X-ra

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Extracting information from published crystallographic data

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published crystallographic data

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Steps in solving a structureX-ra

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clone gene ray crysta

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purify

express protein

#1

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crystallize

purify#1

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measure X-ray diffraction

calculate electron density

BMB695a

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calculate electron density

build atomic model, #4

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atomic model, interpret and

publish

#4

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TopicsX-ra

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Case 1: the refolding loop

� Missing atoms, B-factors and multiple crystal forms as a measure of flexibility

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forms as a measure of flexibility

� What is the role of crystal contacts?

� Looking at electron density

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� Looking at electron density

Case 2: How do we get from A to B?

� Superimposing similar structures 2008, K

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� Superimposing similar structures

� Looking for clashes

Case 3: How careful was I in 1999?

� Checking your favorite part of a protein structure

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� Checking your favorite part of a protein structure for possible alternative interpretations , U

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Case 1: from Selase’s PhD defenseX-ra

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• Proteolytic assay to probe for transition between transcription initiation and elongation in T7 RNA

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transcription initiation and elongation in T7 RNA polymerase

• Protein conformation of both initiation and

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• Protein conformation of both initiation and elongation state are known (1QLN, 1MSW)

• Question from the audience: “Does the refolding 2008, K

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• Question from the audience: “Does the refolding loop really refold, or does it transition from

unfolded to folded?”

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unfolded to folded?”

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Background info (straight from the thesis)X-ra

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Non-template DNA strand Intercalating valineloop

Template DNAstrandRefolding loopAT-rich recognition loop

Template DNA strand

Non-template DNA strand

A B

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Intercalating valine loop

Template DNA strand

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AT-rich recognition loop

Specificity loop

Specificity loop

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RNA

Specificity loop

Refolding loop

N-terminal 20 residues

Thumb

RNAExtended N-terminal

20 residues

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Thumb

Thumb

Figure 1.1.0: Labeled regions of the crystal structures of T7 RNA polymerase initiation and elongation complexes.

(A) Initiation complex (1QLN); (B) Elongation complex (1MSW); Part of the C-terminal domain (residues 72-152 and 204-258) iscolor coded in grey, while the rigid N-terminal domain is colored forest green. In the elongation complex, the N-terminal 20 residues

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color coded in grey, while the rigid N-terminal domain is colored forest green. In the elongation complex, the N-terminal 20 residuesgets extended by residues 44-60 to 38 residues and the entire helix is colored yellow.

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The proteolytic assayX-ra

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Figure 2.6.0: Partial tryptic

digest of transcription

complexes;

(A) SDS gel electrophoresis of

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(A) SDS gel electrophoresis ofcomplexes halted at the indicatedposition and then exposed to 1.2equivalents of trypsin for 30 secondsprior to quenching in a 5X SDS gel

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prior to quenching in a 5X SDS gelloading buffer (0.225M Tris HCl, 5%SDS, 50% glycerol and 0.05%bromophenol blue) at roomtemperature. ‘E’ shows the

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temperature. ‘E’ shows thepolymerase only, without proteolysis;(B) Plot of sink challenge data fromfigure 2.4.0 (□) compared to plot ofthe relative band intensity

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the relative band intensity(I80kDa/(I80kDa+I88kDa+I98kDa)) at eachstall position in “A” (○).

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Case 1: list of tasksX-ra

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1. Check for missing atoms/residues

2. Look at B-factors

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2. Look at B-factors

3. Check for crystal contacts

4. Look at other crystal forms

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4. Look at other crystal forms

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The Protein Database (PDB)X-ra

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• All journals require deposition of coordinates (sometimes instantly, sometimes after 6 mo)

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(sometimes instantly, sometimes after 6 mo)

• Not all journals require deposition of raw data

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• Some information is easily available directly from the PDB, but some questions require downloading the crystallographic model and looking at it with

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the crystallographic model and looking at it with specialized tools

• T7 polymerase during initiation: 1QLN

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• T7 polymerase during initiation: 1QLN

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ToolsX-ra

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• Firstglance in Jmol

• Electron density server (only if raw data available) ray crysta

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– Download density

– How well do atoms fit to map?

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– Many other aspects

• What if? (you can upload your own coordinates) 2008, K

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• What if? (you can upload your own coordinates)

– Crystal contacts to 5 Å distance

– Atomic clashes

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– Atomic clashes

• Helixweb, NIH

– B-factor plot

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– B-factor plot

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B-factors and disorderX-ra

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• Synonyms: Temperature factors, atomic displacement

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• Synonyms: Temperature factors, atomic displacement factors

• B-factors describe how the electron density of an atom is broadened by static and dynamic disorder in

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atom is broadened by static and dynamic disorder in the crystal

B-factor Displacement

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B-factor Displacement20 Å2 0.25 Å40 Å2 0.51 Å

Static disorder: distinct atomic positions in different unit cells of the crystal

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40 Å2 0.51 Å80 Å2 1.01 Å

of the crystalDynamic disorder: changes in conformation over time during the measurement

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the measurement

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Disorder makes interpretation more difficult

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Shading represents noise

more difficult

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Phosphorous

represents noise due to 1) errors inexperimental data

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Carbon

data 2) errors in phases derived from model

B

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CarbonB

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The asymmetric unitX-ra

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• Crystal symmetry duplicates/triplicates etc. molecules placed into the unit cell

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molecules placed into the unit cell

• The unique volume of the unit cell not related by crystal symmetry is called asymmetric unit

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asymmetric unit

• Non-crystallographic symmetry (NCS)– the asymmetric unit often contains more than

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– the asymmetric unit often contains more than one copy of the protein

– the operations superimposing these multiple copies are called non-crystallographic symmetry

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copies are called non-crystallographic symmetry operations

– NCS-related molecules aren’t identical and have different crystal environments

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different crystal environments

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List of structures (from Uniprot)X-ra

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1ARO, X-ray, 2.80 Å, P=1-883. 1CEZ, X-ray, 2.40 Å, A=1-883.

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1ARO, X-ray, 2.80 Å, P=1-883. 1CEZ, X-ray, 2.40 Å, A=1-883.1H38, X-ray, 2.90 Å, A/B/C/D=1-883.1MSW, X-ray, 2.10 Å, D=1-883.1QLN, X-ray, 2.40 Å, A=1-883.

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1QLN, X-ray, 2.40 Å, A=1-883.1S0V, X-ray, 3.20 Å, A/B/C/D=1-883.1S76, X-ray, 2.88 Å, D=1-883.1S77, X-ray, 2.69 Å, D=1-883.

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1S77, X-ray, 2.69 Å, D=1-883.2PI4, X-ray, 2.50 Å, A=6-883.2PI5, X-ray, 2.90 Å, A=6-883.4RNP, X-ray, 3.00 Å, A/B/C=1-883.

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4RNP, X-ray, 3.00 Å, A/B/C=1-883.

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Case 2X-ra

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• Selase found that the refolding loop is protected from proteolysis before the promoter falls off

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from proteolysis before the promoter falls off

• Can we build a model in which the refolding loop is in the elongation conformation while the rest of

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is in the elongation conformation while the rest of the N-terminal part of T7 RNAP is still in its initiation conformation? 2

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initiation conformation?

• Tasks

– Superimpose the two structures

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– Superimpose the two structures

– Look for clashes

– Modify the resulting model to remove clashes

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– Modify the resulting model to remove clashes

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Interpretation of electron densityX-ra

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Building a model into the electron density involves interpretation and prior knowledge

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involves interpretation and prior knowledge

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• Protein/solvent regions

• C-alpha trace

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• main chain, peptide direction

• sequence assignment 2008, K

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• side chain conformations

• disulfides, metals, glycosylation and other

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• disulfides, metals, glycosylation and other surprises

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Pop quiz: interpreting electron density

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Pop quiz: interpreting electron density

1) Which pairs of amino acids have very similar electron

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1) Which pairs of amino acids have very similar electron density and are thus difficult to distinguish crystallographically? Asp/Glu ; Thr/Val ; Leu/Ile ; Lys/Met ; Asp/Asn ; Leu/Asp ; Glu/Gln

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Lys/Met ; Asp/Asn ; Leu/Asp ; Glu/Gln

2) Which amino acids other than histidine have two side chain conformations resulting in almost identical electron 2

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chain conformations resulting in almost identical electron density? What could help to distinguish the two possible conformations?

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N

NH , U

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NH

N

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