf ^ «p ^ rffc 4% Ate Si*- 'V' w; w# jgfe rffc rigfe I&i? V*- S>*- Si*-' Si*-' fei*- wS SEE Crystal • ' Diffraction pattern .i'r> «,*> ®g •£» «ws gffi* •S.*- *<«*• •SfJ? fifel giBe |«g®S .P> *SP >-«*• 4&f gte $M «p «p Jb #te ign fern *sM Crystal Diffraction pattern Patterson map of Heavy atoms Heavy atom derivatives + Heavy atom + Heavy atom Co-crystallization Optimization: pH, substance, time of soaking, concentration Soaking 1. Non-specific binding 2. Change of the unit cell parameters 3. Disruption of the crystal Problems with binding of HA Anomalous scattering Anomalous scattering Absorbtion of X-ray Reemiting alternated phases Heavy atoms Fe, Mn, Se (SeMet) 10 000 Dispersive term Absorption term 12 000 14 000 X-ray energy (eV) t Se K edge = 0.9795 A 16 000 Anomalous scattering //'< [Fp(-)\ \. r • Friedels law false • Structure factors are alternated • The most common: SeCys • Finding Heavy atoms by Patterson map! Electron density maps Model building & resolution 3.0 A 4.0A Refinement of the model Main objective of the refinement process - build a model that explains diffraction data as good as possible and without violation of chemistry Update the model using the density in COOT i Update (Peak - refinement in REFMAC i Density improves Ramachandran diagram The Ramachandran Plot. Geometrical parameters B Bond Length r B Bond Angle 8 Dihedral Angle RMSD= /^=i^n ^2 https://www.webmo.net/link/rielp/AdiustTool.ritml Validation (Reciprocal space) R = I*hki\\Fo\- k\Fc\\ Y*hki\Fo\ • 95 % of the reflections • Model bias • Overfitting Random structure: R = 0.65 Validation (Reciprocal space) ? = Zhki\\Fo\- k\Fc\\ • 95 % of the reflections • Model bias • Overfitting = I*hkieT\\Fo\~k\Fc\ ^ ^hklET\Fo\ • 5 % of the reflections • No model bias!! Validation (Reciprocal space) R = Zhki\\Fo\~k\Fc R \\F0\-k\Fc free 'hklET \F0 95 % of the reflections Model bias Overfitting Good structure: • Resolution * 10 • R-Rfree=4-6% 5 % of the reflections No model bias Validation | Val 1 HID About Ligand Extension □ Rarnachandran Plot ► H Kleywegt Plot... Incorrect Chiral Volumes... Unrnodelled blobs... Difference Map Peaks... Check/Delete Waters... Li Geometry analysis ► Peptide omega analysis Temp. fact, variance analysis Ji Average Temp. fact, analysis GLN and ASN B-factor Outliers Rotamer analysis Density fit analysis Probe clashes NCS Differences Highly coordinated waters... Pukka Puckers...? Alignment vs PIR... WinCoot Page 2 Preliminary Pull wwPDB X-ray Structure Validation Report D_9100019218 1 Overall quality at a glance (T) The following experimental techniques were used to determine the structure: X-RA Y DIFFRACTION The reported resolution of this entry is 1.65 A. Percentile scores (ranging between 0-1O0) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on whicb the scores axe based. Ml-ii-ii Pentnflt Hunks Value (I: m ln,l-«r<- ■ ttuma^hund rail nutliLTH ^^^b SUtchiiiTHHiClitn Mí? trk: Whole archive (#Entries) Similar resolution (#Entries, resolution range(A)) 111664 2539 (1.66-1.62) Clashscore 122136 2672 (1.66-1.62) Rarnachandran outliers 120033 2629 ; 1.66-1.62) Sidechain outliers 120020 2629 (1.66-1.62) RSRZ outliers 10S0SS 249S (1.66-1.62) The table below summarises the geometric issues observed across the polymeric chains and their fit to the electron density. The red, orangen yellow and green segments on the lower bar indicate the fraction of residues that contain outliers for >=;}, 2, 1 and 0 types of geometric quality criteria. A grey segment represents the Fraction of residues that are not modelled. The numeric value for each fraction is indicated below the corresponding segment, with a dot representing Fractions <—h% The upper red bar (where present] indicates the fraction of residues that have poor 6t to the electron density. The numeric value is given above the bar. Mol Chain Length Quality of chain AAA 348 jV:. 1 911 a% - 8 PDB validation system Structural database America WORLDWIDE e RCSB PDB Deposit -■ Search ▼ Visualize ▼ Analyze - Download *■ Learn ■» More » Documentation ▼ Careers Pt>6-loi SPDB • PlUTtlN DA I A IANI V I'lMItadfetxinv ■.ill] '■UCIEIC aciD MIIUH 4 ProttM Data Bank n*oo Structure Summary 3D View Annotations Experiment Sequence Genome Ligands Versions E Biological Assembly 1 0 © 3D View: Structure | Electron Density | Validation Report | Ligand Interaction Global Symmetry: Cyclic - C3 Ö (3D View) Global Stoichiometry: Homo 3-mer - A3 0 2BT9 Ii Display Files » © Download Files r Lectin from Ralstonia solanacearum complexed with Me-fucoside DOI: 10.2210/pdb2BT9/pdb Classification: LECTIN Organism(s): Ralstonia solanacearum Expression System: Escherichia coli BL21(DE3) Mutation(s): No 0 Deposited: 2005-05-27 Released: 2005-06-02 Deposition Author(s): Mitchell, E.R, Kostlanova, N., Wimmerova, M., Imberty, A. Experimental Data Snapshot Method: X-RAY DIFFRACTION Resolution: 0.94 A R-Value Free: 0.120 wwPDB Validation 0 Metric Clashscore Ramachandran outliers Sidechaln outliers RSRZ outliers © 3D Report Full Report Percentile Ranks Mm I Pvctntil« rvlitiv* to afl X-ray ituxtum 0 FcrctnU* fMni U I-rti tmactixn of um« rnoluuoA Ligand Structure Quality Assessment 0